CA2841097A1 - Albumin and g-csf fusion proteins - Google Patents

Abstract

The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of treating, preventing, or ameliorating diseases, disorders or conditions using albumin fusion proteins of the invention.

Description

Albumin Fusion rroteins BACKGROUND OF THE INVENTION
[00011 The invention relates generally to Therapeutic proteins (including, but not limited to, at least one polypeptide, antibody, peptide, or fragment and variant thereof) fused to albumin or fragments or variants of albumin. The invention encompasses polynucleotides encoding therapeutic albumin fusion proteins, therapeutic albumin fusion proteins, compositions, pharmaceutical compositions, formulations and kits. Host cells transformed with the polynucleotides encoding therapeutic albumin fusion proteins are also encompassed by the invention, as are methods of making the albumin fusion proteins of the invention using these polynucleotides, and/or host cells.

[0002] Human serum albumin (BSA, or HA), a protein of 585 amino acids in its mature form (as shown in Figure I (SEQ ID NO:1038)), is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. At present, HA for clinical use is produced by extraction from human blood. The production of recombinant HA (rHA) in microorganiims has been disclosed in EP 330 451 and EP 361 991.

[0003] Therapeutic proteins in their native state or when recombinantly produced, such as interferons and growth horrnaes, are typically labile molecules exhibiting short shelf-lives, particularly when formulated in aqueous solutions. The instability in these molecules when formulated for administration dictates that many of the molecules must be - lyophilized and refrigerated at all times during storage, thereby rendering the molecules difficult to transport and/or store. Storage problems are particularly acute when pharmaceutical formulations must be stored and dispensed outside of' the hospital environment.

[0004] Few practical solutions to the storage problems of labile protein molecules have been proposed. Accordingly, there is a need for stabilized, long lasting formulations of proteinaceous therapeutic molecules that are easily dispensed, preferably with a simple formulation requiring minimal post-storage manipulation: =

=
SUMMARY OF THE INVENTION
An object of the present invention is to provide albumin fusion proteins. In accordance with an aspect of the present invention, there is provided an albumin fusion protein comprising a member selected from the group consisting of:
(a) a Therapeutic protein:X and albumin comprising the amino acid sequence of SEQ ID NO:1038;
(b) a Therapeutic protein:X and a fragment or a variant of the amino acid sequence of SEQ ID NO:1038, wherein said fragment or variant has albumin activity;
(c) a Therapeutic protein:X and a fragment or a variant of the amino acid sequence of SEQ NO:1038, wherein said fragment or variant has albumin activity, and further wherein said albumin activity is the ability to prolong the shelf life of the Therapeutic protein:X compared to the shelf-life of the Therapeutic protein:X in an infused state;
(d) a Therapeutic protein:X and a'fragment or a variant of the amino acid sequence of SEQ ID NO:1038, wherein said fragment or variant has albumin activity, and further wherein the fragment or variant comprises the amino acid sequence of amino acids 1-387 of SEQ ID NO:1038;
(e) a fragment or variant of a Therapeutic protein:X and albumin comprising the amino acid sequence of SEQ ID NO:1038, wherein said fragment or variant has a biological activity of the Therapeutic protein:X;
. (f) a Therapeutic protein:X, or fragment or variant thereof, and albumin, or fragment or variant thereof, of (a) to (c), wherein the Therapeutic protein:X, or fragment or variant thereof, is fused to the N-terminus of albumin, or the N-terminus of the fragment or variant of albumin;
(g) a Therapeutic proteiniX, or fragment or variant thereof, and albumin, or fragment or variant thereof, of (a) to (e), wherein the Therapeutic protein:X, or fragment or variant thereof, is fused to the C-terminus of albuminr or the C-terminus of the fragment or variant of albumin;
(h) a Therapeutic proteni:X, or fragment or variant thereof; and albumin, or fragment or variant thereof, of (a) to (e), wherein the Therapeutic protein:X, or fragment or variant thereof, is fused to the N- terminus and C-teridinus of albumin, or the N-terminus and the C-terminus of the fragment or variant of albumin;
(i) a Therapeutic protein:X, or fragment or variant thereof, and albumin, or fragment or variant thereof; of (a) to (e), which comprises a first Therapeutic protein:X, or fragment or variant thereof, and a second Therapeutic protein:X, or fragment or variant thereof, wherein said first Therapeutic protein:X, or fragment or variant thereof, is different from said second Therapeutic protein:X, or fragment or variant thereof;
(j) a Therapeutic protein:X, or fragment or variant thereof; and albumin, or fragment or variant thereof, of (a) to (i), wherein the Therapeutic protein:X, or fragment or variant thereof, is separated from the albumin or the fragment or variant of albumin by a linker; and (lc) a Therapeutic protein:X, or fragment or variant thereof, and albumin, or fragment or variant thereof, of (a) to (j), wherein the albumin fusion protein has the following formula:
= R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, =
and further wherein RI is Therapeutic prntein:X, or fragment or variant thereof; L is a peptide linker, and R2 is albumin comprising the amino acid sequence of SEQ
110:1038 or a fragment or variant of albumin. s, In accordance with another aspect of the invention, there is provided an albumin fusion protein comprising a Therapeutic protein:X, or fragment or variant thereof, inserted into an albumin, or fragment or variant thereof, comprising the amino acid sequence of SEQ
ID NO:1038 or fragment or variant thereof.
In accordance with another aspe4 of the invention, there is provided an albumin fusion protein comprising a Therapeutic protein:X, or fragment or variant thereof, inserted into an albumin, or fragment or variant thereof, comprising an amino acid sequence selected from the group consisting of:
(a) amino acids 54 to 61 of SEQ ID NO:1038;
(b) amino acids 76 to 89 of SEQ ID 110:1038;
(c) amino acids 92 to100 of SEQ ID NO:1038;
(d) amino acids 170 to 176 of SEQ 110:1038;
(e) amino acids 247 to 252 of SEQ ID NO:1038;
2a =

=
(f) amino acids 266 to 277 of SEQ ID NO:1038;
(g) amino acids 280 to 288 of SEQ ID NO:1038;
(h) amino acids 362 to 368 of SEQ ID NO:1038;
(i) amino acids 439 to 447 of SEQ ID NO:1038;
(j) amino acids 462 to 475 of SEQ ID NO:1038;
(k) amino acids 478 to 486 of SEQ ID NO:1038; and (I) amino acids 560 to 566 of SEQ NO:1038.
In accordance with another aspect of the invention, there is provided a method of extending the shelf life of Therapeutic protein:X, or fragment or variant thereof, comprising the step of fusing the Therapeutic protein:X, or fragment or variant thereof, to albumin, or fragment or variant thereof, sufficient to extend the shelf-life of the Therapeutic protein:X, or fragment or variant thereof, compared to the shelf-life of the Therapeutic protein:X, or fragment or variant thereof, in an unfused state.
The present invention encompasses albumin fusion proteins comprising a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragment or variant thereof) fused to albumin or a fragment (portion) or variant of albumin The present invention also encompasses polynucleotides comprising, or alternatively consisting of;
nucleic acid molecules encoding a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragment or variant thereof) fused to albumin or a fragment (portion) or variant of albumin.
The present invention also encompasses polynucleotides, comprising, or alternatively consisting of, nucleic acid molecules encoding proteins comprising a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragment or variant thereof) fused to albumin or a fragment (portion) or variant of albumin, that is sufficient to prolong the shelf life of the Therapeutic protein, and/or stabilize the Therapeutic protein and/or its activity in solution (or in a pharmaceutical composition) in vitro and/or in vivo. Albumin fusion proteins encoded by a polynucleotide of the invention are also encompassed by the invention, as are host cells transformed with polynucleotides of the invention, and methods of making the albumin fusion proteins of the invention and using these polynucleotides of the invention, and/or host cells.
2b . .

In a preferred aspect of the invention, albumin fusion proteins include, but are not limited to, those encoded by the polynucleotides described in Table 2.
The invention also encompasses phatmaceutical formulations comprising an albumin fusion protein of the invention and a pitarrnaceutically acceptable diluent or carrier.
Such formulations may be in a kit or container. Such kit or container may be packaged with instructions pertaining to the extended shelf life of the Therapeutic protein.
Such formulations may be used in methods of treating, preventing, ameliorating or diagnosing a disease or disease symptom in a patient, preferably a mammal, most preferably a human, comprising the step of administering the pharmaceutical formulation to the patient.
In other embodiments, the present invention encompasses methods of preventing, treating, or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the "Preferred Indication: Y" column of Table 1 comprising administering to a patient in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a Therapeutic protein or portion corresponding to a Therapeutic 2c protein (or fragment or variant thereof) disclosed iri`the "Therapeutic Protein: X" column of Table 1 (in the same row as the disease or disorder to be treated is listed in the "Preferred Indication: Y" column of Table 1) in an amount effective to treat, prevent or ameliorate the disease or disorder.
[0009] In one embodiment, an albumin fusion protein described in Table 1 or 2 has extended shelf life.
[00101 In a second embodiment, an albumin fusion protein described in Table 1 or 2 is more stable than the corresponding unfused Therapeutic molecule described in Table 1.
[0011] The present invention further includes transgenic organisms modified to contain the nucleic acid molecules of the invention (including, but not limited to, the polynucleotides described in Tables 1 and 2), preferably modified to express an albumin fusion protein of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0012] Figure IA-IT) shows the amino acid sequence of the mature form of human albumin (SEQ ID NO:1038) and a polynucleotide encoding it (SEQ ID NO:1037).
[0013] Figure 2 shows the restriction map of the pPPC0005 cloning vector ATCC
deposit PTA-3278.
[0014] Figure 3 shows the restriction map of the pSAC35 yeast S.
cerevisiae expression vector (Sleep et al., BioTechnology 8:42 (1990)). .
[0015] Figure 4 shows the effect of various dilutions of EPO albumin fusion proteins encoded by DNA comprised in Construct ID NOS. (hereinafter CID) 1966 and 1981 and recombinant human EPO on the proliferation of TF-1 cells (see Examples 8 and 9). Cells were washed 3X to remove GM-CSF and plated at 10,000 cells/well for 72 hours in the presence of 3-fold dilutions of CID 1966 protein or Cill 1981 protein.
Concentrations used were calculated based on the weight of Epo alone,. not HSA plus Epo.
Recombinant human Epo (rhEpo) was used as the positive control and serially diluted 3 fold from 100 ng/ml to 0.01 ng/ml. Cells were exposed to 0.5 mCi/well oi3H-thymidine for an additional 18 hours.
(0) rhEpo; (V) IISA-Epo 1981; (*) Epo-}ISA 1966..
[0016] Figure 5 is a dose response analysis and shows the effect of various doses of recombinant human EPO and EPO albumin fusion proteins encoded by DNA comprised in CID 1966 and 1981 on the percent change in hematocrit from day 0 to day 7 (see Examples 8 and 9). 48 eight-week old female DBA/2Nlisd mice were divided into 12 groups of 4 animals each. Recombinant human Epo (rhEpo) was administered subcutaneously at 0.5, 1.5, 4.5 and 12 Ag/kg on days 0, 2, 4, and 6. Epo albumin fusion proteins made from constructs CID 1966 and CID 1981 were administered subcutaneously at 2, 6, 18, and 54 lig/kg on days 0, 2, 4, and 6. The higher doses of the Epo albumin fusion proteins allows a rough equimolar comparison with recombinant human Epb (note that the weight of the fusions is about 4.35 times the weight of non-glycosylated Epo). On days 0 and 7 of the experiment, the animals were bled via a tail vein and the hematocrit was determined by centrifugation.
(is) rhEpo; (o) CID 1981; (A) CID 1966.
[0017] Figure 6A shows the effect of various subcutaneous administrations of Epo albumin fusion proteins encoded by DNA comprised in CID 1966 and 1997, respectively, on the percent change in hematocrit from day 0 to day 8 (see Examples 8 and 10).
*, p<0.005 compared to rhEpo as determined by Mann-Whitney nonparametric analysis (n=6).
[0018] Figure 6B shows the effect of subcutaneous administrations of Epo albumin fusion proteins encoded by DNA comprised in CID 1997 and 1966 on the percent change in hematocrit from day 0 to day 14 (see Examples 8 and 10). *, p<0.005 compared to rhEpo as determined by Mann-Whitney nonparametric analysis (n=6); **, p<0.05 compared to rhEpo as determined by Mann-Whitney nonparametric analysis (n5).
100191 Figure 7 shows the effect of various dilutions albumin fusion proteins encoded by DNA comprised in CID 1981 and 1997, respectively, on the proliferation of TF-1 cells (see Examples 9 and 10). Cells were washed 3X to remove GM-CSF and plated at 10,000 cells/well for 72 hours in the presence of 3-fold dilutions of Epo albumin fusion proteins encoded by CID 1981 or 1997. Equimolar amounts of rhEpo were used as a positive control (4.35 times less protein added since weight of non-glycosylated Epo is 20 kd, while Epo albumin fusion proteins are 87 kd). Cells were exposed to 0.5 liCi/well of 311-thymidine for an additional 24 hours. (a) rhEpo Standard; (A) OD 1981 (CHO); (o) CID 1997 (NSO).
[0020] Figure 8 shows the effect of various doses of recombinant human EPO
(rhEpo) and EPO albumin fusion protein encoded by DNA comprised in construct 1997 (CID
1997) on the percent change in hematocrit from day 0 to day 8 (see Example 10). (A) =
rhEpo, (0) = CID 1997.
[0021] Figure 9 shows the effect of various dilutions of 11,2 albumin fusion proteins encoded by DNA comprised in CID 1812 (see Example 15) on CTLL-2 proliferation.
lx 104 cells/well were seeded in a 96-well plate= in a final volume of 200 ul of complete medium . 4 containing the indicated amount of 1L2 albumin fusion protein (CID 1812). All samples were run in triplicate. The cells were incubated for 40 hours at 37 C, then 20 ul of Alamar Blue was added and cells incubated for 8 hours. Absorbance at 530/590 was used as a measure of proliferation. EC50 = 0.386 0.021. (d) = CDD 181.
[0022] Figure 10 shows the effect of 1L2 albumin fusion protein encoded by DNA
comprised in CID 1812 on RENCA tumor growth at day 21 (see Example 15). BALB/c mice (n=10) were injected SC (midflank) with 105 RENCA cells. 10 days later mice received 2 cycles (Day 10 to Day 14 and Days 17-21) of daily (QD) injections of rIL2 (0.9 mg/kg), IL2 albumin fusion protein (CID 1812 protein; 0.6 mg/kg), or PBS (Placebo) or injections every other day (QOD) of CID 1812 protein (0.6 mg/kg). The tumor volume was determined on Day 21 after RENCA inoculation. The data are presented in scatter analysis (each dot representing single animal). Mean value of each group is depicted by horizontal line. *, p=0.0035 between placebo control and CID 1812 protein. The number in parentheses indicates number of mice alive over the total number of mice per group. (0) =
Placebo; (*) =
1L2; ) = CID 1812 protein (QD); (0) = OD 1812 protein (QOD).
[0023] Figure 11 shows the effect of various dilutions of GCSF albumin fusion proteins encoded by DNA comprised in CID 1642 and 1643 on NES-60 cell proliferation (see Examples 19 and 20). (11I) = CID 1642; (A) = C.ID 1643; (0) = HSA.
[0024] Figure 12 shows the effect of recombinant human GCSF (Neupogen) and GCSF albumin fusion protein on total white blood cell count (see Example 19), Total WBC
(103 cells/ul) on each day are presented as the group mean d SEM. GCSF albumin fusion protein was administered se at either 25 or 100 ug/kg every 4 days x 4 (Q4D) , or at 100 ug/kg every 7 days x 2 (Q7D). Data from Days 8 and 9 for GCSF albumin fusion protein 100 ug/kg Q7 are presented as Days 9 and 10, respectively, to facilitate comparison with other groups. Controls were saline vehicle administered SC every 4 days x 4 (Vehicle Q4D), or Neupogen administered SC daily x 14 (Neupogen 5 ug/kg QD). The treatment period is considered Days 1-14, and the recovery period, Days 15-28.
[0025] Figure 13 shows the effect of various dilutions of IFNI) albumin fusion proteins encoded by DNA comprised in CID 2011 and 2053 on SEAP activity in the ISRE-SEAP/293F reporter cells (see Example 25). Proteins were serially diluted from 5e-7 to le-14 g/m1 in DMEM/10% EBS and used to treat ISRE-SEAP/293F reporter cells. After hours supernatants were removed from reporter cells and assayed for SEAP
activity. IFNb albumin fusion protein was purified from three stable clones: 293F/#2011, CH0/#2011 and NS0/42053. Mammalian derived IFbib, Avonex, came from Biogen and was reported to have a specific activity of 2.0e5 1U/ug.
[0026] Figure 14 illustrates the steady-state levels of insulin mRNA in INS-1 (832/13) cells after treatment with GLP-1 or GLP-1 albumin fusion protein encoded by construct ED
3070 (CU) 3070 protein). Both GL?-1 and the CID 3070 protein stimulate transcription of the insulin gene in INS-1 cells. The first bar (black) represents the untreated cells. Bars 2-4 (white) represent cells treated with the indicated concentrations of GLP-1.
Bars 5-7 (gay) represent cells treated with the indicated concentrations of CID 3070 protein.
[0027] Figure 15 compares the anti-proliferative activity of IFN albumin fusion protein encoded by CID 3165 (CU) 3165 protein) and recombinant IFNa (rIFNa) on Hs294T
melanoma cells. The cells were cultured with v.arying concentrations of either protein or rIFNa and proliferation was measured by BrdU incorporation after 3 days of culture. ClD 3165 protein caused measurable inhibition of cell proliferation at concentrations above 10 ng/ml with 50% inhibition achieved at approximately 200 ng/ml. (it) =

protein, (*) = rIFNa.
[0028] Figure 16 shows the effect of various dilutions of IFNa albumin fusion proteins on SEAP activity in the 1SRE-SEAP/293F reporter cells. One preparation of IFNa fused upstream of albumin (*) was tested, as well as two different preparations of IFNa fused downstream of albuniin. (A) and (U).
[0029] Figure 17 shows the effect of time and dose of IFNa albumin fusion protein encoded by DNA comprised in construct 2249 (CU) 2249 protein) on the mRNA
level of OAS (p41) in treated monkeys (see Example 31). Per time point: first bar =
Vehicle control, 2nd bar b =30 ug/kg OD 2249 protein day 1 iv, third bar =30 ug/kg CID 2249 protein day 1 Sc, 4th =
oar 300 ug/kg CID 2249 protein day I se, 5th bar = 40 ug/kg recombinant IFNa day 1,3 and 5 sc.
[0030] Figure 18 shows the effect of various dilutions of insulin albumin fusion proteins encoded by DNA comprised in constructs 2250 and 2276 on glucose uptake in 3T3-Li adipoeytes (see Examples 33 and 35).
[0031] Figure 19 shows the effect of various GCSF albumin fusion proteins, including those encoded by CID #1643 and #2702 (L-171, see Example 114), on NFS cell proliferation. The horizontal dashed line indicates the minimum level of detection.
DETAILED DESCRIPTION
' 6 =
=
Definitions [00321 The following definitions are provided to facilitate understanding of certain terms used throughout this specification. ' [00331 As used herein, "polynucleotide" refers to a nucleic acid molecule having a nucleotide sequence encoding a fusion protein comprising, or alternatively consisting of, at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one Therapeutic protein X (or fragment or variant thereof); a nucleic acid molecule having a nucleotide sequence encoding a fusion protein comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NO:Y (as described in column 6 of Table 2) or a fragment or variant thereof; a nucleic acid molecule having a nucleotide sequence comprising or alternatively consisting of the sequence shown in SEQ ID NO:X; a nucleic acid molecule having a nucleotide sequence encoding a fusion protein comprising, or alternatively consisting of, the amino acid sequence of SEQ NO:Z; a nucleic acid molecule having a nucleotide sequence encoding an albumin fusion protein of the invention generated as described in Table 2 or in the Examples; a nucleic acid molecule having a nucleotide sequence encoding a Therapeutic albumin fusion protein of the invention, a nucleic acid molecule having a nucleotide sequence contained in an albumin fusion construct described in Table 2, or a nucleic acid molecule having a nucleotide sequence contained in an albumin fusion construct deposited with the ATCC (as described in Table 3).
100341 As used herein, "albumin fusion construct!' refers to a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of a Therapeutic protein (or fragment or variant thereof); a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of a Therapeutic protein (or fragment or variant thereof) generated as described in Table 2 or in the Examples; or a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least -one polynucleotide encoding at least one molecule of a Therapeutic protein (or fragment or variant thereof), further comprising, for example, one or more of the following elements: (1) a functional self-replicating vector (including but not limited to, a shuttle vector, an expression vector, an integration vector, and/or a replication system), (2) a region for =

initiation of transcription (e.g., a promoter region, such as for example, a regulatable or inducible promoter, a constitutive promoter), (3) a region for termination of transcription, (4) a leader sequence, and (5) a selectable marker. The polynucleotide encoding the Therapeutic protein and albumin protein, once part of the albumin fusion construct, may each be referred to as a "portion," "region" or "moiety" of the albumin fusion construct.
[0035] The present invention relates pnerally to polynucleotides encoding albumin fusion proteins; albumin fusion proteins; and methods of treating, preventing, or ameliorating diseases or disorders using albumin fusion proteins or polynucleotides encoding albumin fusion proteins. As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a Therapeutic protein (or fragment or variant thereof). An albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albtanin, which are associated with one another by genetic fusion (i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a Therapeutic protein is joined in-frame with a polynucleotide encoding all or a portion of albumin). The Therapeutic protein and albumin protein, once part of the albumin fusion protein, may each be referred to as a "portion", "region" or "moiety" of the albumin fusion protein (e.g., a "Therapeutic protein portion" or an "albumin protein portion"). In a highly preferred embodiment, an albumin fusion protein of the invention comprises at least one molecule of a Therapeutic protein X or fragment or variant of thereof (including, but not limited to a mature form of the Therapeutic protein X) and at least one molecule of albumin or fragment or variant thereof (including but not limited to a mature form of albumin).
[0036] in a further preferred embodiment, an albumin fusion protein of the invention is processed by a host cell and secreted into the surrounding culture medium.
Processing of the nascent albumin fusion protein that occurs in the secretory pathways of the host used for expression may include, but is not limited to signal peptide cleavage;
formation of disulfide bonds; proper folding; addition and processing of carbohydrates (such as for example, N- and 0- linked glycosylation); specific proteolytic cleavages; and assembly into multimeric proteins. An albumin fusion protein of the invention is preferably in the processed form. In a most preferred embodiment, the "processed form of an albumin fusion protein"
refers to an albumin fusion protein product which has undergone N- terminal signal peptide cleavage, herein also referred to as a "mature albumin fusion protein".

[00371 In several instances, a representative clone containing an albumin fusion construct of the invention was deposited with the American Type Culture Collection (herein referred to as "ATCC)"). Furthermore, it is possible to retrieve a given albumin fusion construct from the deposit by techniques known in the art and described elsewhere herein.
The ATCCe is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCCO deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
[00381 In one embodiment, the invention provides a polynucleotide encoding an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein and a serum albumin protein. In a further embodiment, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein and a serum albumin protein. In a preferred embodiment, the .invention provides an albumin fusion protein comprising, or alternatively consisting of, 4 Therapeutic protein and a serum albumin protein encoded by a polynucleotide described in Table 2. In a further preferred embodiment, the invention provides a polynucleotide encoding an albumin fusion protein whose sequence is shown as SEQ ID NO:Y in Table 2. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of a Therapeutic protein and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of a Therapeutic protein and a serum albumin protein. In preferred embodiments, the serum albnmin protein component of the albumin fusion protein is the mature portion of serum albumin. The invention further encompasses polynucleotides encoding these albumin fusion proteins.
[00391 In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein, and a biologically active and/or therapeutically active fragment of serum albumin. In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein and a biologically active and/or therapeutically active variant of serum albumin. In preferred embodiments, the Therapeutic protein portion of the albumin fusion protein is the mature portion of the Therapeutic protein. In a further preferred embodiment, the Therapeutic protein portion of the albumin fusion protein is the extracellular soluble domain of the Therapeutic protein. In an alternative embodiment, the Therapeutic protein =

portion of the albumin fusion protein is the active form of the Therapeutic protein. The invention further encompasses polynucleotides encoding these albumin fusion proteins.
[0040] In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment or variant of a Therapeutic protein and a biologically active and/or therapeutically active fragment or variant of serum albumin. In preferred embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, the mature portion of a Therapeutic protein and the mature portion of serum albumin. The invention further encompasses polynucleotides encoding these albumin fusion proteins.
Therapeutic proteins [0041] As stated above, a polynucleotide of the invention encodes a protein comprising or alternatively consisting of, at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human, serum albumin, which are associated with one another, preferably by genetic fusion.
[0042] An additional embodiment includes a polynucleotide encoding a protein comprising or alternatively consisting of. at least. a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are linked with one another by chemical conjugation.
[0043] As used herein, "Therapeutic protein" refers to proteins, polypeptides, antibodies, peptides or fragments or variants thereof, having one or more therapeutic and/or biological activities. Therapeutic proteins encompassed by the invention include but are not limited to, proteins, polypeptides, peptides, antibodies, and biologics. (The terms peptides, proteins, and polypeptides are used interchangeably herein.) It is specifically contemplated that the term "Therapeutic protein" encompasses antibodies and fragments and variants thereof. Thus a protein of the invention may contain at least a fragment or variant of a Therapeutic protein, and/or at least a fragment or variant of an antibody.
Additionally, the term "Therapeutic protein" may refer to the endogenous or naturally occurring correlate of a Therapeutic protein.
[0044] By a polypeptide displaying a "therapeutic activity" or a protein that is "therapeutically active" is meant a polypeptide that possesses one or more known biological and/or therapeutic activities associated with a therapeutic protein such as one or more of the Therapeutic proteins described herein or otherwise known in the art. As a non-limiting example, a "Therapeutic protein" is a protein that is useful to treat, prevent or ameliorate a disease, condition or disorder. As a non-limiting eiample, a "Therapeutic protein" may be one that binds specifically to a particular cell type (normal (e.g., lymphocytes) or abnormal e.g., (cancer cells)) and therefore may be used to target a compound (drug, or cytotoxic agent) to that cell type specifically.
[0045] For example, a non-exhaustive list of "Therapeutic protein"
portions which may be comprised by an albumin fusion protein of the invention includes, but is not limited to, erythropoietin (EPO), 11,2, G-CSF, Insulin, Calcitonin, Growth Hormone, IFN-alpha, IFN-beta, PTH, TR6 (International Publication No. WO 98/30694), BLyS, BLyS
single chain antibody, Resistin, Growth hormone releasing factor, VEGF-2, KGF-2, D-SLAM, KDI, and TR2, GLP-1, Extendin 4, and GM-CSF.
100461 Interferon hybrids may also be fused to the amino or carboxy terminus of albumin to form an interferon hybrid albumin fusion protein. Interferon hybrid albumin fusion protein may have enhanced, or alternatively, suppressed interferon activity, such as antiviral responses, regulation of cell growth, and modulation of immune response (Lebleu et al., PNAS USA, 73:3107-3111 (1976); Gresser et al., Nature, 251:543-545 (1974); and Johnson, Texas Reports Biol ilded, 35:357-369 (1977)). Each interferon hybrid albumin fusion protein can be used to treat, prevent, or ameliorate viral infections (e.g., hepatitis (e.g., HCV); or HIV), multiple sclerosis, or cancer.
100471 In one embodiment, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon alpha-interferon alpha hybrid (herein referred to as an alpha-alpha hybrid). For example, the alpha-alpha hybrid portion of the interferon hybrid albumin fusion protein consists, or alternatively comprises, of interferon alpha A fused to interferon alpha D. In a further embodiment, the. A/D hybrid is fused at the common 13gIII
restriction site to interferon alpha D, wherein the N-terminal portion of the A/D hybrid corresponds to amino acids 1-62 of interferon alpha A and the C-terminal portion corresponds to amino acids 64-166 of interferon alpha D. For example, this A/D
hybrid would comprise the amino acid sequence:

SILAVICKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQEMaRKE (SEQ ID
NO:1326), wherein the X.1 is R or K and the X2 is A or V (see, for example, Construct ID
#2875). In an additional embodiment, the A/D hybrid is fused at the common PvIIM

restriction site, wherein the N-terminal portion of the A/D hybrid corresponds to amino acids 1-91 of interferon alpha A and the C-terminal portion corresponds to amino acids 93-166 of interferon alpha D. For example, this A/D hybrid would comprise the amino acid sequence:
CDLP QTHSLGSRRTLMLLAQMRXIISLFS CLKDRHDFGFPQEEFGNQFQKAEIIPVLHE

SILAVKKYFRRITLYLTEKKYSPCAWEVVRAErMRSLSLSTNLQERLRRKE (SEQ ID
NO:1311), wherein the X1 is R or K and the second X2 is A or V (see, for example, Construct ID #2872). These hybrids are further described in 'U.S. Patent No. 4,414,510.
[00481 hi an additional embodiment, the alpha-alpha hybrid portion of the interferon hybrid albumin fusion protein consists, or. alternatively comprises, of interferon alpha A fused to interferon alpha F. In a further embodiment, the A/F hybrid is fused at the common Pvulif restriction site, wherein the N-terminal portion of the A/F hybrid corresponds to amino acids 1-91 of interferon alpha A and the C-terminal portion corresponds to amino acids 93-166 of interferon alpha F. For example, this AfFlybrid would comprise the amino acid sequence:
CDLPQTHSLGSRRTLMLLAQMRXISLFSCLKDRIIDFGFPQEEFGNQFQKAETIPVLHE
MIQQIFINTLFSTKDSSAAWDETLLDKFYTELYQQLNDMEACVIQEVOVEETPLMNVDS
ILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQERLRRKE (SEQ ID
NO:1321), wherein X is either R or K (see, for example, Construct ID #2874).
These hybrids are further described in U.S. Patent No. 4,414,510.
In a further embodiment, the alpha-alpha hybrid portion of the interferon hybrid albumin fusion protein consists, or alternatively comprises, of interferon alpha A fused to interferon alpha B. In an additional embodiment, the AM hybrid is fused at the common Pvu111 restriction site, wherein the N-terminal portion of the AM hybrid corresponds to amino acids 1-91 of interferon alpha A and the C-terminal portion corresponds to amino acids 93-166 of interferon alpha B. For example, this A/B hybrid would comprise an amino acid sequence:
CDLPQTHSLGSRRTLMLLAQMRXIISLFSCLICDRHDFGFPQEEFGNQFQKAETIPVLHE

DSILAVRKYFQRITLYLTEKKYSSCAWEVVRAEIMRSFSLSINLQKRLKSKE (SEQ ID
NO:1316), wherein the X1 is R or K and X2 through X5 is SCVM or VLCD (see, for example, Construct ID #2873). These hybrids are further described in U.S. Patent No.
4,414,510.

[0049] In another embodiment, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon beta-interferon alpha hybrid (herein referred to as a beta-alpha hybrid). For example, the beta-alpha hybrid portion of the interferon hybrid albumin fusion protein consists, or alternatively comprises, of interferon beta-1 fused to interferon alpha D (also referred to as interferon alpha-1). In a further embodiment, the beta-1/alpha 1) hybrid is fused wherein the N-terminal portion corresponds to amino acids 1-73 of interferon beta-1 and the C-terminal portion corresponds to amino acids 74-167 of interferon alpha D. For example, this beta-1/alpha D hybrid would comprise an amino acid sequence:
MSYNLLGFIQRSSNFQCQKLLWQINGRLEYCIXDRMNFDIPEEIKQLQQFQKEDAAL
TIYEMLQNIFAIFRQDSSAA'WDEDLLDKFCTELYQQLNDLEACVMQEERVGETPL1VJIN
XDSILAVKICYFRRITLYLTEKKYSPCAWEVVRAE1MRSLSLSTNLQERLRRKE (SEQ
ID NO:2130), wherein X is A or V. These hybrids are further described in U.S.
Patent No.
4,758,428.
[0050] In another embodiment, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon alpha-interferon beta hybrid (herein referred to as a alpha-beta hybrid). For example, the alpha-beta hybrid portion of the interferon hybrid albumin fusion protein consists, or alternatively comprises, of interferon alpha D (also referred to as interferon alpha-1) fused to interferon beta-1. In a further embodiment, the alpha D/beta-1 hybrid is fused wherein the N-terminal portion corresponds to amino acids 1-73 of interferon alpha D and the C-terminal portion corresponds to amino acids 74-166 of interferon beta-1. For example, this alpha 1)/beta-1 hybrid would have an amino acid sequence:
MCDLPETHSLDNRRTLMLLAQMSRISPSSCLIVIDRIMFGFPQBEFDGNQFQICAPAISV
LIIELIQQIFNLFTTKDSSSTGWNETIVENLLANVYHQINHLKTVLEEICLEICEDFTRGKL
MSSLIILKRYYGRILHYLKAKEYSHCAWTIVRVE.tIRNFYFINRLTGYLRN (SEQ JD
NO:2131). These hybrids are further described in U.S. Patent No. 4,758,428.
[0051] In farther embodiments, the interferon hybrid portion of the interferon hybrid albumin fusion proteins may comprise additional combinations of alpha-alpha interferon hybrids, alpha-beta interferon hybrids, and beta-,alpha interferon hybrids. In additional embodiments, the interferon hybrid portion of the interferon hybrid albumin fusion protein may be modified to include mutations, substitutions, deletions, or additions to the amino acid sequence of the interferon hybrid. Such modifications to the interferon hybrid albumin fusion =

proteins may be made, for example, to improve levels of production, increase stability, increase or decrease activity, or confer new biological properties.
[0052] The above-described interferon hybrid albumin fusion proteins are encompassed by the invention, as are host cells and vectors containing polynucleotides encoding the polypeptides. In one embodiment, a interferon hybrid albumin fusion protein encoded by a polynucleotide as described above has extended shelf life. In an additional embodiment, a interferon hybrid albumin fusion protein encoded by a polynucleotide described above has a longer serum half-life and/or more stabilized activity in solution (or in a phaintaceutical composition) in vitro and/or in vivo than the corresponding unfused interferon hybrid molecule.
[0053] In another non-limiting example, a "Therapeutic protein" is a protein that has a biological activity, and in particular, a biological activity that is useful for treating, preventing or ameliorating a disease. A non-inclusive, list of biological activities that may be possessed by a Therapeutic protein includes, enhancing the immune response, promoting angiogenesis, inhibiting angio genesis, regulating endocrine function, regulating hematopoietic functions, stimulating nerve growth, enhancing an immune response, inhibiting an immune response, or any one or more of the biological activities described in the "Biological Activities" section below and/or as disclosed for a given Therapeutic protein in Table I (column 2).
0054j As used herein, "therapeutic activity" or "activity" may refer to an activity whose effect is consistent with a desirable therapeutic outcome in humans, or to desired effects in non-human mammals or in other species or organisms. Therapeutic activity may be measured in vivo or in vitro. For example, a desirable effect may be assayed in cell culture.
As an example, when EPO is the Therapeutic protein, the effects of EPO on cell proliferation as described in Example 8 may be used as the endpoint for which therapeutic activity is measured. Such in vitro or cell culture assays are commonly available for many Therapeutic proteins as described in the art. Examples of assays include, but are not limited to those described herein in the Examples section or in the "Exemplary Activity Assay"
column (column 3) of Table I.
[0055] Therapeutic proteins corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, such as cell surface and secretory proteins, are often modified by the attachment of one or more oligosaccharide groups. The modification, referred to as glycosylation, can dramatically affect the physical properties of proteins and can be important in protein stability, secretion, and localization. Glycosylation occurs at specific locations along the polypeptide backbone. There are usually two major types of glycosylation:
glycosylation characterized by 0-linked oligosaccharides, which are attached to serine or threonine residues; and glycosylation characterized by N-linked oligosaccharides, which are attached to asparagine residues in an Asn-X-.Ser or Asn-X-Thr sequence, where X can be any amino acid except proline. N-acetylneuramic acid (also laiown as sialic acid) is usually the terminal residue of both N-linked and 0-linked oligosaccharides. Variables such as protein structure and cell type influence the number and nature of the carbohydrate units within the chains at different glycosylation sites. Glycosylation isomers are also common at the same site within a given cell type.
[0056] For example, several types of human interferon are glycosylated. Natural human interferon-a2 is 0-glycosylated at threonine 106, and N-glycosylation occurs at asparagine 72 in interferon-04 (Adolf et al., J. Biochem 276:511 (1991); Nyman TA et al., J. Biochem 329:295 (1998)). The oligosaccharides at asparagine 80 in natural interferon-nla may play an important factor in the solubility and stability of the protein, but may not be essential for its biological activity. This permits the production of an unglycosylated analog (interferon-13 lb) engineered with sequence modifications to enhance stability (Hosoi et al., J.
Interferon Res. 8:375 (1988; Karpusas et al, Cell Mol Life Sci 54:1203 (1998);
Knight, J.
Interferon Res. 2:421 (1982); Runkel et al., Pharm Res 15:641 (1998); Lin, Dev. Biol. Stand.
96:97 (1998)). Interferon-y contains two N-linked oligosaccharide chains at positions 25 and 97, both important for the efficient formation of the bioactive recombinant protein, and having an influence on the phannacokinetic properties of the protein (Sareneva et al., Eur. I.
Biochem 242:191 (1996); Sareneva et al,. Biochem J. 303:831 (1994); Sareneva et al., J.
Interferon Res. 13:267 (1993)). Mixed ()linked and N-linked glycosylation also occurs, for example in human erythropoietin, N-linked glycosylation occurs at asparagine residues located at positions 24, 38 and 83 while*O-linked glycosylation occurs at a serine residue located at position 126 (Lai et al., J. Biol. Chem. 261:3116 (1986); Broudy et al, Arch.
Biochem. Biophys. 265:329 (1988)).
[0057]
Glycosylation of EPO albumin fusion proteins may influence the activity and/or stability of the EPO albumin fusion proteins. The EPO portion of the albumin fusion protein may contain 3 N-linked sites for glycosylation, each of which can carry one tetra-antennary structure. When the EPO albumin fusion protein is glycosylated, the half-life of the molecule may be increased. In one embodiment, the EPO albumin fusion protein is glycosylated. In another embodiment, the EPO albumin fusion protein is hyperglycosylated.
[0058] One type of sugar commonly found in oligosaccharides is sialic acid. Each tetra-antennary structure of the N-linked glycosylafion sites of EPO may carry four sialic acid residues. Accordingly, in a preferred embodiment, the EPO albumin fusion protein is glycosylated with a carbohydrate group containing sialic acid. In an additional embodiment, the EPO albumin fusion protein comprises a fully sialylated EPO protein containing four sialic acid residues per tetra-antennerary structure pr site with a molar ratio of sialic acid to protein 12:1 or greater. In alternative embodiments, the EPO albumin fusion protein comprises a hypersialylated EPO protein wherein one, two, or three sialic acid residues are attached at each tetra-antennerary structure per site with a molar ratio of sialic acid to protein less. than 12:1. =
100591 Two types of sialic acid that may be used in the sialylation of the EPO
albumin fusion protein are N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). In a preferred embodiment, hypersialylated EPO albumin fusion proteins contain Neu5Ac. More preferably, the total sialic acid content of hypersialylated EPO
albumin fusion proteins is at least 97% Neu5Ac. Most preferred are EPO albumin fusion protein structures with little or no Neu5Gc.
100601 Preferably, the albumin EPO fusion protein has at least 4 moles of sialylation, and more preferably, at least 8-9 moles of sialylation. An additional embodiment comprises an albumin EPO fusion protein with 4 moles of sialylation, 5 moles of sialylation, 6 moles of sialylation, 7 moles of sialylation, 8-9 moles of sialylation, 8 moles of sialylation, 9 moles of sialylation, 10 moles of sialylation, 11 moles of sialylation, or 12 moles of sialylation.
[0061] The degree of sialylation of a protein changes the charge of the protein and its retention time on a chromatography column. Therefore, certain chromatography steps used in the purification process may be used to monitor or enrich for hypersialylated EPO albumin fusion proteins. In a preferred embodiment, the amount of sialylation may be monitored by HPLC chromatography. In an additional embodiment, steps in the purification process of EPO albumin fusions may be used to enrich for hypersialylated EPO albumin fusion proteins.
In a preferred embodiment the purification steps that may be used to enrich for hypersialylated EPO albumin fusion proteins comprise the butyl-sephanDeFF
purification step to remove virus particles by high ammonium salt and the hydroxyapatite chromatography at pH 6.8 for the final purification step.
[0062] Therapeutic proteins corresponding to a Therapeutic protein portion of an =

albumin fusion protein of the invention, as. well as analogs and variants thereof, may be modified so that glycosylation at one or more sites is altered as a result of manipulation(s) of their nucleic acid sequence, by the host cell in which they are expressed, or due to other conditions of their expression. For example, glycosylation isomers may be produced by abolishing or introducing glycosylation sites t e.g., by substitution or deletion of amino acid residues, such as substitution of glutamine for asparagine, or unglycosylated recombinant proteins may be produced by expressing the proteins in host cells that will not glycosylate them, e.g. in E coil or glycosylation-deficient yeast. These approaches are described in more detail below and are known in the art.
[0063] Therapeutic proteins, particularly those disclosed in Table 1, and their nucleic acid and amino acid sequences are well known in the art and available in public databases such as Chemical Abstracts Services Databases (e.g., the CAS Registry), GenBank, and subscription provided databases such as GenSeq (e.g., Derwent). Exemplary nucleotide sequences of Therapeutic proteins which may be used to derive a polynucleotide of the invention are shown in column 7, "SEQ ID NO:X," of Table 2. Sequences shown as SEQ ID
NO:X may be a wild type polynucleotide sequence encoding a given Therapeutic protein (e.g., either full length or mature), or in some instances the sequence may be a variant of said wild type polynucleotide sequence (e.g., a polynucleotide which encodes the wild type Therapeutic protein, wherein the DNA sequence of said polynucleotide has been optimized, for example, for expression in a particular species; or a polynucleotide encoding a variant of the wild type Therapeutic protein (i.e., a site directed mutant; an allelic variant)). It is well within the ability of the skilled artisan to use the sequence shown as SEQ ID
NO:X to derive the construct described in the same row. For example, if SEQ ID NO:X
corresponds to a full length protein, but only a portion of that protein is used to generate the specific CID, it is within the skill of the art to rely on molecular biology techniques, such as PCR, to amplify the specific fragment and clone it into the appropriate vector.
[0064] Additional Therapeutic proteins corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention include, but are not limited to, one or more of the Therapeutic proteins or peptides disclosed in the "Therapeutic Protein X" column of Table 1 (column 1), or fragment or variable thereof.
[0065] Table I. provides a non-exhaustive list of Therapeutic proteins that correspond to a Therapeutic protein portion of an albumin fusion protein of the invention, or an albumin fusion protein encoded by a polynucleotide of the invention. The first column, "Therapeutic 17 .
.=

Protein X," discloses Therapeutic protein molecules that may be followed by parentheses containing scientific and brand names of proteins that comprise, or alternatively consist of, that Therapeutic protein molecule or a fragment or variant thereof.
"Therapeutic protein X"
as used herein may refer either to an individual Therapeutic protein molecule, or to the entire group of Therapeutic proteins associated with a given Therapeutic protein molecule disclosed in this column. The "Biological activity" column (column 2) describes Biological activities associated with the Therapeutic protein molecule. Column 3, "Exemplary Activity Assay,"
provides references that describe assays which may be used to test the therapeutic and/or biological activity of a Therapeutic protein:X or an albumin fusion protein comprising a Therapeutic protein X (or fragment thereof) portion.
The fourth column, "Preferred Indication: Y," describes diseae, disorders, and/or conditions that may be treated, prevented, diagnosed, and/or ameliorated by Therapeutic protein X or an albumin fusion protein comprising a Therapeutic protein X (or fragment thereof) portion. The "Construct ID" column (column 5) provides a link to an exemplary albumin fusion construct disclosed in Table 2 which encodes an albumin fusion protein comprising, or alternatively consisting of the referenced Therapeutic Protein X (of fragment thereof) portion.
=
=
=

-Table 1 Therapeutic Biological Activity ,Exernplary Activity Preferred lndication:Y
... __ _ Construct ID Therapeutic ' Proteiti:X Assay t .
Protein:Z
- ..
EPO Stimulates cellular Cell proliferation assay Anemia; Anemia in Renal Disease; Anemia in 1772, 1774, 1781, 1783, See Table 2, ' . (Erythropoietin; differentiation of bone- using a erythroleukemic cell Oncology Patients; Bleeding Disorders; Chronic 1793, 1794, 1925, 1926, SEQ ID NO:Z
Epoetin alfa; ,marrow stem cells at an line TF-1. (Kitamura at al Renal Failure; Chronic Renal Failure in Pre- 1966, 1969, 1980, 1981, for particular Epoetin beta; :early stage of 1989 J.Cell. Physiol.
Dialysis Patients; Renal Disease; End-Stage 1994, 1995, 1996, 1997, construct.
Gene-activated erythropoiesis; 140:323) Renal Disease; End-Stage Renal Disease in 2047, 2102, 2283, 2284, I terythropoietin; accelerates the Dialysis Patients; Chemotherapy; Chemotherapy 2287, 2289, 2294, 2298, Darbepoetin- proliferation and in Cancer Patients;
Anemia in zidovudine-treated 2310, 2311, 2325, 2326, alpha; NESP; maturation of terminally HIV patients; Anemia in zidovudine-treated 2344, 2363, 2373, 2387, Epogen; Procrit; differentiating cells into 'patients; Anemia in HIV patients; Anemia in 2414, 2441, 2603,2604, Cl . Eprex; Erypo; erythrocytes; and premature infants;
Surgical patients (pre and/or 2605, 3194, 3195, 3196, Espo; Epoimmun; modulates the level of ' post surgery);
Surgical patients (pre and/or post 1..) ..
EPOGIN; circulating erythrocytes, surgery) who are anemic; Surgical patients (pre c 0.
= NEON.ECORMO. , . , ad/or post surgery) who are undergoing elective 'N; HEMOLINK; " am-pry; Surgical patients (pre and/or post ko .4 ',--8 Dynepo; surgery) who are undergoing elective, non- 1..) !ARANESP) ' - = cardiac surgery;
Surgical patients (pre and/or 1-.
, post surgery) who are undergoing elective, non- 1 :
1-.
,.: cardiac, non-vascular surgery; Surgical patients 1..) E (pre and/or post surgery) who are undergoing ko elective, non-vascular surgery; Surgical patients (pre and/or post surgery) who are undergoing cardiac and/or vascular surgery; Aplastic anemia;
Refraaary anemia; Anemia in Inflammatory Bowel Disease; Refractory anemia in , Inflammatory Bowel Disease; Transfusion , avoidance;
Transfusion avoidance for surgical patients; Transfusion avoidance for elective , = i surgicalpatients;
Transfusion avoidance for , elective orthopedic surgical patients; Patients , who want to Increase Red Blood Cells.
f G-CSF /Stimulates the Proliferation of =trine Chemoprotection; Adjunct to Chemotherapy; 1642, 1643, 2363, 2373, ,. See Table 2, (Granulocyte proliferation and NFS-60 cells (Weinstein et Inflammatory disorders;
Cancer; Leukemia; 2387, 2414, 2441, 2702, 1SEQ ID NO :Z :
colony- differentiation of the al Proc Nat! Acad Sci U S Myelocytc leukemia;
Neutropenia, Primary 2637, 2700, 2701,2703, Lfor partidatax s _ , Table 1 Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic Protein:X AssayProtein :Z :
stimulating factor; progenitor cells for A 1986; 83, pp5010-4) -neutroperias (e.g.; Kostrnann syndroMe).; 2886, 2887,2888, 2889, construct, Crranulolcine; granulocytes and Secondary neutropenia; Prevention of 2890, KRN 8601; monocytes-macrophirga. neutropenia;
Prevention and treatment Of Filgrastim; neutropenia in HIV-infected patients; Prevention.
Lcnograstirn; and treatment of neutropenia associated with t Meograstim; 'chemotherapy;
Infections associated with Nartograstim; neutropenias;
Myelopysplasia; Autoinuuutie o Neupogen; disorders; Psoriasis;
Mobilization of P
NOPIA; Gran; hematopoietic progenitor cells; Wound Healing; 0 N) GRANOCYTE; Autoimmune Disease;
Transplants; Bone marrow = 0 p.
Granulokine; , transplants; Acute myelogeneous leukemia;

Neutrogin; Neu- Lymphoma, Non-Hodgkin's lymphoma; Acute ko = .
.
¨.1 up; Neutronaax) = = . = lymphoblastic leukemia; Hodgkin's disease;.. , =
' i Accelerated myeloid recovery; Glycogen storage N) c) _______________________________________________________ :disease.
LA.) C) s...a.._ GM-CSF ;Regulates hernatopoietic Colony Stimulating Assay: Bone Marrow Disorders; Bone marrow 1697, 1699, 2066, and 2067.
See Table 2, 1¨`
I
- (Granulocyte-cell differentiation, gene Testa, N.G., et al., "Assays transplant; Chemoprotection; Hepatitis C; HIV SEQ ID NO:Z N) macrophage expression, growth, and for hematopoietic growth Infections; Cancer; Lung Cancer; Melanoma; for particular ko colony- .function. factors." Ballcwill FR (edt) Malignant melanoma; Mycobacterium avium construct.
stimulating factor; Cytokines, A practical complex;
Mycoses; Leukemia; Myeloid rhuGM-CSF; BI Approach, pp 229-44; IRL Leukemia;
Infections; Neonatal infections;
61012; Prokine; Press Oxford 1991. Neutropenia;
Mucositis; Oral Mucositis; Prostate Iviolgrarnostim; Cancer; StemCell Mobilization; Vaccine Sargramostim; i Adjuvant; Ulcers (such as Diabetic, Venous GM-CSF/IL 3 . Stasis, or Pressure Ulcers); Prevention of fusion; neutropenia; Acute myelogenous leukemia; , :
Ivlilodistinn Hematopoietic progenitor cell mobilization;
LeucotroPin; Lymphoma; Non-Hodgkin's lymphoma; Acute .
PROKINE; Lymphoblastic Leukemia; Hodgkin's disease; , LEUKOMAX; ; Accelerated myeloid recovery; Transplant Interberia; Rejection;
Xenotransplant Rejection.
Leukine; Leukine ' , Liquid; Pixykine) -Table I
_______________________________________________________________________________ _______________________________________ -'Therapeutic Biological Activity 7Exernplary Activity ' Preferred Indication :Y Construct ID Therapeutic Protein:X _ Assay i Protein:Z
Human growth Binds to two GHR '-Ba/F3-hGlIR proliferation Acromegaly;
Growth failure; Growth hormone 13163, 2983, See Table 2, hormone molecules and Induces assay, a novel specific replacement; Growth hormone deficiency; SEQ ID
NO:Z
(Pegvisamont; signal transduction bioassay for serum human Pediatric Growth Hormone Deficiency; Adult for particular Somatrern; through receptor growth hormone. I Clin Growth Hormone Deficiency; Idiopathic Growth construct.
1Somatropin; dirnerization Endoerinol Metab 2000 Hormone Deficiency; Growth retardation;
TROVERT; Nov;85(11):4274-9 Prader-Willi Syndrome; Prader-Willi Syndrome PROTROPIN; Plasma growth hormone in children 2 years or older; Growth deficiencies;
BIO-TROPM; ' (GH) immunoassay and Growth failure associated with chronic renal = IIIIMATROPE; tibial bioassay, App! insuffi6ency; Osteoporosis; Postmenopausal NUTROPIN; ' Physiol 2000 osteoporosis;
Osteopenia, Osteoclastogenesis; ri - . NUTROPlN AQ; Dec;89(6):2174-8 burns; Cachexia;
Cancer Cachexia; Dwarfism;
. NUTROPHIN; Growth hormone (bGH) Metabolic Disorders; Obesity;
Renal failure; c) n.) NORDITROPIN; . receptor mediated cell Tumefs Synciromea Fibronryalgia; Fracture = co .,b.
GENOTROPIN; mediated proliferation, treatment; Frailty, AIDS wasting; Muscle SAIZEN; Growth Horm IGF Res 2000 Wasting;
Short Stature; Diagnostic Agents; 0 ..i SEROSTIM) - . = Oct;10(5):248-55 Female Infertility; lipodystrophy. .
I.) . .
' ' . =Intornational standard for =
. = = = .. . 1-, .
L.,.) growth hormone, Horm Res =
. 1 199951 Suppl 1:7-12 I-, =

Insulin (Human Stimulates glucose uptaire-lInsulin activity may be Hyperglycemia; Diabetes; Diabetes Insipidus; 2250, 2255, 2276, 2278, See Table 2, - to insulin; Insulin and promotes assayed in vitro using a (3- Diabetes mellitus; Type 1 diabetes; Type 2 ,2656, 2668, 2669, 2671, SEQ ID NO:Z ko aspart; Insulin glycogenesis and HI-glucose uptake assay. (3 diabetes; Insulin resistance; Insulin deficiency; 2821, 2822, 2832, 2877, for particular Glargine; Insulin lipogerteas. Biol Chem 1999 Oct 22;
Hyperlipidernia; Hyperketonemia; Non-insulin 2878, 2882, 2885, 2891, construct.
lispro; Lys-B28 274(43)30864-30873). dependent Diabetes Mellitus (NIDDM); Insulin- 2897, 2930, 2931, 2942, Pro-B29; lyspro; dependent Diabetes Mellitus (1DDM); A '2516, 3025, 3133, 3134, `
LY 275585; Condition Associated With Diabetes Including, 3197, 3198, 2726, 2727, diarginylinsulim But Not Limited To Obesity, Heart Disease, 2784, 2789 Des-B26-B30- ' Hyperglycemia, Infections, Retinopathy, And/Or , insulin-B25- Ulcers; Metabolic Disorders; Immune Disorders; .
amide; Insulin , Obesity; Vascular Disorders; Suppression of .
deternir; LABI; , Body Weight Suppression of Appetite;
NOVOLIN; Syndrome X.
1NOVORAPID; = .
- -Table 1 _ _______________________________________________________________________________ _____________________________ Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic Protein:X Assay Protein:Z
HUMULIN;
NOVOMLX 30;
VELOSULIN;
NOVOLOG;
LANTUS;
ILETLN;
HUMALOG;
MACRULIN;

EXUBRA;
. cn INSUMAN;
1..) co ORALIN;
0.
1-.
ORALGEN;
' 0 HUMAIIALE; . ^ -4, ..-. " - = - . ' -.
....1 *
HUMAHALIN) .
"
____ cn .
.
Interferon alfa Confers a range ef Anti-viral assay: Rubinstein Viral infections; HIV Infections; Hepatitis;
2249, 2343, 2366, 2381, See Table 2, w (Interferon alfa- 'icFlhilar responses S, Familletti PC, Pestka S. Chronic Hepatitis; Hepatitis B; chronic Hepatitis 2382, 2410, and 3165. SEQ ID NO=2 1 1-. .
.
. 1-.
2b; recombinant; including antiviral, ' .(101)-Convenient assay for B; Hepatitis C; Chronic Hepatitis C; Hepatitis D; ' for particblar 1 1..) Interferon alfa-nl; antiproliferative, interferons. I. Virol.
Chronic Hepatitis D; Human Papillomavirus; construct.
ko 'Interferon alfa-n3; antitumor and 37(2):755-8; Anti- Herpes Simplex Virus Infection; External Peginterferon immunomodulatory proliferation assay: Gao Y, Condylomata Acuminate; HIV; HIV Infection;
alpha-2b; activities; stimulate at al (1999) Sensitivity of Oncology; Cancer; Solid Tumors; Melanoma; , Ribavirin and production of two an epstein-barr virus-Malignant Melanoma; Renal Cancer (e.g., Renal interferon alfa-2b; enzymes: a protein 1dnase positive tumor line, Daudi, Cell Carcinoma); Lung Cancer (e.g,. Non-Small Interferon and an oligoadettylate to alpha interferon correlates Cell Lung Cancer or Small Cell Lung Cancer) alfacon-1; synthetase. with expression of a GC- Colon Cancer; Breast Cancer; Liver Cancer;
interferon rich viral transcript. Mol Prostate Cancer;
Bladder Cancer; Gastric Cancer;
consensus; Ylvl Cell Biol. 19(11):7305-13. Sarcoma; AIDS-Related Kaposi's Sarcoma;
643; CIFN; Lymphoma; T Cell Lymphoma;
Cutaneous T- , , interferon -alpha Cell Lymphoma; Non-Hodgkin's Lymphoma;
consensus; Brain Cancer; Glioma;
Glioblastoma Multiform , recombinant Cervical Dysplasia; Leukemia;
Preleukernia;
'metbionyl 1 Bone Marrow Disorders; Bone Disorders; Hairy .
consensus _ , _ Cell Leukemia; Chronic Myelogeonus Leukemia;
- =

Table 1 Therapeutic Biological Activity ,Exemplary Activity iPreferred Indication:Y
Construct ID i Therapeutic 1 :
=
ProtXAssay Protein:Z
A ' s interferon; 'Hematological Malignancies; Hematological recombinant : 4:Disorders;
Multiple Iviyeloma; Bacterial consensus infections;
Chemoprotection; Thrombocytopenia;
interferon; COP ! Multiple Sclerosis;
Pulmonary Fibrosis; Age-35269; RO Related Macular Degeneration; Macular 253036; RO Degeneration;
Crohn's Disease; Neurological 258310; IN ______ IRON Disorders;
Arthritis; Rheumatoid Arthritis;
A; PEG- Ulcerative Colitis;
Osteoporosis, Osteopenia, INTRON; Off; Osteoclastogenesis;
Fibromyalgia; Sjogren's o OMNIFEROlt Syndrome; Chronic Fatigue Syndrome; Fever; 0 . PEG- . Hemmorhagic Fever;
Viral Hernmorhagic Fevers; iv OMNIFEROlt Hyperglycemia;
Diabetes; Diabetes Insipidus; Ø
1-, VELDONA; ): Diabetes mellitus;
Type 1 diabetes; Type 2 - 0 ko .
. ..:
'PEG- diabetes; Insulin resistance; Insulin deficiency;
t,s2, REBETRON; Hyperlipidenria;
IlyperIcetonemia; Non-insulin iv ROFERON A; ' dependent Diabetes Mellitus (NIDDIVI); Insulin- = 1-, w ..

WELLFERON; .! dependent Diabetes Mellitus (fDDM); A : = . 1-, ALFERON Condition Associated With Diabetes Including, 1-, N/LDO; ; But Not Limited To Obesity, Heart Disease, iv ko REBETRON; : Hyperglycemia, Infections, Retinopathy, And/Or = ALTEM.OL; ' ' Ulcers;
Metabolic Disorders; Immune Disorders;
VIRAFERONPF Obesity; Vascular Disorders; Suppression of .
G; PEGASYS; Body Weight;
Suppression of Appetite;
' VIRAFERON; Syndrome X.
VIRAFON;
AMPLIGEN;
INFERGEN;
INFAREX;
, ORAGEN) Calcitonin Regulates levels of ,Hypocalcernic Rat Bioassay,: Bone Disorders; Fracture prevention;
1833, 1834, 1835, 1836, 'See Table 2, (Salmon 'calcium and phosphate in bone resorbing assay and the.Hypercalcemia; Malignant hypercalcemia; 2447, 2513, 2806, 2915 SEQ
ID NO:Z
-Calcitonirt serum; causes a reduction pit assay, CT receptor lOsteoporosis; Paget's disease; Osteopenia, for particular ' ,(Salcatonin); in serum calcium--an binding assay, CAMP
lOsteociastogenesis; osteolysis; osteomyelitis:
--=-.:
construct. ._ _ , Table I
,..., .
Therapeutic Biological Activity Exemplary Activity Prefened Indication:Y
Construct II) Therapeutic -Protein:X Assay 'Protein:Z , , Calcitonin effect opposite to that of stimulation assay: J Bone losteonecrosis; periodontal bone loss;
human-salmon human parathyroid Miner Res 1999 osteaardritis; rheumatoid arthritis; osteopetrosis;
hybrid; hormone. Aug;14(8):1425-31 periodontal, lytie, or metastatic bone disease;
Forcaltonin; osteoclast differentiation inhibition; bone , Farlical; disorders; bone healing and regeneration:
Calcitonin;Calcito i 'dna Alrnirall;
' (-) Calcitonixta Hubber;
o iv Cakimar;Calsyna =
co Ø
r; Calogen; =
= 1-, ,. Miacaleic;
to -.1 . MiacaleiN . ' iv SB205614;

1-, Mactitonin=
w = Cibacalein;
. = = . = - 1-, -.
, == Cibacaltina; , iv Cibacalcine;
, Salmocalcin;
PowderJect Calcitonin) , (CAS-21215-62-3) Interferon beta 'Modulates MHC antigen Anti-viral assay: Rubinstein , Multiple Sclerosis; Oncology; Cancer; Solid 1778, 1779, 2011, 2013, See Table 2, (Interferon beta- expression, NK cell S, Familletti PC, Pestka S. Tumors;
Melanoma; Malignant Melanoma; Renal 2053, 2054, 2492, 2580, 'SEQ ID NO:Z
la; Interferon activity and IFNg (1981) Convenient assay for Cancer (e.g., Renal Cell Carcinoma); Lung 2795, 2796, 2797. Zr particular beta lb; production and IL12 interferons. J. Vito].
Cancer (e.g,. Non-Small Cell Lung Cancer or µ construct.
Interferon-beta- production in monocytes. 37(2):755-8; Anti- Small Cell Lung Cancer) Colon Cancer; Breast serine; SH 579; proliferation assay: Gao Y, Cancer; Liver Cancer; Prostate Cancer; Bladder ZK 157046; et al (1999) Sensitivity of Cancer;
Gastric Cancer; Sarcoma; AIDS-Related [' BCDF; beta-2 IF; an epstein-barr virus- Kaposi's Sarcoma; Lymphoma; T Cell Interferon-beta-2; positive tumor line, Daudi, Lymphoma;
Cutaneous T-Cell Lymphoma; Non- .
rhIL-6;,S.10031; = . to alpha interferon correlates Hodgkin's Lymphoma; Brain Cancer; Glioma;
., Table 1 Therapeutic Biological Activity Exemplary Activity - Preferred Indication :Y
Construct II) Therapeutic Protein:X Assay Protein:Z
, DL 8234; 1 with expression of a GC- 'Glioblastoma Multiforme; Cervical Dysplasia;
PERON; IFNbeta; rich viral transcript. Mol Leukemia;
Preleukemia; Bone Marrow BETASERON; Cell Biol, 19(11):7305-13. Disorders; Bone Disorders; Hairy Cell Leukemia;
AVONEX; Chronic Myelogeonus Leukemia;
Hematological flUBIF; Malignancies; Hematological Disorders; Multiple BETAFERON; Myeloma; Bacterial Infections;
SIGOSIX) Chemoprotection;
Thrombocytopenia; Viral infections; IITV Infections; Hepatitis; Chronic o Hepatitis; Hepatitis B; Chronic Hepatitis B;
Hepatitis C; Chronic Hepatitis C; Hepatitis D;

iv Chronic Hepatitis D; Human Papilloma 'virus;
! co 0.
Herpes Simplex Virus Infection; External Condylornata Acuminata; Hly.; HIV Infection;
ko .4 Pulmonary Fibrosis; Age-Related Macular Degeneration; Macular Degeneration; Crohn's r = . Disease; Neurological Disorders; Arthritis; , ' Rheumatoid Arthritis;
Ulcerative Colitis;
, 1-, . Osteoporosis, Osteopenia, Osteoclastogenesis; iv , Fibromyalgia; Sjogren's Syndrome; Chronic ko Fatigue Syndrome; Fever; Herranorhagic Fever;
Viral Hemmorhagic Fevers; Hyperglycemia;
Diabetes; Diabetes Insipidus; Diabetes mellitus;
Type 1 diabetes; Type 2 diabetes; Insulin :
, resistance; Insulin deficiency; Hyperlipidemia;
, .....
, , Ilyperketoucuila; Non-insulin dependent Diabetes Mellitus (NIDDIvI); Insulin-dependent ' Diabetes Mellitus (IDDM); A Condition ' Associate4 With Diabetes Including, But Not Limited To Obesity, Heart Disease, , Hyperglycemia, Infections, Retinopathy, And/Or : Ulcers; Metabolic Disorders;
Immune Disorders;
Obesity; Vascular Disorders; Suppression of , Body Weight; Suppression of Appetite;
A
:

, Table 1 _ _______________________________________________________________________________ ______________________________________ iTherapeutic 'Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID !Therapeutic Protein:X Assay ' Protein:Z
, ,.
I
, Syndrome X.
, õ
,Growth hormone Acts on the anterior Growth hormone-releasing Acromegaly;
Growth failure; Growth hormone 1747 and 1748, 'See Table 2, Ireleasing factor; pituitary to stimulate the peptides (GHRPs) are replacement; Growth hormone deficiency; SEQ
ID NO:Z
IGrowth hormone production and secretion known to release growth Pediatric Growth Hormone Deficiency; Adult for particular releasing of growth hormone and hormone (GH) in vivo and Growth Hormone Deficiency; Idiopathic Growth constmct.
hormone exert a trophie effect on in vitro by a direct action on Hormone Deficiency; Growth retardation; .
(Senuorelin the gland receptors in anterior Prader-Willi Syndrome; Prader-Willi Syndrome c) acetate; pituitary cells. Biological in children 2 years or older; Growth deficiencies;
, Pralmorelin; activity can be measured in Growth failure associated with chronic renal 0 1..) ,Somatorelin; cell lines expressing growth insufficiency; Osteoporosis; Osteopenia, 0 0.
Somatoliberin; hormone releasing factor Osteoelastogenesis; Postmenopausal c:.
Geref; Gerel; receptor (Mol Endominol osteoporosis;
bums; Cachexia; Cancer Cachexia; , ko ..3 'Groliberin) 1992 Oct6(10):1734-44, Dwarfism; Metabolic Disorders; Obesity; Renal . === .=
. .
1..) Molecular Endocrinology, failure; Turner's Syndrome; Fibromyalgia;
1-.
'vol 7, 77-84). 'Fracture treatment;
Frailty, AIDS wasting; . w ..: - -. 1-. Muscle Wasting;
Short Stature; Diagnostic , . = 1-.
' .
. . .... Agents; Female Infertility; lipodystrophy: 1 . ' .

, NJ
, l0 11-2 Promotes the growth of B 'T cell proliferation assay Cancer; Solid Tumors; Metastatic Renal Cell 1757, 1758, 1812,1813, See Table 2, (Aldesleukin; and T cells and augments "Biological activity of Carcinoma; Metastatic Melanoma; Malignant 1952, 1954, 2030, and 2031, SEQ ID NO:Z
interlerkin-2 NK cell and cm cell recothbinait hoeoan Melanoma; Melanoma; Renal Cell Carcinoma; for particular fusion toxin; T killing activity, interieukin-2 produced in Renal Cancer; Lung Cancer (e.g,. Non-Small Cell construct.
cell growth factor; Escherichia coli." Science Lung Cancer or Small Cell Lung Cancer); Colon , PROLEUK1N; 223: 1412-1415, 1984. Cancer;
Breast Cancer; Liver Cancer, Leukemia;
IMIVIUNACE; natural killer (NK) cell and Preleukemia; Hematological Malignancies;
CELEUK; Cli. cytotoxicity assay genmtological Disorders; Acute Myeloid ONCOLIPIN 2; . "Control of homeostasis of Leukemia;
Melanoma; Malignant Melanoma;
MACROLIN) CD8+ memory T cells by Non-Hodgkin's Lymphoma; Ovarian Cancer;
opposing cytolcines. Science Prostate Cancer; Brain Cancer; Glioma;
288: 675-678, 2000; cru- Glioblastoma Multiforme; Hepatitis; Hepatitis C;
2 Proliferation: Gillis et al Lymphoma; HIV Infection (AIDS);
(1978) J. branunol. 120, Inflammatory Bowel Disorders; Kaposi's 2027 Sarcoma; Multiple Sclerosis; Arthritis;
Rheumatoid Arthritis; Transplant Rejection;
' Table 1 , _______________________________________________________________________________ ___________________________ .
IliMrapetitie Biological Activity Exemplary Activity Preferred Indication :Y
Construct ID Therapeutic ProteitrA Assay Protein :Z
Diabetes; Type 1 Diabetes Mellitus; Type 2 ' ,..Diabetes.
-Parathyroid Acts in conjuction with Adenylylcyclase Bone Disorders; Fracture prevention; 1749, 1750, 1853, 1854, See Table 2, hormone; calcitonin to control -stimulation in rat Hypercakemia; Malignant hypercalcemia; '1889, 1906, 1907, 1914, SEQ ID NO:Z
parathyrin (PTH; calcium and phosphate losteosarcoma cells, OSteoporosis; Paget s disease; Osteopenia, 1932, 1938, 1941, 1949, for particular ' Ostabolin; ALX1- metabolism; elevates lovariectoniized rat model of Osteoclastogenesis; osteolysis; osteomyelitis; 2021, 2022, 2023, 2428, construct.
11; hPTH 1-34; blood calcium level; osteoporosis; IUBM13 Life osteonecrosis; periodontal bone loss; 2714, 2791, 2965, 2966, LY 333334; MN stimulates the activity of 2000 Feb;49(2):131-5 osteoarthritis; rheumatoid arthritis; osteopetrosis;
ci 10T; parathyroid osteocytes; enhances j periodontal, lytic, or metastatic bone disease;
hormone (1-31); absorption of Ca+/Pi fr osteoclast differentiation inhibition; bone o . .
iv = FORTEO; small intestine into blood; disorders; bone healing and regeneration. co Ø
PARATHAR) promotes reabsorption of 1-=
. .

'Ca+ and inhibits Pi=by 1 - -. . .
.
ai kidney tubules, , i .-1..) t3 R.esistrt Mediates insulin Ability of resistin to 'Hyperglycemia; Diabetes; Diabetes Insipidus; 2295, 2296, 2297, 2300, See Table 2, o . i 1-=
. . = = _ resistance in Type ll influence type II diabetes ' Diabetes mellitus; Type 1 diabetes;
Type 2 . = and 2309, SEQ ID NO:Z w . . .
I
diabetes;
is ii insulin- can be determined using 'diabetes; Insulin resistance; Insulin deficiency; for particular 1-=
1-=
stimulated glucose assays known in the art:
ilyperlipidemia; Hyperketonemia; Non-insulin construct. 1 iv uptake Pontoglio et al., I Clin dependent Diabetes Mellitus (NIDDIVI); Insulin- , ko Invest 1998 May dependent Diabetes Mellitus (IDDM); A
15;101(142215-21 Condition.
Associated With Diabetes including But Not Limited To Obesity, Heart Disease, .
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;
, ,Obesity; Vascular Disorders; Suppression of =
, Body Weight;
Suppression of Appetite;
Syndrome X.
TR6 (DcR3; Inhibits Fas Ligand and ellular apoptosis can be Fas Ligand or LIGHT induced apoptotic 1520, 1537, 1545, 1546, See Table 2, Decoy Receptor AIM-2 (TM, LIGHT) s. asured by annexin disorders:
hepatitis; liver failure (including 1568, 1570, 1622, 1623, SEQ ID NO:Z
3; FASTR) mediated apoptosis.
taining, TUNEL staining, , fulminant liver failure); graft versus host 1645, 1700, 1702, 1703, for particular asurement of caspase disease; graft rejection; myelodysplastic 1704, 1891, 1892, 1912, construct.
evels. Inhibition of cell syndrome; renal failure; insulin dependent and 1913.
= owth can also be directly _ diabetes mellitus', rheumatoid arthritigi , .

Table 1 ,.
_______________________________________________________________________________ ________________________________ Therapeutic Biological Activity !Exemplary Activity Preferred Indication:Y
IConstruct ID Therapeutic ,Protein:XjAssay Protein:Z
, ' easured, for example by inflammatory bowel disease; autoimmune IALOMAR Blue staining, disease; toxic epidermal necrolysis; multiple Assay refs: cytotoxicity sclerosis.
ssay on human ' ibrosarcoma (Epsevik and issen-Meyer, 1986, 3.
mmunol. methods).
DcCAF (D- Inhibits proliferation )eCAF activity can be B cell and/or.T cell mediated immune 1657, See Table 2, SLAM; BCM- and differentiation of B . etermined using assays disorders; Immunodeficiency (e.g., Common SEQ ID
NO:Z o iv . like membrane cells; Antagonize BLyS ,nown in the art, such as for Variable Immunodeficiency, Selective IgA for particular co Ø
protein; activity -xample, those described in Deficiency) construct, BLAME (B xamples 32-33 of , ko .
..]
.
, ' lymphocyte nternational Pilblication -. iv activator o. W00111046.

1-.
=
macrophage w , = expressed)) . ., = 1-.
-=
. .
1-.
' ' = BLyS (B Promotes proliferation, 1:LyS activity can be B cell and/or ...f cell mediated immune 1680, 2095, and 2096.
See Table 2, iv Lymphocyte differentiation and i etemiined using assays disorders, particularly immune system SEQ
ID NO:Z ko Stimulator; survival of B cells; .., own in the art, such as, disorders associated with low B cell numbers for particular Neutrokine Promotes or example, the , or low serum immunoglobulin; construct.
alpha; TL7; immunoglobulin stimulatory proliferation Immunodeficiency (e.g., Common Variable BAFF; TALL- production by B cells. ssay and other assays Immunodeficiency, Selective IgA Deficiency), , I; THANK; .' closed by Moore et al., Radiolabeled forms: lymphoma, non-Hodgkins E, radiolabeled ' 1999, Science, lymphoma, chronic lymphocytic leukemia, BLyS) '85(5425):260-3. multiple myeloma.
.
Anti-BLyS Agonize or antagonize :LyS agonist or antagonist B cell and/or T cell mediated immune 1821, 1956, 2501, 2502, . See Table 2, single chain BlyS activity. ctivity can be determined disorders; Autoimmune disorders, particularly 2638. SEQ ID NO:Z
antibody (sc sing assays known in the autoimmune diseases associated with the for particular Evil 16A01, , such as, for example, a production of autoantibodies; Rheumatoid construct.
scEvI050B11, . odified version the Arthritis, Systemic Lupus Erythrrratosus; , savI006D08) stimulatory proliferation SjOgren's Syndrome, cancers expressing Blys , and others. = ssay disclosed by Moore et as an autocrine growth factor, e.g. certain 1., 1999, Science, chronic lymphocytic leukemias. ______________ -..

Table 1 .
Therapeutic I Biological Activity Exemplary Activity Preferred ludication:Y
Construct ID Therapeutic Protein:X ' Assay , .
,Protein:Z
, 85(5425):260-3, in which t =rt lyS is mixed or =,preincubated with the anti-: lyS antibody prior to = eing applied to the , esponder B lymphocytes.
_ MPIF-1 , Inhibits myeloid '1F-1 activity can be Chemoprotection; Adjunct to Chemotherapy; 1681, 3166, 3167, 3168, See Table 2, (Myeloid progenitor cells; and easured using the Inflammatory disorders; Cancer; Leukemia; SEQ
ID NO:Z
Progenitor activates monocytes $ eloprotection assay and Myelocytic leukemia; Neutropenia, Primary for particular cr Inhibitoryhemotaxis assay described neutropenias (e.g.; Kostmann syndrome);
construct. 0 , iv Factor; OK US patent 6,001,606.
. Secondary neutropenia;
Prevention of co Ø
beta-8; neutropenia;
Prevention and treatment of = 1-, Mirostipen). 1 neutropenia in HIV-infected patients' .
' . -. tri= ko Prevention and treatment of neutropenia iv t..) s.0 associated with 'chemotherapy; Infections 0 1-, . associated with neutropenias; Myelppysplasia; w .

. . = . .. Autoimmune disorders; Psoriasis; Mobilization , .
1-, of hematopoietic progenitor cells; Wound iv Healing; Autoirnmune Disease; Transplants;
ko =
, Bone marrow transplants; Acute myelogeneous leukemia; Lymphoma, Non-Hodgkin's , lymphoma; Acute lymphoblastic leukemia;
, . Hodgkin's disease;
Accelerated myeloid recovery; Glycoon storage disease.
KIM Inhibits bone marrow *I activity can be Multiple sclerosis; Hepatitis; Cancer; Viral 1746. See Table 2, (Keratinocyte proliferation; and shows easured using the antiviral infections, HIV infections, Leukemia. SEQ ID NO:Z
Derived antiviral activity.
nd cell proliferation assays ` for particular Interferon; d escribed in Examples 57-construct.
Interferon .3 of International , Kappa ublication No.
, Precursor) 00107608.
- TNER2 (p75) Binds both TNFa and -cell proliferation can be Autoimmune disease; Rheumatoid Arthritis; 1777 and 1784. = See Table 2, ' . (BNBREL) TNFb; mediates T-cell easured using assays Psoriatic arthritis; Still's Disease;
Ankylosing 1 SEQ ID NO:Z

, Table 1 Therapeutic Biological Activity Exemplary Activity Preferred Indication :Y
Construct ID Therapeutic ProteintX Assay Protein:Z t proliferation by TNF; known in the art. For =
Spondylitis; Cardiovascular Diseases; i for particular reduces signs and example, "Lymphocytes: a Vasulitis; Wegener's granulomatosis; I construct.
, structural damage in practical approach"
edited Amyloidosis; Systemic Lupus Erythematosus, patients with by: SL Rowland, A5 Insulin-Dependent Diabetes Mellitus;
moderately to severly McMichael ¨chapter 6, Immunodeficiency Disorders; Infection;
active rheumatoid pages 138-160 Oxford Inflammation;
Inflammatory Bowel Disease;
arthritis (RA). University Press (2000); Chrohn's Disease; Psoriasis; AIDS; Graft .
o and "Current Protocols on Rejection; Graft Versus Host Disease.
, CD-ROM" section 3.12 'Proliferation Assays for T-co .
0.
cell Function John Wiley t.gc Sows, Inc. (1999).
. ________________ , .. , Keratinocyte Stimulates epithelial KGF-2 activity can be Stimulate Epithelial Cell Proliferation; 1785;1786; 1916, 1917, !
See Tab'le 2, = =
1..) growth factor 2 cell growth. measured using the wound Stimulate Basal Keratiriocytes; Wound 2498, 2499, 2552, 2553, 1 SEQ
ID NO:Z
1-.
(Repifermin; tealing assays and Healing; Stimulate, Hair Follicle Production; 2584, 2607, 2608, 2606, 1 for particalar w KGF-2; pithelial cell proliferation Healing Of Dermal Wounds. Wound Healing; 2630 construct 1-.
' Fibroblast' = = ssays described hi US
Eye Tissue Wounds, Dental Tissue Wounds, 1 1..) Growth Factor- ,,, atent 6,077,692.
Oral Cavity Wounds, Diabetic Ulcers, Dermal ko ' 10; FGF-10) Ulcers, Cubitus Ulcers, Arterial Ulcers, Venous = Stasis Ulcers, Burns Resulting From Heat Exposure Or Chemicals, or Other Abnormal -= Wound Healing Conditions such as Uremia, Malnutrition, Vitamin Deficiencies or , Complications Associated With Systemic Treatment With Steroids, Radiation Therapy or Antineoplastic Drugs or Antimetabolites;
, . Promote Dermal Reestablishment Subsequent To Dermal Loss; Increase the Adherence Of Skin Grafts To A Wound Bed; Stimulate Re-.
. Epithelialization from The Wound Bed; To Promote Skin Strength; Improve The r = Appearance Of Aged Skin; Proliferate Hepatocytes, Lung, Breast, Pancreas, Stomach, I
_________________ -Table 1 _______________________________________________________________________________ ______________________________ ...
Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic Proteiu:X Assay Protein:Z
Bladder, Small Intestine, Large Intestine;
Sebocytes, Hair Follicles, Type Il ' Pneurnocytes, Mucin.-Producing Goblet Cells, or Other Epithelial Cells, Endothelial Cells, Keratinocytes, or Basal Keratinocytes (and =. Their Progenitors) Contained Within The Skin, Lung, Liver, Bladder, Eye, Salivary Glands, or Gastrointestinal Tract; Reduce The Side . Effects Of Gut Toxicity That Result From ci ' Radiation, Chemotherapy Treatments Or Viral Infections; Cytoprotector, especially of the , 1.) Small Intestine Mucosa or Bladder; Mucositis co 0.
(Mouth Ulcers); Regenecation Of Skin; Full 1-=
.
c:.
= ¨ = =
'. . ko and/or Partial Thickness Skin Defects, . ' ...3 t.,..) ..= ' including Burns, (e.g., Repopulation Of flair 1.) Follicles, Sweat Glands, And Sebaceout = c:.
1-=
=
Glands); Psoriasis; Epidermolysis Bullosa; w .
.
, ' =, 1-=
Blisters; Gastric and/or Doudenal Ulcers;
1-=
I
Reduce Scarring; Inflarnamatory Bowel N.) L Diseases; Crohn's Disease;
Ulcerative Colitis; ko Gut Toxicity; Lung Damage; Repair Of Alveoli ;
And/or Brochiolax Epithelium; Acute Or , ' Chronic Lung Damage; Emphysema, A.RDS;
i Inhalation Injuries;
Hyaline Membrane , Diseases; Infant Respiratory Distress Syndrome; 13ronchopulmonaty Displasia In Premature Infants; Fulminant Liver Failure;
Cirrhosis, Liver Damage caused by Viral - Hepatitis and/or Toxic Substances; Diabetes =. Mellitus; Inflammation.
TR2 (and Inhibits B cell Co-stimulation B-cell Herpes; immune disorders; autoimtnune 1788 and 2129, ' See Table 2, E
TR2svl, proliferation, and Proliferation assay and Ig disease; graft versus host disease; graft SEQ ID NO:Z
TR2SV2; mediates and inhibits production assay (Moore et _., rejection; variable immunodeficiency; for particular , Table 1 ._ 'Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic TroteinA Assay Protein:Z
TNFRSF14; Herpes Simplex Virus =: I., 1999, Science, immunodeficiency syndromes; cancer.
construct.
HVEM; Herpes (HSV) infection. 285(5425):260-3.). HSV-1 Virus Entry =nd HSV-2 Infectivity Mediator; ' ssay: International , ATAR) *ublication No. WO

=
Macrophage Chemotactic for emokine activities can be Inflammatory diseases; wound healing; 1809, 2137, 2474, 2475, See Table 2, derived monocyte-derived 4eterrnin' ed using assays angiogenesis; AIDS infection. 2476, and 2477. SEQ ID
NO:Z (') = chemokine, dendritic cells and 1L-2- sown in the art:
Methods for particular 0 MDC (Ckbeta- activated natural killer ' Molecular Biology, construct. iv co 13) cells. 000, vol. 138: Chemolcine Ø
1-, ' rotocols. Edited by: A.E.I. .

ko . -. . , roudfoot, T.N.C. Wells, 1 " - =

. .
iv Id C.A. Power. C Htunana tµ.) ' ress Inc., Totowa, NJ
.... = w 1-, HAGDG59 = Activates MIP 1 a II eudritic cell assays are Immune disorders; cancer; viral infection; 1830 and 183 L
See Table 2, , (Retinal short- release in Dendritic ell known in the art. For inflammation; sepsis; arthritis; asthma.
SEQ ID NO:Z iv ko ' chain Cells. - mple, I. Immunol.
for particular dehydrogenase) 158:2919-2925 (1997); J.
construct.
ukoc. Biol. 65:822-828 1999).
-_______________________________________________________________________________ ____________________________________ .
GriRH Promotes release of nRH is known to cause Infertility; Kallmarm's syndrome or other foams 1862 arid 1863, See Table 2, (Gonadotropin follicle-stimulating , e release of follicle .. of hypergonadotropic hypergonadism (failure SEQ ID NO:Z , Releasing hormone and luteini7ing timulating hormone (FSH) to go through puberty naturally), for particular Hormone) hormone from anterior -nd/or luteini7ing hormone . construct.
pituitary. LH) in vivo by a direct -ction on receptors in nterior pituitary _onadotropes. GnRH
- ctivity can be determined sy measuring FSH levels in le medium of cultured _ = 14 Table 1 Therapeutic Biological Activity En/apiary Activity Preferred Indication:Y
Construct ID Therapeutic Protein:X . ssay Protein:Z
c =Metopes before and i ' 1 r &RH
i .. splementation. For ' .mple, Baker et at Biol 1 eprod 2000 .., __________________________________ ... ep;63(3):865-71.
./
., Teprotide Inhibits angiotensin . "bition of ACE can be ' Hypertension; congestive heart failure. 1866, 1867, 2025, and i See Table 2, converting enzyme . 4 ." .,. using assays 2026, I SEQ ID NO:Z
ci (ACE). t' own in the art. For for particular maple, Anzenbacherova construct. o .
1..) al., J.Pharma Biomed co 0.
2001 Mar; 24(5-1-, = , - ' 1' .
. :1151-6. 0 ke e -=4 . Human Involved in ernakine activities can be Autoumuune disorders; Immunity; Vascular 1933, 1934, 1947, 1948, ! See Table 2, = 1..) chemokine inflainniation, allergy, . etennined using assays and Inflammatory disorders; HIV; AIDS; 1955, 1998, 2355, 2412, SEQ ID NO:Z 0 .
1-, . HCC-1. (ckBeta- tissue rejection, viral .... 'warn the art: Methods infectious disea,ses. 2449, 2837, 2838, 2839, for particular w ...1 1; HWFBD) infection, and tumor , . Molecular Biology, 2840, 2841, 2842, 2843, , construct.
1-, biology; enhances t2000, vol. 138: Chemokine 2844, 2845, 2849, 2947, , 1..) proliferation of CD34+ protocols. Edited by: A.E.I.
3066, 3105, 3124, 3125, ke myeloid progenitor Proudfoot, T.N.C. Wells, 3139, 3152, 3153, 3154, cells. .4mtl CA. Power. 0 Htunana ' 3155, 3156, 3169, 3170, , . Press Inc., Totowa, NJ
3202, 3203, 3204, 3205, õ
-3206, 3207, 3272 - ___________________ i . ACM inhibitor Inhibits production of [inhibition of angiotensin Treatment for elevated angiotensin II and/or 1989, 2000, 2001, and See Table 2, (DX512) angiotensinll which an be determined using aldosterone levels, which can lead to 2002. I SEQ ID NO:Z
induces aldosterone 6ssays known in the art.
vasoconstriction, unpaired cardiac output for particular , production, arteriolar or example, in vitro using and/or hypertension; Cardiovascular Disease; construct.
smooth muscle proliferation assay with Cardiac Failure; Diabetes; Type II Diabetes;
vasoconstriction, and at cardiac fibroblasts as Proteinuria; Renal disorders, congestive heart , . proliferation of cardiac escribed in Naunyn failure.
fibroblasts, Induces chmiedebergs Arch angiogenesis; an hamaac.o11999 , i enzyme that converts ay;359(5):394-9.
, =

Table 1 -Therapeutic Biological Activity Exemplary Activity 1Preferred Indication:Y
Construct ID Therapeutic Protein:X Assay Protein:Z
¨ , angiotensin I to , angiotensin1-9; also cleaves des-Arg, bradylcinin and , neurotensin. .
' TRI (OCIF; Inhibits Coculture Assay for Osteoporosis;
Paget's disease; osteopenia; 2016, 2017, 2085, 2086, See Table 2, Osteoclastogene osteoclastogenesis and Osteoclastogenesis, Bone osteolysis; osteomyelitis; osteonecrosis; 2529, 2530, 2531,2532, SEQ ID NO:Z
o sis inhibitory bone resorption, and resorption assay using fetal periodontal bone loss; osteoarthritis;
2555, 2556, 2557, and for particular factor; induces fibroblast long-bone organ culture rheumatoid arthritis; osteopetrosis;
periodontal, 2558., construct. 0 1..) oospteoprtumoteogrerin, proliferation. isystem, dentine resorption lytic, or metastatic bone disease; osteoclast G
ce 0.
- , 'assay, and fibroblast . differentiation inhibition; bone disorders; bone 1-=
c:.
= necrosis factor proliferation-assays are healing and regeneration; organ calcification; 'µ ko .
, * . -.4 - receptor = each described in Kwon et vascular calcification.
1..) superfamily al,, FASEB J. 12: 845-854 1-=
member 11B 1998).
w . yrecursos) - - .. =
1-=
.
- - 1-=
Human = Chemotactic for both semokine activities can be Cancer, Wound healing; Inflammatory 2101, 2240, 2241, 2245, See Table 2, 1 1..) chemokine activated (CD3+) T . etermnied using assays disorders; Immrnunoregulatory disorders; 2246, 2247, and 2248.
SEQ ID Nal ko Ckbeta-7 cells and nonactivated = own in the art:
Methods Atherosclerosis; for particular (CD14-) lymphocytes 'n Molecular Biology, Parasitic Infection; Rheumatoid Arthritis; construct.
and (CD4+) and 000, vol. 138: Chemokine Asthma;
Autoimmune disorders.
(CD 8+) T lymphocytes 1* rotocols. Edited by: A.E.I.
and (CD45RA+) T cells i'roudfoot, T.N.C. Wells, ..d C.A. Power. C Humana = ress Inc., Totowa, NJ
CKbeta4 Attracts and activates Chemokine activities can be Cancer; Solid Tumors; Chronic Infection; 2141, 2330, 2335, 2336, See Table 2, (HGBAN46; microbicidal'determined using assays Autoimmune Disorders; Psoriasis; Asthma; 2337, 2338, and 2348.

HE9DR66) leukocytes; Attracts known in the art:
Methods Allergy; Hematopoiesis; Wound Healing; Bone for particular CCR6-expressing in Molecular Biology, Marrow Failure; Silicosis; Sarcoidosis; Hyper-construct.
immature dendritic cells l00O, vol. 138: Chemokine Bosinophilic Syndrome; Lung Inflammation;
and memory/effector T ro.tbccth. Edited by: A.E.L Fibrotic Disorders;
Atherosclerosis; Periodontal cells; B-cell roudfoot, T.N.C. Wells, diseases;
Viral diseases; Hepatitis.
chemotmds; inhibits ind CA. Power. C Humana ¨........¨.....J ______________________ =
, ..

. , Table 1 Therapeutic Biological Activity .Exemplary Activity Preferred Indication:Y
Construct ID 'Therapeutic Protein:X __________________________ Assay .
.Protein:Z
proliferation of myeloid Press Inc., Totowa, NJ
;
, progenitors; chemotaxis I of PBMC's. .
Leptin Controls obesity 6 vivo modulation of food Hyperglycemia; Diabetes; Diabetes Insipidus; 2146, 2184, 2186, and , See Table 2, :t= through regulation of intake, reduction in body Diabetes mellitus; Type 1 diabetes; Type 2 2187, SEQ ID NO:Z
'= appetite, reduction of hweight, and lowering of diabetes; Insulin resistance; Insulin deficiency;
for particular body weight, and ' . ulin and glucose levels in I
Hyperlipidernia; Hyperketonetnia; Non-insulin construct.
. lowering of insulin and biob mice, 1 dependent Diabetes Mellitus (NIDDM);
i glucose level. adioinmumoassa.y (RIA) insulin-dependent Diabetes Mellitus (IDDM); a=

.nd activation of the leptin Condition Associated With Diabetes Including, . =

, ceptor in a cell-based But Not Limited To Obesity, Heart Disease, , iv co .ssay. Protein Expr Purif t Hyperglycemia, Infections, Retinopathy, Ø
1-, 998 Dec;14(3):335-42 1, And/Or Ulcers;
Metabolic Disorders; Immune 0 ko 1 Disorders; Obesity; Vascular Disorders;
' i. - = . ..]
i....i t.ii = Suppression of Body Weight; Suppression of iv . .

=.... . Aplattite; Syndrome X; Immunological . = ' . .
1-, _ .., .
w Disorders; InummosuppressiOn=

, , 1-, _ .
; IL-1 receptor Binds HA receptor 1) Competition for IL-1 Autoimmune Disease; Arthritis; Rheumatoid 2181, 2182, 2183, and Ste Table 2, antagonist without activating the oinding to IL-1 receptors in Arthritis; Asthma;
Diabetes; Diabetes Mellitus; 2185. SEQ ID NO:Z iv ko (Anakinra; target cells; inhibits the IrT4,\ICI or C311/HeI cells GVHD; Inflammatory Bowel Disorders; for particular soluble binding of ILI-alpha ',Carter et al., Nature 344: Chron's Disease; Ocular Inflammation;
construct.
interleukin-1 and IL1-beta; and p13-638, 1990);
Psoriasis; Septic Shock; Transplant Rejection;
receptor; IRAP; neutraii7es the biologic I Inflammatory Disorders; Rheumatic Disorders;
KINERET; activity of ILl-alpha 2) Inhibition of IL-I-Osteoporosis; Postmenopausal Osteoporosis;
, ANTRIL) and IL1-beta. ,:nduced endothelial cell- Stroke.
=leukocyte adhesion (Carter lit al., Nature 344: 633-638, 990);
, . 3) Proliferation assays on .
375-C6 cells, a human naelanoma cell line highly [
kiisce_ptible to the .

Table 1 Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID !Therapeutic :
Protein:X Assay Protein :Z
' ¨
ntiproliferative action of -1 (Mural T at al., J. Biol.
'hem. 276: 6797-6806, ______________________________________ 2001). , TREM-1 Mediates activation of Secretion of cytokines, Inflammation; Sepsis; bacterial infection; 2226 and 2230. See Table 2, (Triggering neutrophil and chemokines, degranulation, autoimmune diseases; GVHD. SEQ ID NO:Z
Receptor monocytes; Stimulates and cell surface activation for particular Expressed on neutropbil and markers can be determined construct. 0 Monocytes 1) monocyte-mediated using assays described in iv inflammatory response; Bouchpn et al, J Immunol co Promotes secretion of 2000 May Ø
1-, .
=
TNF, IL-8, and MCP-I;
15;164(10):4991-5. 0 .
.
.. . Induces neutrophil .
.. . .
- ..
. .
degranulation, Ca2+
iv t.,.) mobilization and 1-, cr.
w =
tyrosine 1-, .
. .
=
, phosphorylation of 1-, , iv extracellular signal-Hko related kinase 1 (ERK1), ERK2 and phospholipase C-gamma.
HCNCA73 Induces T-cell FMAT can be used to Autoimrnune disorders; Inflammation of the 2244 and 2365. See Table 2, activation- expression measure T-cell surface gastrointestinal tract; Cancer; Colon Cancer; SEQ ID NO:Z
of CD152 marker; markers (CD69, CD152, Allergy; Crohn's disease, for particular Stimulates release of 071, HLA-DR) and T-cell construct.
TNF-a and MIP-la cytolcine production (e.g., from immature, "iFNg production). .1. of monocyte-derived Biomo1. Screen. 4:193-204 dendritic cells; 1999). Other T-cell Promotes maturation of roliferation assays: .
,.
dendritic cells. rLymphocytes: a practical 4pproach" edited by: SL
.
_____________________ , _ Table 1 ) _______________________________________________________________________________ ___________________________ Therapeutic Biological Activity Exemplary Activity Preferred Indicatiou:Y
Construct ID Therapeutic Protein :XAssay Protein:Z
_ ....
Rowland, AJ McMichael:
Chapter 6, pages 138-160 Oxford University Press (2000); WO 01/21658 Exarnpks 11-14, 16-17 and , _______________________________________________ VEGF-2 Promotes endothelial VEGF activity can be Coronary artery disease; Criticailimb Iselin-a: 2251, 2252, 2256, and See Table 2, (Vascular cell proliferation. determined using assays Vascular disease; proliferation of endothelial 2257. SEQ ID NO:2 Endothelial lcnown in the art such as cells, both vascular and lymphatic. Antagonists for particular Growth Factor- those disclosed in may be useful as anti-angiogenic agents; construct. o , 2; VEGF-C) International Publication Cancer*. 0 No. %Gt./045835, for tv co "
.
H
, -HCHI4F25 Activates MIPla Dendritic cell assays t 'are Immune disorders; cancer, 2271, 2280, and 2320, See Table 2, 0 to (jumping Release in Dendritic well known in the art For SEQ ID NO:Z -..1 translocation Cells. example, J. Rommel.
for particular I.) breakpoint) 158:2919-2925 (1997); J. .
construct.
w Leukoc. Biol. 65:822-828 i 1-.
_1199). 9 1-, i .
_ , I.) IILD01118 Activates L6/GSK3 Assays for activation of Hyperglycemia; Diabetes; Diabetes Insipidus; 2328, 2340, 2350, 2351, ee Table 2, , lD
(Bone kinase assay. GSK3 kinase activity are Diabetes mellitus;
Type 1 diabetes; Type 2 2359, 2362, 2367, 2369, EQ ID NO:Z
Morphogenic well known in the art For diabetes; Insulin resistance, Insulin deficiency 2370, 2473, )r particular Protein 9 ' example, Biol. Chem.
Hyperlipidemia; Hyperketoneznia; Non-insulin 2623, 2624, ,onstruct.
(BMP9); 379(8-9): (1998) 1101-dependent Diabetes Mellitus (NIDDM); Insulin-2625, 2631, r Growth 1110.; Biochem J. 1993 dependent Diabetes Mellitus (IDDM); A 2632, 2633.
differentiation' Nov 15;296 ( Pt 1)15-9. Condition Associated With Diabetes Includings factor-2 But Not Limited To Obesity, Heart Disease, , precursor Hyperglycemia, Infections, Retinopathy, (GDP-2 And/Or Ulcers; Metabolic Disorders; Immune precursor)) Disorders; Obesity; Vascular Disorders; , Suppression of Body Weight; Suppression of - Appetite; Syndrome 2C.
_ -, Glucagon-Like- Stimulates the synthesis G121 activity may be Ilypetglrernia; Diabetes; Diabetes Insipiclus; 2448õ2455, 2456 2457, See Table 2, , , Table I
Therapeutic Biological Activity 'Exemplary Activity Preferred Indication:Y
Construct ID 'Therapeutic `Protein:X Assay Protein:Z
, Peptide I (G1.21; and release of insulin; lassayed in vitro using a r3-Diabetes mellitus; Type 1 diabetes; Type 2 2803, 2804, 2900, 2904, SEQ
ID NO:Z
Insulinotropin) enhances the sensitivity ofilij-glucose uptake assay.
(I' diabetes; Insulin resistance; Insulin deficiency; 2945, 2964, 2982, 3070, for particular adipose, muscle, and liver B161 Chem 1999 Oct 22; Hyperlipideinia;
Hyperketonemia; Non-insulin 12802, 3027, 3028, 3045, 'construct.
tissues towards insulin; 274(43):30864-39873), (dependent Diabetes Mellitus (NIDDM); Insulin- 3046, 3069, 3071, 3072, stimtilates glucose uptake; i'dependent Diabetes Mellitus (IDDM); A 3085, 3086, 3087, 3140, = slows the digestive 1Condition Associated With Diabetes Including, 3309 = process; suppresses But Not Limited To Obesity, Heart Disease, o = appetite; blocks the Hyperglycemia, Infections, Retinopathy, And/Or co secretion of glucagon. ' Ulcers; Metabolic Disorders; Immune Disorders; iv co ". 'Obesity; Vascular Disorders; Suppression of Ø
1-, Body Weight; Suppression of Appetite;
co ko ..3 Syndrome X.
, Exendin-4 (AC- Stimulates the synthesis 'Exendhi-4 activity may be Hyperglycemia; Diabetes; Diabetes Insipidus; '2469 and 2470.
'See Table 2, iv ,2.993) and release of insulin;
assayed in vitro using a [3- Diabetes mellitus;
Type 1 diabetes; Type 2 SEQ ID NO:Z co 1-, . w t.,.) . oo enhances the sensitivity of H)-glucose uptake assay.
(I 'diabetes; Insulin resistance; Insulin deficiency; : for particular 1-, .adipose, muscle, and liver Biol Chem 1999 Oct 22; Hyperlipidernia;
iiyperkeronemia; Non-insulin , construct.
.. :. . :
. . 1 tissues towards instal; 2721(43):30864-30873.). dependent Diabetes Mellitus (N1DDM); Insulin- =iv ko stimulates glucose uptake; dependent Diabetes Mellitus (IDDM); A
slows the digestive Condition Associated With Diabetes Including, , , process; suppresses But Not Limited To Obesity, Heart Disease, appetite; blocks the Hyperglycemia, Infections, Retinopathy, And/Or = secretion of glucagon. Ulcers; Metabolic Disorders; Immune Disorders;:
Obesity; Vascular Disorders; Suppression of Body Weight; Suppression of Appetite;
=Syndrome X ' ¨
T20 (T20 HIV a peptide from residues Virus inhibition assays as HIV; AIDS; SIV (simian immunodeficiency 7777, 2672, 2673 iSee Table 2, inhibitory 643-678 of the HIV gp41 described in Zhang et al., virus) infection. ISEQ ID NO:Z .
peptide, DP178; transmembrane protein Sept. 262002, for particular DP178 HIV ectodomak which binds Sciencexpress .
construct.
inhibitory to gp41 in its resting state (www.sci6ncevress.org).
peptide) and prevents transformation to the ' fusogt.tt,stee , _ Table I
._ _______________________________________________________________________________ ______________________________________ Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic .
Protein:X Assay ' ,Protein:Z
= 1'1249 (T1249 a second generation HIV Virus inhibition assays as HIV; AIDS; SIV (simian immunodeficiency 9999, 2667, 2670, 2946 See Table 2, HIV inhibitory :fusion inbitor described in Zhang et al., virus) infection SEQ ID NO:Z
= peptide; '11249 Sept. 26 2002, for particular anti-HIV peptide) Sciencexpress construct.
' (wwW.sciencexpress.org).
.
¨ ¨
Interferon Confers a range of Anti-viral assay: Rubinstein Viral infections; HIV Infections;
Hepatitis; 2875, 2872, 2876, 2874, See Table 2, Hybrids, cellular responses

5, Familletti PC, Pestka S. Chronic Hepatitis;
Hepatitis B; Chronic Hepatitis 2873. SEQ ID NO:Z
specifically including antiviral, (1981) Convenient assay for B; Hepatitis C; Chronic Hepatitis C; Hepatitis D;= , for particular .
ci preferred: antiproliferative, .. interferons. J.
Vim!. Chronic Hepatitis D; Human Papillomavirus;
construct.
'antitumor and =37(2):755-8; Anti- Herpes Simplex Virus Infection; External 0 iv III'llalpha Alt) irumunomoduiatorj proliferation assay: Gao Y, Candylornata Acuminata; HIV; HIV Infection; co !hybrid (BglII activities; stimulate : et al (1999) Sensitivity of Oncology; Cancer; Solid Tumors; Melanoma; ' 4.
1-, . .

. version) production of two an epstein-barr virus-Malignant Melanoma; Renal Cancer (e.g., Renal .
= . ke .= .
- ., . ..]
IFNalpha'AfD enzymes: a protein kinase !positive tumor line, Daudi, Cell Carcinoma); Lung Cancer (e,g,. Non-Small - = , = .
iv = 1/4? i 2, hybrid (Pvull and an oligoadenylate to alpha interferon correlates Cell Lung Cancer or Small Cell Lung Cancer) 0 1-, - , version) . spathetase. Also, with expression of a GC- Colon Cancer; Breast Cancer;
Liver Cancer; w . .

TFNalpha AF modulates MHC antigen rich viral transcript. Mol Prostate Cancer; Bladder Cancer; Gastric Cancer;
hybrid expression, NK cell Cell Biol. 19(147305-13.
Sarcoma; AIDS-Related Kaposi's Sarcoma;

iv IFNalpha A/B 'activity and 1FNg Lymphoma; T Cell Lymphoma; Cutaneous T- ke hybrid production and IL12 Cell Lymphoma; Non-Hodgkin's Lymphoma;
IFNbeta 1/alpha production in monocytes. " Brain Cancer;
Glioma; Glioblastoma Multiforme;
D hybrid Cervical Dysplasia; Leukemia; Preleukemia;
(IFNbeta- I /alpha- . Bone Marrow Disorders; Bone Disorders; Hairy I hybrid) . Cell Leukemia;
Chronic Myelogeonus Leukemia;
IFNalpha/beta Hematological Malignancies; Hematological hybrid . Disorders;
Multiple Myeloma; Bacterial Infections; Chemoprotectio-n; Thrombocytopenia;
, Multiple Sclerosis; Pulmonary Fibrosis; Age-Related Macular Degeneration; Macular Degeneration; Crohn's Disease; Neurological , Disorders;
Arthritis; Rheumatoid Arthritis;
, k ,Ulcerative Colitis; Osteoporosis, Osteopeuia, ' ,e=p m Osteoclastagenesis; Fibroinyalgia; Siogren's _ Af .......................II¶ft. ¨

Table 1 Therapeutic Biological Activity Exemplary Activity Preferred Indication :Y
Construct ID Therapeutic Protein:X ,Assay Protein:Z
='Syndrome; Chronic Fatigue Syndrome; Fever;
Hemmorhagic Fever; Viral Hemmorhagic Fevers;
= Hyperglycemia; Diabetes; Diabetes Insipidus;
= Diabetes mellitus; Type 1 diabetes; Type 2 diabetes; Insulin resistance; Insulin deficiency;
Hyperlipidernia; Hyperketonemia; Non-insulin = dependent Diabetes Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A
= Condition Associated With Diabetes Including, But Not Limited To Obesity, Heart Disease, co =
Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;

= = = -\
Obesity; Vascular Disorders; Suppression of = =
Body Weight; Suppression of Appetite;
Syndrome X.
=
B-type natriuretic stimulates smooth muscle.linhibition of angiotensin can Congestive heart failure; cardiac volume 3119,.8888. See Table 2, peptide (BNP, relaxation and be determined using assays overload;
cardiac decompensation; Cardiac SEQ ID NO:Z
brain natriuretic vasodilation, known in the art, for Failure; Left Ventricular Dysfunction; Dyspnea for particular 1\=) peptide) natriuresis,and example using an in vitro = construct.
suppression of renin- proliferation assay with rat angiotensin and cardiac fibroblasts as endothelin. described in Naunyn Schmiedebergs Arch Pharmacol 1999 May;359(5):394-9.
Vasodilation can be measured in animals by imeasuring the myogenic responses of small renal arteries in an isobaric .
= arteriograph system (see Am J Physiol Regal Integr Comp Physiol 2002 = _________________________ Table 1 _______________________________________________________________________________ ______________________________________ ....
- , ' Therapeutic , Biological Activity -Exemplary Activity Preferred Indication:Y
,Construct ID Therapeutic Protein :X ' assay Protein :Z

Aug;283(2):R349-R355).
Natriuesis is determined by measuring the amount of sodium in the urine.
a-defensin, Suppression of HIV Virus inhibition assays as }Irv, AIDS; ARC. 3208, 3209, 3210, , See Table 2, ' including alpha 1 replication; active against described in Zhang et al, SEQ ID NO:Z
,defensin, alpha 2 bacteria, fungi, and Sept. 26 2002, for particular .
ce - defensin, alpha 3 enveloped viruses.
Sciencexpress construct . defensin (www.sciencexpress.org).
o 1..) (myeloid-related co 0.
defensan; DEFA I;
o neutrophil-ko .
-.3 ,specific defbnoilt , . = -. .
... .
CAFD
' 0 1-.
41. -e-. rhosphaADnin.' =R.egulation of phosphate Blood phosphate levels can Hyperphosphatemia.; Hyperphosphittemia in 3218. See Table 2, w .

(matrix ,.. = metabolise. .
he measured using methods chromic renal failure;
hypophosphatemia; SEQ ID NO:Z
1-.
extracellular 'known in the art Arch as the Osteomalacia; Rickets; X-linked dominant ' for particular 1..) phosphogiyeopto( Hypophosphatemic Rat hypophosphatenaic rickets/osteomalacia (XLH);
construct. ko rein; IVIEPE) ', Bioassay. Zoolog Sci 1995 autosomal dominant hypophosphatemic Oct;12(5):607-10. ricketslosteomalacia (ADHR); tumor-induced , ' ricketsfosteoroalacia (TIM
, - -, _________ -Plpal-l2 Regulation of protease- Platelet aggregation can be Protection against systemic platelet activation, 13274. See Table 2, (pepducin, activated receptor (PAR) measured using methods thrombus, heart attack, stroke, and/or coagulation ' SEQ II) NO:Z
PARI -based signal tTansduction and known in the art such as disorders. , for particular pepducin) thrombin-mediated described in Nature construct.
aggregation of human Medicine 2002 Oct; 8(10):
ylatelsts. 1161-1165.
, -P4pal-10 Regulation of protease- Platelet aggregation can be Protection against systemic platelet activation, 3275, See Table 2, (pepducin, PAR4- activated receptor (PAR) measured using methods thrombus, heart attack, stroke, and/or coagulation ,SEQ ID NO:Z
based pepducin) signal fransduction and known in the art such as disorders, (for particular thrombin-mediated described in Nature tonstruet.
t a;gregation of human _Medicine 2002 Oct; 8(10): .
________ _ _ , Table 1 , _______________________________________________________________________________ ____________________________________ Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
t Construct ID Therapeutic , Protein:X Assay 1 Protein:Z
, platelets. '1161-1165.
HRDFD27 Involved in the T-cell proliferation can be Chemoprotection; Adjunct to Chemotherapy; 2361 See Table 2, proliferation of T cells; measured using assays Inflammatory disorders; Cancer; Leukemia; SEQ ID NO:Z
Production of known in the art. For Myelocytic leukemia; Neutropenia, Primary for particular = TNFgamma.
example, "Lymphocytes: a neutropenias (e.g.;
Kostmamn syndrome); construct.
, practical approach" edited Secondary neutropenia; Prevention of by: SL Rowland, AJ neutropenia;
Prevention and treatment of = McMichael ¨ chapter

6, neutropenia in HIV-infected patients; Prevention c) pages 138-160 Oxford and treatment of neutropenia associated with o iv University Press (2000); and chemotherapy; Infections associated with . co Ø
,"Current Protocols on CD- neutropenias; Myelopysplasia; Autoinanune ROM" section 3.12 disorders;
Psoriasis; Mobilization of 0 ke ...]
.Proliferation Assays for T- hematopoietic progenitor cells; Wound Healing;
..
.
iv cell Function John Wiley & Autoimmune Disease; Transplants; Bone marrow 4=2.

. IV Soncs, Inc. (1999). transplants;
Acute infelogeneous leukemia; w .
.
..
.

= -= = - Lymphoma, Non-Hodgkin's lymphoma; Acute 1-.
lymphoblastic leukemia; HOdgIcin's disease;

iv Accelerated myeloid recovery; Glycogen storage ke disease .
, HWHGZ51 Stimulates an immune :The ability to affect Skeletal diseases and disorders;
Musculoskeletal 2407, 2408 See Table 2, =
(CD59; response and induces chondrocyte differentiation diseases and disorders; Bone fractures and/or SEQ ID NO:Z
Metastasis- inflammation by inducing can be measured using :breaks; Osteoporosis (postmenopausal, senile, or for particular associated GPI- mononuclear cell, methods known in the art, idiopathic juvenile); Gout and/or pseudogout; construct.
adhered protein eosinophil and PMN such as described in Bone Paget's disease; Osteoarthritis; Tumors and/or homolog) infiltration; Inhibits (1995) Sep; 17(3):279-86.
cancers of the bone (osteochondromas, benign growth of breast cancer, chondromas, chondroblastomas, chondromyxoid ovarian cancer, leukemia, fibromas, osteoid osteomas, giant cell tumors, and melanoma; multiple myelomas, osteosarcomas, = Overexpressed in colon, fibrosarcornas, malignant fibrous histiocytomas, lung, breast and rectal chondrosarcomas, Ewing's tumors, and/or , tumors; Regulates glucose., malignant lymphomas); Bone and joint infections and/or FFA update by (osteomyelitits and/or infectious arthritis);
adipocytes and skeletal Charcot's joints;
Heel spurs; Sever's disease;
, ,, Table 1 !Therapeutic Biological Activity Exemplary Activity Preferred Indication :Y
Construct ID Therapeutic ,Proteitt:X Assay Protein :Z , muscle; Induces Sport's injuries;
Cancer; Solid Tumors;
redifferentiation of Melanoma; Malignant Melanoma; Renal Cancer chondrocytes (e.g., Renal Cell Carcinoma); Lung Cancer (e.g,, Non-Small Cell Lung Cancer or Small Cell Lung Caner) Colon Cancer; Breast Cancer; Liver Cancer; Prostate Cancer; Bladder Cancer; Gestic Cancer; Sarcoma; AIDS-Related Kaposfs Sarcoma; Lymphoma; T Cell Lymphoma;
Cutaneous T-Cell Lymphoma; Non-Hodgkin's Lymphoma; Brain Cancer; Glioma; Glioblastoma Muldforrne; Cervical Dysplasia; Leukemia;
co Preleulcemia; Bone Marrow Disorders; Bone =
= = = Disorders;
Hairy Cell Leukemia; Chronic = Myelogeortus Leukemia; Hematological c.k.) Malignancies;
Hematological Disorders; Multiple = = . Myelorna;
Kidney diseases and disorders;
= = Shonlein-Henoch purpura, Berger disease, celiac disease, dermatitis herpetifonnis, Citron disease;
Diabetes; Diabetes Insipidus; Diabetes mellitus;
rs Type I diabetes; Type 2 diabetes; Insulin resistance; Insulin deficiency; Ilyperlipidemia;
Hyperketenetniaõ. Non -insulin dependent Diabetes Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A Condition Associated With Diabetes Including, But Not Limited To Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;
Obesity; Vascular Disorders; Suppression. of Body Weight; Suppression of Appetite;
Syndrome X; Kidney disorders;
Hyperinsulinemia; Hypoinsulinemia;
,hrimunole_gical disorders (el& arthritis, asthma, Table 1 ITherapeutic Biological Activity Exemplary Activity PreferredIadication:Y
iConstruct II) Therapeutic TroteitEX Assay Protein:Z
, , sitieiramodoennadefilency diseases, AIDS, rheumatoid arthritis, granulomatous disease, inflammatory bowl disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, T-cell mediated cytotoxicity, host-versus-graft disease, .autoimmunity disorders, demyelination, systemic .
lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjorgren's disease, A .
I;C 1 _ C17 (cytokine¨JInhibits glucose and/or Proliferation of kidney Kidney diseases and disorders; Shonlein-Henoch :2489, 2490 See Table 2, co Ø
like protein C17) PFA uptake by mesangial cells can be purpura, Berger disease, celiac disease, SEQ ID NO:Z

adipocytes; Induces assayed using techniques dermatitis heipetiformis, Chron disease;. for particular . ko .
--]
. ,proliferation of kidney described in J. Ltivestig.
: Diabetes; Diabetes Insipidus; Diabetes mellitus;
construct. iv Imesangial cells; Med. (1998) Aug; Type I diabetes.; Type 2 diabetes; Insulin 0 1-, -4. ;Regulation of cytokine 46(6):297-302.
resistance; Insulin deficiency; Hyperlipidernia; .
_ w 4:u.
:, . 1 .
Iproduction and antigen . = '1-, = = Ilyperketonernia; Non-insulin dependent I .
! ' 'presen . tation Diabetes Mellitus (NIDDM); Insulin-dependent 1 iv Diabetes Mellitus (IDDM); A Condition ko Associated With Diabetes Including, But Not Limited To Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or = Ulcers; Metabolic Disorders; Immune Disorders;
Obesity; Vascular Disorders; Suppression of Body Weight; Suppression of Appetite;
Syndrome X; Kidney disorders;
Hyperinsulinernia; Hypoinsulinemia;
= , Hcmatopoietic disorders; Immunological diseases and disorders; Developmental diseases : and disorders; Hepatic diseases and disorders;
'Cancer (particularly leukemia); Immunological , =
disorders (e.g. arthritis, asthma, immunodeficiency diseases, AIDS, rheumatoid arthritis, granulomatous disease, inflammatory ( ., Table 1 4.
_______________________________________________________________________________ ___________________________________ Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct II) Therapeutic I
Protein:X
¨_ t. Assay Proteiu:Z 1 bow! disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, T-cell Mediated cytotoxkity, host-versus-graft disease, autoirnmunity disorders, demyelination, systemic lupus erythematosis, drug induced hemolytic ' anemia, rheumatoid arthritis, Siorgren's disease, ,scierodenna) -HDPBQ71 Regulates production and Such assays that may be Blood disorders and infection (e.g., viral 2515, 2545 See Table 2, o secretion of TENgamma; used or routinely modified infections, tuberculosis, infections associated SEQ ID NO:Z
Activation of myeloid to test irrnnunomodulatory with chronic granulornatosus disease and for particular 0 iv cells and/or activity of polypeptides of malignant osteoporosis); Autoinmiune disease construct co Ø
=
hematopoietic cells the invention (including (e.g., rheumatoid arthritis, systemic lupus c:.
ko antibodies and agonists or erythematosis, multiple sclerosis); , antagonists of the invention) Inununodeficiency, boosting a T cell-mediated iv .
.P.0 include the assays disclosed immune response, and suppressing a T cell-1-, t..n i.
w . - . - in Miraglia eta!. I
mediated immune response;
Inflammation and , .- 1-, = . iontolecular Screening inflammatory disorders; Idiopathic pulmonary 4:193-204 (1999); Rowland fibrosis; Neoplastic diseases (e.g., leukemia, 1..) et al., ""Lymphocytes: a lymphoma, melanoma);
Neoplasms and cancers, ko practical approach"" such as, for example, leukemia, lymphoma, Chapter 6138-160 (2000); melanoma, and prostate, breast, lung, colon, = Gonzalez et al., 3 Clin Lab pancreatic, esophageal, stomach, brain, liver and =. Anal 8(5):225-233 (1995); urinary cancer;. Benign dysproliferative disorders . :.
Mien etal., Ann NY Acad and pre-neoplastic conditions, such as, for .
Sci 856:22-32 (1998); example, hyperplasia, metaplasia, and/or Boehm et al., Annu Rev dysplasia; Anemia;
Pancytopenia; Leukopenia;
Innurnel 15:749-795 Tfnornbocytopenia;
Hodgkin's disease; Acute (1997), and Rheumatology lymphocytie anemia (ALL); Plasmacytornas;
(Oxford) 38(3):214-20 Multiple myeloma; Bm-kitt's lymphoma;
(1999) Arthritis; AIDS;
Granulomatous disease;
Inflammatory bowel disease; Sepsis;
, ,Neutropenia;
Neutrophilia; Psoriasis;
Suppression of immune reactions to tramplaMed i Table 1 Therapeutic t- Biological Activity Exemplary Activity 1Preferred Indication:Y
Construct ID Therapeutic protein:XAssay Pr otein:Z
, _ _______________ organs and tissues; Hemophilia;
Hypercoagulation; Diabetes mellitus;=
, - ,Endocarditis;
Meningitis; Lyme Disease;
Asthma; Allergy Oscar (osteoclast- Regulator of osteoclast Assay to detect osteoclast Skeletal diseases and disorders; Musculoskeletal 2571, 2749 See Table 2, as.sociated differentiation; regulator differentiation is described diseases and disorders; Bone fractures and/or SE,'Q ID NO2 receptor isoform- of innate and adaptive in J. Exp. Med. (2002) Jan breaks; Osteoporosis (postmenopausal, senile, or for particular o -3) immune responses 21; 195(2):201-9.
idiopathic juvenile); Gout and/or pseudogout;
construct.
Paget's disease; Osteoarthritis; Tumors and/or cancers of the bone (osteochondromas, benign co Ø
chondroraas, chondroblastomas, chondromyxoid .
c:.
fibromas, osteoid osteomas, giant cell tumors, - ke -.3. , =
multiple myelornas, osteosarcomas, .
1..) fibrosairomas, malignant fibrous histiocytomas, ....
1-, chondrosarcornas, Evving's tumors, and/or w ..
. -= . malignant lymphomas);
Bone and joint infections 1-1 1-, L (osteomyelitits and/or infectious arthritis); 1 1..) Charcot's joints; Heel spurs; Sever's disease;
ke Sport's injuries õ
..
Turnstatin (T5, Inhibits angiogenesis;
A tumor cell proliferation Cancer; Solid Tumors; Melanoma; Malignant 2647,2648, 2649, 2650, See Table 2, T7 or T8 peptide; Inhibits tumor growth;
assay is described in J. Biol. Melanoma; Renal Cancer (e.g., Renal Cell 2943, 2944, 3047, 3048 SEQ ID NO:Z
a3(111)NC1) Inhibits protein synthesis ' Chem. (1997) 272:20395-Carcinoma); Lung Cancer (e.g,. Non-Small Cell I for particular 20401. Lung Cancer or Small Cell Lung Cancer) Colon construct.
Protein synthesis can be Cancer; Breast Cancer;
Liver Cancer; Prostate , : measured as described in Cancer;
Bladder Cancer; Gastric Cancer;
Science (2002) Jan 4; Sarcoma; AIDS-Related Kaposi's Sarcoma;
i 295(5552):140-3. Lymphoma; T Cell Lymphoma; Cutaneous T-, Cell Lymphoma; Non-Hodgkin's Lymphoma;
Brain Cancer; Glioma; Glioblastoma Multiforme;
, . Cervical Dysplasia;
Leukemia; Preleukemia;
. Bone Marrow Disorders;
Bone Disorders; Hairy , Cell Leukemia; Chronic Myelogeonus Leukemia;
HematologicaIMalignancies; Hematological .
---, Table l , Therapeutic Biological Activity Exemplary Activity Preferred ludication:Y
Construct ID Therapeutic Protein:X AssayProtein:Z
_ ) IhSerderl;
Iviu%pie Warms; Anogeriesis CNTF (Ciliary Enhances myelin Regulation of myelin Neurological and neural diseases and disorders, 2724, 2725, 3171, 3172 See Table 2, neurotrophic formation; Reduces formation can be assayed as particularly diseases and disorders associated SEQ ID NO:Z
factor) photoreceptor described in J. Neurosci.
with myelin and demyelination, such as, for for particular degredation; Regulates (2002) Nov. 1; 22(21):9221- example, ALS, multiple sclerosis, Huntington's construct.
' calcium currents 7. disease; NeurOnal and spinal cord injuries;
Disorders of the eye, such as, for example, retinitis pigmentosa, blindness, color-blindness, k macular degeneration.
, _______________________________________________________________________________ ______________________________________________ c.
Sornatostatin Inhibits growth hormone, Inhibition of growth Cancer; Metastatic carcinoid tumors; Vasoactive 2798, 2825, 2830, 2831, See Table 2, iv (Octreotide; ,glucagons and insulin;
hormone release in humans Intestinal Peptide secreting adenomas; Diarrhea 2902 SEQ ID NO :Z co Ø
1-, octreotide acetate; Suppresses LF response by somatostatin can be and Flushing; Prostatic disorders and cancers;
for particular , 0 .
ko Sandostating to GriRH; Decreases measured as described in J. Breast cancer;
Gastrointestinal disorders and construct .
. ...3 .

LARS) splanchnic blood flow; Chn. Endocrinol, Metab. cancers; Cancers of the endocrine system; Head iv ..ta.c.
---) Inhibits release of (1973) Oct; 37(4):632-4.
and neck paragangliomas; Liver disorders and ' =
1-, w ' serotonin, gastrin, Inhibition of insulin' = cancers;
Nasopharyngeal cancers; Thyroid 1 .
vasoactive intestinal . ecretion by somatosiatin disorders and cancers; Acromegaly; Carcinoid .---'peptide, secretin, motilin, can be measured as Syndrome;
Gallbladder disorders, such as ko and pancreatic described in the Lancet 'gallbladder contractility diseases and abnormal ,polypeptide. (1973) Dec. 8; bile secretion;
Psoriasis; Diabetes; Diabetes 2(7841):1299-1301. lbsipidus; Diabetes mellitus; Type I diabetes;
Type 2 diabetes; Insulin resistance; Insulin deficiency; HyperLipidentia; Hyperketonemia;
. Non-insulin dependent Diabetes Mellitus (N1DDM); Insulin-dependent Diabetes Mellitus (IDDM); A Condition Associated With Diabetes balding, But Not Limited To Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; Inanune . Disorders;
Obesity; Vascular Disorders;
Suppression of Body Weight; Suppression of Appetite; Syndrome X; Kidney disorders;
Neurolo; "cal disorders and diseases includin t -Table I ,.
Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic Protein:X Assay , Protein:Z
_ Alzheiroers Disease, Parkinson's disease and = dementia; Neuropsychotic disorders, including Bipolar affective disorder; Rheumatoid arthritis;
= Hypertension; Intracranial hypertension;
Esophageal varices; Graves' disease; Seizures;
. Epilepsy; Gastritis;
Angiogenesis;
1L-22 (I122, Stimulates glucose uptake 1L-22 activity may be Hyperglycemia; Diabetes; Diabetes Insipidus; 2901, 2903 See Table 2, interleukin-22; in skeletal muscle cells; :assayed in vitro using a [3-Diabetes mellitus; Type 1 diabetes; Type 2 SEQ ID NO:Z 0 =IL17D, IL27) increases skeletal muscle IlIghicose uptake assay. (I
iliabetes; Insulin resistance; Insulin deficiency; for particular 0 insulin sensitivity.- Biol Chem 1999 Oct 22;
Hyperlipidemia; Hyperketonemia; Non-insulin ' construct. "
co 274(43):30864-30873). dependent Diabetes Mellitus (NIDDM); Insulin- Ø
1-, ' dependent Diabetes Mellitus (IDDM); A . ke == =
-..3 . Condition Associated With Diabetes Including, = iv But Not Limited To Obesity, Heart Disease, 4:.
.
1-, co= Hyperglycemia, Infections, Retinopathy, And/Or ' w - t , = . cers; Metabolic Disorders; Immune Disorders; - . = = - = .. - =
*
. - = === -1-, , - Obesity; Vascular Disorders; Suppression of , iv Body Weight; Suppression of Appetite;
ke ' Syndrome X.
.
liCE1P80 Stimulates glucose uptake ;HCE1P80 activity may be Byperglycemia; Diabetes; Diabetes Insipidus; 2908, 3049, 3050, 3051, , See Table 2, in; increases insulin assayed in vitro using a [3- -Diabetes mellitus; Type 1 diabetes; Type 2 3052 SEQ ID NO:Z
sensitivity. HI-glucose uptake assay. (1, diabetes;
Insulin resistance; Insulin deficiency; for particular Biel Chem 1999 Oct 22; Hyperlipidernia;
Hyperketonemia; Non-insulin construct.
274(43):30864-30873). dependent Diabetes Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A
Condition Associated With Diabetes Including, But Not Limited To Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;
= Obesity; Vascular Disorders; Suppression of t. Body Weight; Suppression of Appetite;
Syndrome X, , .= -0--HDRMI82 Stimulates glucose HDRMI82 activity may be Hyperglycemia; Diabetes;
Diabetes Insipidus; _2909 See Table 2, , .
õ

Table 1 , _______________________________________________________________________________ ______________________________________ , Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic ' Protein:X Assn Protein:Z
uptake; increases insulin assayed in vitro using a [3- Diabetes mellitus; Type 1 diabetes; Type 2 'SEQ ID NO:Z
sensitivity. *glucose uptake assay. (I diabetes;
Insulin resistance; Insulin deficiency; for particular Biol Chem 1999 Oct 22; Hyperlipidetnia;
Hyperketonernia; Non-insulin construct.
274(43):30864-30873). dependent Diabetes Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A
Condition Associated With Diabetes Including, . .. But Not Limited To Obesity, Heart Disease, , Hyperglycemia, Infections, Retinopathy, And/Or (-) Ulcers; Metabolic Disorders; Immune Disorders;
Obesity; Vascular Disorders; Suppression of iv = = .
, . Body Weight; Suppression of Appetite; 03 0.
=
Syndrome X. 1-=
cp .HDALV07 Modulates insulin action Insulin activitymv be Diabetes; Diabetes Insipidus; Diabetes mellitus;
' 3053, 3055, 3056 'See Table 2, ke=
. <

(adiponectin; assayed in vitro using a [3- Type 1 diabetes; Type 2 diabetes; Insulin SEQ ID NO:Z iv t . gelatin-binding *glucose uptake assay. (I resistance;
Insulin deficiency; Hyperlipidemia; for particular 0 1-=
.. = 28k protein - . . Biol. Chen-11999 Oct 22;
Hyperketonemia; Non-insulin dependent =
construct. w precurson; ' 274(43):30864-30873).
Diabetes Mellitus (NIDDM);
Insulin-dependent 1-=
1-=
adipose most Diabetes Mellitus (IDDM); A Condition ' iv abundant gene Associated With Diabetes Including, But Not ke=
transcript; APM- õ Limited To Obesity, Heart Disease, 1; 0BP28; Hyperglycemia,.
Infections, Retinopathy, And/Or ACRP30; Ulcers; Metabolic Disorders; Immune Disorders;
ADIPOQ) Obesity; Vascular Disorders; Suppression of Body Weight; Suppression of Appetite;
Syndrome X; Hyperglycemia; Familial combined . hyperlipidemia;
Metabolic syndrome;
. Inflammatory disorders; Atberogertic disorders .
C Peptide An insulin precursor C-peptide concentrations Diabetes; Diabetes Insipidus;
Diabetes mellitus; 3088, 3149 See Table 2, involved in insulin can be measured using Type 1 diabetes; Type 2 diabetes; Insulin SEQ
ID NO:Z
regulation assays well known in the art, resistance; Insulin deficiency; Hyperlipidemia; for particular :
such as the one described in Hyperketonemia; Non-insulin dependent construct.
PNAS (1970) Sep; Diabetes Mellitus (NIDDM); Insulin-dependent 6701148-55 Diabetes Mellitus (1DDM); A Condition Table 1 __________________________________________________________ _ __________________________________________________________ Therapeutic Biological Activity Exemplary Activity Preferred Iudication:Y
Construct ID Therapeutic 1 ,Proteiti:X Assay Protein:Z ' Associated With Diabetes Including, But Not , ,Limited To Obesity, Heart Disease, ' Hyperglycemia, Infections, Retinopathy, AndlOr ' Ulcers; Metabolic Disorders; Immune Disorders;
. Obesity; Vascular Disorders; Suppression of it Body Weight;
Suppression of Appetite;
Syndrome X; Hyperglycemia; Familial combined c) hyperlipidemia; Metabolic syndrome ..=
HCBOG68 Controls proliferation/ Activation of cAIVIP- Treatment of Obesity; treatment of Diabetes; 3106, 3270 See Table 2, o (enteric differentiation or mediated transcription in suppression of body weight gain;
suppression of SEQ ID NO:Z 1..) co .
0.
adipokine; Fat metabolism/ adipocytes can be assayed appetite.
Hyperglycemia; Diabetes; Diabetes for particular SID; praline rich physiology/pathology/ of using methods lmown in the Insipidus; Diabetes mellitus; Type I diabetes; construct. ke -.3 acidic protein) adipocytes and adipose art (Berger et al., Gene Type 2 diabetes; Insulin resistance; Insulin 1..) tissue in response to 66:1-10(1998); Cullen and deficiency;
Hyperlipidemia; Hyperketonemia;
=
c, dietary conditions.
Maim, Methods hi Enzymol Non-insulin dependent Diabetes Mellitus w . - = . .. 216362-368 (1992); = (NIDDM);
Insulin-dependeht Diabetes Mellitus =.,. .
.
1-, ' Bentham et al., Proc Nati (IDDM); A
Condition Associated With Diabetes 1 1..) Aced Sci USA 85:6342- Including, But Not Limited To Obesity, Heart ke 6346 (1988); Reusch et al., Disease, Hyperglycemia, Infections, Retinopathy, .
Mol Cell Biol 20(3):1008- And/Or Ulcers; Metabolic Disorders; Immune 1020 (2000); and Klemm et Disorders; Obesity; Vascular Disorders;
al., J Biol Chem 273:917- Suppression of Body Weight; Suppression of , 923 (1998)). Appetite; Syndrome X.
Other indications for antibodies and/or antagonists, include treatment of weight loss;
treatment of AIDS wasting; appetite stimulant;
i treatment of cachexia. i __________________________ -, 'PYY (Peptide Decreases appetite;
Appetite and food intake can Most preferred:
Treatment of Obesity; treatment 3108, 3109, 3281, 3117, See Table 2, =YY), including increases satiety; be can be measured by of Diabetes; suppression of body weight gain; 3118, 3282.
SEQ ID NO:Z
PYY3-36 decreases food intake, methods known in the art 'suppression of appetite. for particular (amino acid (Batterham et al. Nature Hyperglycemia; Diabetes; Diabetes Insipidus;
construct.
residues 31-64 of t 2002; 418:650654) Diabetes mellitus; Type I diabetes; Type 2 full length PYY, I
, 9 cliabetes- Insulin resistance- Insulin deficiency-..t........--.. -9 =
Table I
Therapeutic Biological Activity Exemplary Activity PrefetTed Indication :Y
Construct ID therapeutic Protein:X Assay =Protein:Z
amino acid Hyperlipidemia;
FIyperketonemia; Non-insulin residues 3-36 of dependent Diabetes Mellitus (NIDDM); Insulin-mature PYY) dependent Diabetes Mellitus (IDDM); A
Condition Associated With Diabetes Including,=
But Not Limited To Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;
Obesity; Vascular Disorders; Suppression of o = Body Weight; Suppression of Appetite;
= Syndrome X.
1..) Other indications for antibodies, antagonists.:
co 0.
1-, treatment of veight loss; treatment of AIDS

=
wasting; appetite stiinaliintl=treatnent of _ cachexia.
1..) ______________________ 4 .

WNT1015 Inhibits adipogenesis. 'WNT10b activity can be Most preferred: Treatment of Obesity; ' 3141 See Table 2, . . . . . Imeasured using suppression of body weight gain; suppression of . SEQ ID NO:Z w 1-, for particular adipogenesis inhibition appetite. =
. . 1-, assays (Ross et al., Science Other indications: Hyperglycemia; Diabetes;
construct. 1..) 2000; 289(5480:950-953 Diabetes hisipidus; Diabetes mellitus; Type I=
ko diabetes; Type 2 diabetes; Insulin resistance;
= Insulin deficiency; Hypalipidemia;
Hyperketonemia; Non-insulin dependent , -5. Diabetes Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM).
.
AVIIT11 Promotes cardiogenesis. \Will activity can be Treatment of Cardiovascular disorders; 3142 See Table 2, ,measured using assays Congestive Heart Failure; Myocardial Infarction. fSEQ ID NO:Z
known in the art, including for particular , cardiogenesis assays construct.
(Eisenberg et al., Dev Dyn , 1999 Sep;216(1):45-581.
=, .
..
narstatin 'inhibits cancer Herstatin activity can be Oncology; Cancer; Solid Tumors; Melanoma; 3143 See Table 2, ,proliferation. measured using cell Malignant Melanoma; Renal Cancer (e.g., Renal SEQ ID NO:Z
i 'proliferation assays known Cell Carcinoma); Lung Cancer (e.1,. Non-Small for par dealer ..
, Table 1 Therapeutic Biological Activity Exemplary Activity Preferred Indication :Y
Construct ID Therapeutic Protein:X , Assay Protein:Z
_ in the art (Doherty et al., Cell Lung Cancer or Small Cell Lung Cancer); ,construct.
PNAS 1999; 96(19):10869- Colon Cancer; Breast Cancer; Liver Cancer;
10874. Prostate Cancer;
Bladder Cancer; Gastric Cancer;
Sarcoma; AIDS-Related Kaposi's Sarcoma;
Lymphonaa; T Cell Lymphoma; Cutaneous T-Cell Lymphoma; Non-Hodgkin's Lymphoma;
= Brain Cancer; Glioma; Glioblastoma Multiforme;
c) Cervical Dysplasia; Leukemia; Preleukemia;
Hairy Cell Leukemia; Chronic Myelogeonus , o iv Leukemia; iHcaelmaDitsoolordgeicrsal; Multiple , co Ø
______________________ -.. _ itignpla:Mciyese.loma.

Adrenomedullin ;stimulates vasodilation; Vasqggation can be Treatment of Congestive Heart Failure; 3144 See Table 2, ko . , -.3 promotes bone growth. Measured usmg assays = Hypertension;
Myocardial Infarction; Septic ;SEQ ID NO:Z
iv known in the art (Ashton at Shock; Osteoporosis; Postmenopausal !for particular o t al. Pharmacology 2000; osteoporosis;
Osteopenia. construct.
t = = = _ 61(2):1Q1-105. The .
promotion of bone growth _ 1 iv can be measured using ko assays known in the art, , such as the osteoblast t proliferation assay (Cornish et al. Am .1 Physiol 1997 Dec;273(6 Pt 1):E1113-20). _ _ .

Nogo Receptor Receptor for the axon The promotion of axon Treatment of Central Nervous System Damage;
3184, 3185 See Table 2, growth inhibitor, Nogo, regeneration and growth can Spinal Cord Injury;
Peripheral Nerve Damage; SEQ ID NO:Z
be measured using assays Neurodegenerative Diseases; Parkinson's for particular , known in the art (Fournier et Disease; Alzheimer's Disease; Huntington's construct. ;
, al. Nature 2001; Disease;
Amyotrophic Lateral Sclerosis;
409(6818):341-346). ;Progressive Supranuclear Palsy; Creutzfeld-Jacob Disease; Motor Neuron Disease.

, , CART (Cocaine- Inhibits food intact and Appetite and food intake can Most preferred: Treatment of Obesity; 3232 See Table 2, and fat storage; promotes lipid be can be measured by suppression of body weight gain; suppression of , SEQ ID NO:Z
Amphetamine-, oxidation. methods known in the art appetite for partictilar __ , Table I. =
Therapeutic Biological Activity 'Exemplary Activity Preferred Indication:Y
Construct ID iTherapeutic Protein:X! Assay ilsPoins'otructeint:Z
.., ' ,,_ , _ Regulated - (Batterham et al. Nature Other indications; Hyperglycemia; Diabetes;
Transcript) 2002; 418;650654) Diabetes Insipidus;
Diabetes mellitus; Type 1 diabetes; Type 2 diabetes; Insulin resistance;
- Insulin deficiency;
Hyperlipidemia;
liyperketonemia; Non-insulin dependent Diabetes Mellitus (NIDDM), Insulin-dependent /Diabetes Mellitus (IDDM).
,RegIV (Colon Stimulates glucose 'Reg/V activity may be Hyperglycemia; Diabetes; Diabetes Insipid/u; 4910. See Table 2, iSpecific Gene; uptalre; increases insulin assayed in vitro using a [3-Diabetes mellitus; Type I diabetes; Type 2 SEQ ID NO:Z
ci 'Colon Specific sensitivity. 11j-glucose uptake assay. (I diabetes;
Insulin resistance; Insulin deficiency; . for particular Protein) Biol Chem 1999 Oct 22;
Hyperlipidemia; Hyperketonemia; Non-insulin construct.
o r..) 274(43):30864-30873). dependent Diabetes Mellitus (NIDDM); Insulin- co .4.
dependent Diabetes Mellitus (IDDM); A
I-, Condition Associated With Diabetes 'wintry:1g, . ke -.3 tr, t.,..) 13rt Not Limited To Obesity, Heart Disease, n.) _ . , , = 'Hyperglycemia, Infections, Retinopathy, And/Or 0 =
Ulcers; Metabolic Disorders; Immune Disorders; w .

Obesity, Vascular Disorders; Suppression of I¨

, 1-, Body Weight; Suppression of Appetite;

N.) ,. SYridrome X, ke Cosyntropin Synthetic cortizotropin; The activity of cosyntropin -'Endocrine; Addison's disease; Cusbing's SEQ ID:
(Cortrosyn) stimulates the release of can be assessed in vivo by syndrome;
pituitary dysfunction; acute adrenal NO:2198 (CAS-16960-16- cortisol. measuring serum cortisol crisis 0) levels. (Frank et al. J. Am.
Vet Med. =Assoc. 1998 212(10);1569-71), , , Pexiganan Disrupts bacterial Pexiganan acetate activity Trtuttment of Infectious Diseases; Treatment of i SEQ ED NO:
Acetate membranes. can be assessed using in Bacterial Infections. 2199 (CAS-172820-23- vitro antibacterial assays 4) known in the art. (Zasloff et al., Antimicrobial Agents and Chemotherapy 1999, r 43:782-788)_ ..., - -Table 1 , ¨
, Therapeutic Biological Activity .Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic ' Protein:X 'Assay Proteiu:Z

,Pramlintide Stows gastric emptying; ,Appetite and food intake can Treatment of Obesity; treatment of Diabetes; SEQ ID NO:
(Arnylin) decreases food intake. ,be can be measured by suppression of body weight gain; suppression of (CAS-151126-32- methods known in the art 'appetite;
treatment of endocrine disorders;
8) 1(Battertiam et al. Nature ,H}perglyzernia; Diabetes; Diabetes Insipidus;
2002; 418:650654) Diabetes mellitus;
Type I diabetes; Type 2 diabetes; Insulin resistance; Insulin deficiency;
Hyperlipidernia; Hyperketonemia; Non-insulin (-) dependent Diabetes Mellitus (NIDDM); Insulin-, dependent Diabetes Mellitus (IDDM); A

:
iv ;Condition Associated With Diabetes Including, co Ø
=
But Not Limited To Obesity, Heart Disease, . Hyperglycemia, Infections, Retinopathy, And/Or ko Ulcers; Metabolic Disorders; Immune Disorders;
iv Obesity; Vascular Disorders; Suppression of 1-, c.h Body Weight;
Suppression of Appetite; w , 1-, Synrome X.
= . d = ¨ = = .- =
Other indications for antibodies, antagonists: iv ko treatment of weight loss; treatment of AIDS
wasting; appetite stironlant; treatment of , cachexia.
, _________ Teriparafide Acts in conjuction with Adenylyl cyclase Bone Disorders; Fracture prevention; , SEQ ID NO:
(CAS-52232-67- calcitonin to control stimulation in rat Hypeicalcernia;
Malignant hypercalcemia; 2201 4) calcium and phosphate osteosarcoma cells, Osteoporosis; Paget's disease; Osteopenia, metabolism; elevates ovariectomized rat model of Osteoclastogenesis; osteolysis; osteomyelitis;
blood calcium level; osteoporosis: TUBMB Life osteonecrosis; periodontal bone loss;
stimulates the activity of ,2000Feb;49(2):131-5 osteoarthritis;
rheumatoid arthritis; osteopetrosis;
osteocytes; enhances periodontal, lytie, or Metastatic bone disease;
absorption of Ca+/Pi from osteoclast differentiation inhibition; bone small intestine into blood; disorders; bone healing and regeneration.
promotes reabsorption of Ca+ and inhibits Pi by ) _kidney tubules.
_______________________________________________________________________________ _______________________ , Table I
-_______________________________________________________________________________ ______________________________________ * Therapeutic Biological Activity Exemplary Activity Preferred.
Indication:Y Construct ID Therapeutic Proteitta Assay ,Protein :Z
Terlipressin Analog of vasopressin; Terlipmsin activity can be Variceal hemorrhage; cirrhosis; portal SEQ ID NO:
(triglycyl lycine induces vasoconstriction. measured using assays of hypertension; hepatorenal syndrome; Blood- '2202 vasopressin) vasoconstriction, such as the related disorders , (CAS-14636-12- isolated arterial ring .
5) preparation (Landstrom at al., Hum Reprod 1999 ai lan;14(1);151-5).
Illaritide Stiendates nairiuresis, Illariiide acivity can be Excretory disorders; Acute renal failure; asthma;
i SEQ II) NO:
(CAS-118812-69- diuresis, arid vasodilation assessed by measming congestive heart failure; hypertension; pulmonary 12203 1-=
) .cGMP accumulation in rat hypertension; cardiovascular disorders c-a ke =
renal cells. (Valentin et al, .4 I.) Hypertension 1993 .
1-=
Apr,21(41:432-81.
La . = , , 1 Aprotiiiin Serine protease inhibitor; Inhibition of thrombin-lalubitiou of fibrinolysis; reduction of blood loss1-, SEQ ID NO:
1-.
II; (Trasylolfm attenuates Systemic induced platelet aggregation during surgery;
Treatment of Inflammation and 2204 i I.) . (CAS-9087-70-i; Inflanatatory Response, can be measured using Inaranar Disorders.
CAS-11061-94-2; fibrinolysis and tirrornbin- toeftiods known in the mt.
' CAS-12407-79-5) induced platelet (PonlHs at al., I Thorae aggregation- Cardiovase Surg 2000 Aug;120(2):370-8).

.--, Aspartocin Arafibeeteria -Aspartocia activity can be Treatnent of Infectious Diseases; treatment of SEQ ID NO:
= (CAS-4117-65-1; , assessed using in vitro bacterial infections. 2205 CAS-1402-89-7) , antibacterial assays known . .
in the art. (Zasioff et at, Antimicrobial Agents and ,Chemotherapy 1999, 41782-788).
., Calciinnin Regulates levels of Hypoetticemic Rat Bioassay, Musculosbelitn1;
Osteroporosis; Paget's disease; SEQ ID NO:
(Calcimar) calciarm and phosphate in bone resorhing assay and the hypercalceinha 2206 (CAS-21215-62- serum; causes a reduction pit assay, CT receptor Bone Disorders; Fracture prevention; Malignant 3) in serum calcium¨an third* assay, CAMP
hypercaloenda; Osteopenia, OsteocIastogenesis;
effect opposite to that of stimulation assay: I Bone osteolysis;
osteomyelitis; osteonecrosis;
human parathyroid 'batter Res 1999 periodontal bone loss; osteoarthritis; rheumatoid .. -..

Table 1 Therapeutic 1 Biological Activity Exemplary Activity Preferred Indication:Y
'Construct ID Therapeutic ,Protein:X Assay iProtein:Z
hormone. 'Aug;14(8):1425-31 arthritis;
osteopetrosis; periodontal, lytic, or metastatic bone disease; osteoclast differentiation inhibition; bone disorders; bone healing and . regeneration.
.
Carperitidc Stimulates natriuresis, Carperitide activity can be Treatment of Heart Failure; Cardiovascular SEQ ID NO:
(HANP; diuresis, and vasodilation. assessed in vitro by disorders; Respiratory disorders; Acute 2207 , recombinant measuring cGMP (respiratory distress syndrome.
r) human atrial accumulation in a number of natriuretic 'cell lines, including PC12 .. 0 iv peptide) ,. cells and cultured human co 0.
, . (CAS-89213-87- glomerular cells. (Medvede . 1-, "
"6) . et al., Life Sci 2001 Aug ko 4 .
=4 4'At A 31;69(15):r783110; Green - - -. -..
iv et al., J Am Soci\lephrol 1-, .1994 Oct;5(4)-1091-8).
w 1 , Desirudin Inhibits thrombin; inhibits Desirudin activity can be Blood-related disorder; Thrombosis;SEQ ID NO:
- . .
1-, ' (recombinant blood clotting, assessed using blood thrombocytopenia; hemorrhages. 2208 1 iv hirudin; Revasc) clotting assays known in the ko , (CAS-120993-53- art, such as in vitro platelet 5) aggragation assays. (Glusa, Haemostasis 1991;21 Suppl 1:116-20).
µ-Emoctakin proinflammatory cytokine ITreatment of Inflammation, Immune disorders, SEQ ID NO:
.(interleukin 8) RSV infection.
2209 .
(CAS-142298-00-8) Felypressin Derivative of Felypressin vasoconstriction 'Treatment of pain; to induce local anesthesia. SEQ ID NO:
(CAS-56-59-7) Vasopressin; Stimulates activity can be measured vasoconstriction; Induces using assays of local anesthesia. vasoconstriction, such as the isolated arterial ring i ' _______________________________________________________________________________ _________________________________ . .

Table 1 7Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic Proteitt:X Assay Protein:Z
_ preparation. (Landstrom et -al., Hum Reprod 1999 JamI4(1):151-5).
, _ Glucagon Induces hyperglycemia. Glucagon activity may be Hypoglycemia; Diabetes; Diabetes Insipidus; SEQ ID NO:
(CAS-16941-32- assayed in vitro using a [3- Diabetes mellitus; Type 1 diabetes; Type 2 p2211 5) 'Hi-glucose uptake assay. (J diabetes; Insulin resistance; Insulin deficiency;
'BRA Chem 1999 Oct 22; Ilyperlipidenia;
Hyperketonemia; Non-insulin 274(43):30864-30873). dependent Diabetes Mellitus (NIDDM); Insulin- . o dependent Diabetes Mellitus (H)DM); A
Condition Associated With Diabetes Including, c:.
1..) But Not Limited To Obesity, Heart Disease, co 0.

Hyperglycemia, Infections, Retinopathy, And/Or ko *Ulcers; Metabolic Disorders; Immune Disorders;
..-3 ' L Obesity; Vascular Disorders; Suppression of 1..) -4 Body Weight;
Suppression of Appetite; 1-w Syndrome X; Endocrine disorders. __ - 1 ....
_______________________________________________________________________________ ___________________________________________ 1-lNagrestipen Inflammation; Immune SEQ ID NO: 1-(CAS-166089-33- .

1..) 4) , ko -_______________________________________________________________________________ _____________________________________ Pentigetide Respiratety, Allergy; Immune SEQ ID NO:
(Pentyde) (CAS-62087-72-3) 1.- ., , Proinsulin Stimulates glucose uptake Insulin activity may be TIlyperglycesni% Diabetes; Diabetes Insipidus;
SEQ ID NO:
(CAS-67422-14- and promotes assayed in vitro using a [3- Diabetes mellitus; Type 1 diabetes; Type 2 2214 4) glYzugenesis and Hi-glucose uptake assay (I diabetes; Insulin resistance; Insulin deficiency;
lipogenesis. Biol. Chem 1999 Oct 22;
Hyperlipidemia; Hyperketonernia; Non-insulin , 274(43):30864-30873). dependent Diabetes Mellitus (NIDDM); Insulin- , t _i dependent Diabetes Mellitus (IDDIv12 ). A
- -Table 1 <
1Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic Protein:X AssayProtein:Z
, .
Condition Associated With Diabetes Including, But Not Limited To Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;
.Obesity; Vascular Disorders; Suppression of , Body Weight; Suppression of Appetite;
Syndrome X.
_ , Becaplennin Promotes wound healing. ,Becaplermin activity can be *Stimulate Epithelial Cell Proliferation; Stimulate SEQ ID NO: P
(Regranex; assessed using animal Basal Keratinocytes; Promote Wound Healing;
2215 , 0 iv recombinant ,wound healing models Stimulate Hair Follicle Production; Healing Of 0 0.
PDGF-BB) 'known in the art. (Saba et Dermal Wounds. Wound Healing; Eye Tissue (CAS-165101-51- at., Ann Plast Surg 2002 Wounds, Dental Tissue Wounds, Oral Cavity ke -.1 - 9) , - 1u149(1)62-6). Wounds, Diabetic Ulcers, Dermal Ulcers, iv Cubitus Ulcers, Arterial Ulcers, Venous Stasis 1-`
00 , Ulcers, Burns Resulting .2..om Heat Exposure Or ' = Chemicals, or Other Abnormal Vound Healing -1-`
Conditions such as Uremia, Malnutrition, iv Vitamin Deficiencies or Complications ke Associated With Systemic Treatment With Steroids, Radiation Therapy or Antineoplastic , , Drugs or Antimetabolites; Promote Dermal Reestablishment Subsequent To Dermal Loss;
, Increase the Adherence Of Skin Grafts To A
Wound Bed; Stimulate Re-Epithelialization from , The Wound Bed; To Promote Skin Stlength;
. Improve The Appearance Of Aged Skin;
, Proliferate Hepatocytes, Lung, Breast, Pancreas, Stomach, Bladder, Small Intestine, Large Intestine; Sebocytes, Hair Follicles, Type II
' . Pneumocytes, Mucin-Producing Goblet Cells, or , Other Epithelial Cells, Endothelial Cells, Keratinocytes, or Basal Keratinocytes (and Their Progenitors) Contained Within The Skin, Lung, ' Table I.
ITherapeutic I Biological Activity Exemplary Activity Preferred Indication :Y
Construct ID Th er apeu tic Protein:X Assay Protein:Z
Liver, Bladder, Eye, Salivary Glands, or Gastrointestinal Tract; Reduce The Side Effects Of Gut Toxicity That Result From Radiation, Chemotherapy Treatments Or Viral Infections;
Cytoproteetor, especially of the Small Intestine Isducoaa or Bladder; Mueositis (Mouth Ulcers);
'Regeneration Of Skin; Full and/or Partial Thickness Skin Defects, including Burns, (e.g., RepopitIation Of Hair Follicles, Sweat Glands, And Sebaceous Glands); Psoriasis;
Epidennolysis Bullosa; Blisters; Gastric and/or - Doutlenal Ulcers; Reduce Scarring;
Inflammatory BoweLDiseases; Crohn's Disease;
Ulcerative Colitis; Gut Toxicity; Lung Damage;
=
Repair Of Alveoli And/or Brochiolar Epithelium Acute Or Chronic Lung Damage; Emphysema, ARDS; Inhalation Injuries; Hyaline Membrane w Diseases; Infant Respiratory Distress Syndrome;
Iiroacbopuknimary Displasia In Premature Infants; Fulminaut Liver Failure; Cirrhosis, Liver Damage caused by Viral Hepatitis and/or Toxic Substances; Diabetes Mellitus; Inflammation;
Cancer; Digestive disorders.
Ghrelin Stimulates release of Appetite and food intake can Endocrine; loss of body weight; loss of body SEQ ID NO:
(Genbank growth hormone from be can be measured by weight associated with cancer or anorexia 2216 AecessionNo anterior pituitary methods known in the art nervosa; loss of appetite; excessive appetite;
AB 029434) Stimulates appetite and (Batterham et al. Nature bodyweight gain; Obesity; Diabetes;
reduces fat burning 2002; 418:650654) Acromegaly; Growth failure;
Growth hormone deficiency; Growth failure and growth retardation Prader-Willi syndrome in children 2 years or older; Growth deficiencies; Growth failure associated with chronic renal insufficiency;
Postmen, ausal oateo*orosis= burns. cachexia.
;

Table 1 , Therapeutic i Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic "
Protein:X Assay ,Proteiti:Z
-cancer cachexia; dwarfism; metabolic disorders;
obesity; renal failure; Turner's Syndrome, , pediatric and adult; fibromyalgia; fracture treatment frailty. AIDS wasting , ...
_ Ghrelin -binding Inhibits growth hormone Appetite and food intake can Endocrine; Obesity; Diabetes; body weight gain;
antibody release in response to be can be measured by excessive appetite; loss of appetite; loss of body including ;Ghrelin; inhibits increase methods known in the art weight. , ri antibody in appetite. (Batterham et al. Nature fragment, or '2002; 418:650654) dominant-co , Ø
negative form of Ghrelin receptor ko - ¨
..]
= - NOGO-66 Neurodegenerative disorders; spinal cord injury; =
SEQ ID .NOt iv peptide fragment neuronal injury;
brain trauma; stroke; multiple 2217 0 1-, (Genbank sclerosis;
demyelin;ting disorders; neural activity w . . .

Accession No. and neurological diseases; neural cell (e.g., 1-, 1-, = neuron, glial cell, and schwami cell) regeneration.
(amino .
iv (amino acids 62- i and/or growth , ko 101)) , Gastric inhibitory Increases nutrient uptake Nutrient uptake and Most preferred: loss of body weight, AIDS , SEQ ID NO:
polypeptide and tryglyceride tryglyceride accumulation wasting, cachexia, loss of apetite. Other: ' 2218 (GIP), including accumulation in can be measured by methods Obesity;
Diabetes; insulin resistance; body GIP fragments adipocytes, which leads to desthed in Miyawaki et al., weight gain; excessive appetite.
(Genbank , obesity and insulin Nat. Medicine, 2002, Vol Accession No. -resistance. 8(7):738-742.
M,4_004123) , , Gastric inhibitory ,Increased use of fat as Fat utilization as an energy 'Obesity; Diabetes; Insulin resistance; body polypeptide predominant energy source can be measured as weight gain.
antibody, or source; decreased described in Miyawaki et antibody accumulation of fat in at., Nat. Medicine, 2002, fragments , adipocytes. Vol 8(7):738-742.
, Table 1 Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID Therapeutic Protein:X Assay _____________________________________________________________ Protein:Z
Gastric inhibitory Increased use of fat as Tat utilization as an energy Most preferred: Obesity;
Diabetes; body weight SEQ ID NO:
peptide receptor predominant energy source can be measured as gain; excessive appetite;
insulin resistance. , 2219 or receptor source; decreased described in Miyawaki et Other: loss of body weight, AIDS wasting, loss of fragments or accumulation of fat in aL, Nat. Medicine, 2002, Iappetite.
variants including adipocytes. Vol 8(7):738-742. , soluble fragments or variants (Genbank Accession , o Number = 0 NM 000164)1..) co , _______________________________________________________________________________ __________________ .
= POMC Activity of POMC-Preferred: resistance to stress; anti-inflammatory SEQ
ID NO:
c:.
(proopiomelanoco , derived fragments are ,activity; analgesic activity; increased skin 2220 ko .
. ..]
rtin), including diverse, and well-known 'pigmentation: increased protein catabolism;
1..) fragments or in the art increased gluconeogenesis;
obesity; diabetes. 0 variants (such as,= ,Other: decreased protein catabolism, decreased 1-.
w .

for example, See, for example, Hadley skin pigmentation, Addison's disease, Cushing's 1-. =
alpha-melanocyte et al., Ann N Y Acad Sci syndrome , 1..) stimulating 1999 Oct 20;885:1-21;
ko hormone, ctMSH, Dotes, Prog Olin l3iol Res gartuna 1990342:22-7; Blalock, , melanocyte Arm N Y Aced Sci. 1999 stimulating Oct 20;885:161-72). , .
hormone, 7MSH, , beta-melanocyte stimulating e .
hormone, 13MSH, adrenocorticotrop ACTH, beta- =
=dolphin, met-enkephalin) (Genbank Atcession No.

. s Table 1 -_______________________________________________________________________________ ___ E
!Therapeutic Biological Activity Exemplary Activity Preferred Iudication:Y
Construct ID Therapeutic Protein:X AssayProtein:Z
_ NM 000930) 1 I
f _ HP 467, HP228 See US Patent No. See US Patent No. Resistance to stress;
anti-inflairunatory activity; ! SEQ ID NO:
i (US Patent No. 6,350,430 6,350,430 analgesic activity;
increased skin pigmentation; 2221 , 6,350,430) increased protein catabolism; increased gluconeogenesis.
. _..
NDP See US Patent No, See US Patent No.
Resistance to stress; anti-inflammatory activity; ---SEQ ID NO: 0 (US Patent No. 6,350,430 6,350,430 analgesic activity;
increased skin pigmentation; 2222 6,350,430) increased protein catabolism; increased 0 1..) co ' gluconeogenesis.
0.
..
1-.

. -________________________________________________________________ Interleukin-21 Immunomodulator; IL-21 activity can be ' Autoirmnune disorders; Inflammatory isorders; 3298 ISEQ ID NO... ...]
(11,21) inhibits interferon gamma assessed by measuring ' ' Treatment of Psoriasis; Rheumatoid Arthritis;
2177 1\.) a, t .. production by Thl cells. interferon gamma Inflammatory bowel diFease. 1-. ...) . w production in Thl cells.
.
1-.
(Wurster et al., : J Exp Med , = 1 2002 Oct 7196(7):969-77) ko Interleukin-4 Immunomodulator; 11-4 activity can be assessed Treatment of Psoriasis; Autoimmune disorders; 3307 SEQ ID NO:
(IL-4) pioinotes the by measuring Thl / Th2 Rheumatoid Arthritis; Inflammatory bowel ,2178 differentiation of T cells cytokine responses of disease; Inflammatory disorders. .
into Th2 phenotype. isolated spleen cells in vitro.
(Waltz et al., Horns Metab Res 2002 Oct;34(10):561-, . 91.
Osteoclast Inhibits osteoclast Ostenclast Inhibitory Lectin Treatment of Bone Disorders; Osteoporosis;
3312 SEQ ID NO:
Inhibitory Lectin formation. activity can be assessed Fracture prevention; Hypercalcernia; Malignant (OCIL) using osteoclast formation hypercakemia;
Paget's disease; Osteopenia, assays known in the art. Osteoclastogenesis;
osteolysis; osteomyelitis; .
/ (Zhou et al., J Biol Chem osteonecrosis;
periodontal bone loss;
- 2002 Dec osteoarthritis;
rheumatoid arthritis; osteopetrosis;
, 13;277(50):48808-15) , periodontal, lytic, or metastatic bone disease;
osteoclast differentiation inhibition; bone healing , Table 1 Therapeutic Biological Activity Exemplary Activity Preferred Indication:Y
Construct ID fl Therapeutic Proteiti:X Assay 1Protebi:Z
,and regeneration.
ci 1.) co i= =
0\

=

Table 2 Fusion Construct Construct Name !Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. - ID 1 Vector ID ID ID ID ID Sequence "
NO:Y NO: NO:Z NO: 1 NO:B
X
A
1 t 1520 pC4:HSA/TR6.V30- Amino acids V30 to 11300 of TR6 pC4 217 1 433 649 650 HSA
11300 (fragment shown as Vi to 11271 of SEQ
ID NO:433) fused downstream of HSA, , pYPG:HSA.TR6coV30- Amino acids V30 to E294 of TR6 pYPGaf 218 2 434 651 652 HSA o E294 (fragment shown as Vito E265 of SEQ

N., ID NO:434) fused downstream of HSA.
co 0.
DNA encoding TR6 has been codon =
loptimized. ,, ko ...3 p. 3 1545 pYPG:HSA.TR6coV30- i Amino acids V30 to L288 of TR6 pYPGaf 219 3.

L288 1 (fragment shown Rs Vi to L259 of SEQ

1-, .
ID NO:435) fused downstreanrof HSA. .
. ---= =-= 1 1-, .
1-, = DNA encoding TR6 has been codon 1 optimized.
ko pYPG:HSA.TR6coV30- Amino acids V30 to R284 of TR6 pYPGaf ' 220 4 436 , R284 (fragment shown as Vito R255 of SEQ
ID NO:436) fused downstream of HSA.
DNA encoding TR6 has been codon oRtimized.
_ 1568 pSAC35:HSA-yTR6 1R6 fused downstream of HSA. DNA" pSAC35 - 221 5 1 437 657 658 HSA/kex2 ..,. , encoding TR6 has been codon sTtimized.
________ 6 1570 pSAC35:TR6-HSA Mature TR6 fused dovhistream of the pSAC35 222 6 438 659 660 HSA/kex2 HSA/kex2 leader and upstream of the mature HSA. , .
¨

7 1622 pC4:synTR6.M1-Synthetic TR6 fused upstream of mature pC4 223 7 439 661 662 !Native H300.HSA HSA, with 2 extra amino acids between I
, the TR6 and HSA portions.t , =
=
Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader I
No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:13 X
A

8 1623 pc4:1-1SA.s-ynTR6.V30- Synthetic mature TR6 fused downstream pC4 224 8 440 663 664 HSA
H300 of FL HSA. Last amino acid HSA
___________________________________ sequence is missing at BSII361 site.

9 1642 pSAC35:GCSF.T31-Mature GCSF cloned downstream of the pSAC35 225 9 441 665 666 HSA/kex2 P204.HSA HSA/kex2 leader and upstream of the mature HSA

1643 pSAC35:1-1SA.GCSF.T3 Mature GCSF cloned downstream of the pSAC35 226 10 442 667 668 HSAikex2 1-P204 mature HSA and HSA/kex2 leader =
sequence.
crs LA 11 1645 IpSAC35:yTR6(N173Q). Mutant mature TR6 cloned upstream of pSAC35 227 443 669 670 HSAikex2 HSA mature HSA and downstream of the HSA/kex2 leader sequence.. .
=
12 1657 pC4.HSA:DeCAF.A23- Amino adds A23to D233 ofDeCAF
pC4 228 111 444 ; 671 672 HSA
D233 fused downstream of full length HSA.
13 1680 pYPG:HSA.BLyS.A134 Amino acids A134 to L285 of BLyS
fused pYPGaf 229 13 445 673 674 HSA
-L285 downstream of FL HSA. Two extra amino acids (Leu, Glu) have been added between the therapeutic protein and HSA
_portions.
14 1681 pYPG.HSA.MPLF.D45- Amino acids D45 to N120 of MET' fused pYPGaf 230 14 446 675 676 HSA
N120 downstream of FL HSA. Two additional amino acids (L and E) have been added between HSA and MPIE.
1697 pSAC35:HSA.GM- Amino acids Al8 to E144 of GM-CSF pSAC35 231 CSF.A18-E144 fused dovvnstream of FL HSA.

Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader -No. ID Vector ID ID
ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
16 1699 pSAC35:GM-CSF.A18- Amino acids A18 to E144 of GM-CSF pSAC35 232 16 448 679 680 HSA/kex2 E144:HSA fused upstream of mature HSA and downstream of HSA/kex2 leader.
17 1700 pSAC35:HSA- Mutant TR6 fused downstream of rnature pSAC35 233 17 449 681 682 HSAikex2 _______________________ yTR6(N173Q) HSA with HSAIkex2 leader s9quence.
18 1702 pYPG:HSA.ek.TR6coV Amino acids V30 to L288 of TR6 pYPGaf 30-L288 (fragment shown as Vito L259 of SEQ
co 11) NO:450) fused downstream of FL
= HSA with an enterolcinase site in between.
' = = DNA encoding TR6 has been codon optimized.
19 1703 pYPG:HSA.ek.TR6coV Amino acids V30 to R284 of TR6 = pYPGaf 30-R284 (fragment shown as V1 to R255 of SEQ =
ID NO:451) fused downstream of HSA
with an enterolcinase site in between.
DNA encoding TR6 has been codon optimized.
20 1704 pYPG:HSA.TR6N30- Amino acids V30 to E294 of TR6 fused pYPGaf E294 downstream of HSA. Two additional amino acids (Len, Glu) are in between HSA and TR6.
21 1746 pYPG:HSA.ek.KDI,L28 Amino acids L28 to 1(207 of KDI fused pYPGaf -K207 downstream of HSA with an enterokinase site in between.
22 1747 pSAC35.HSA.hG1RF. Amino acids yn to L75 of hGlIRF fused pSAC35 -Y32-L75 downstream of HSA.
_ Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID
ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
23 1748 pSAC35.WHRFX32- Amino acids Y32 to L75 of hGERF (see pSAC35 239 455 693 694 HSA/lcex2 L75.HSA also saz rDNO:454) fused upstream of = mature HSA and downstream of HSAficex2 leader se. mice.
24 1749 pSAC35:HSA.PTH.S1- FL HSA fused upstream of amino acids pSAC35 240 456 695 696 F3 S1-F34 of PTH
25 1750 pSAC35:PTH.S1- Amino acids 1-34 of PTH fused upstream pSAC35 241 457 697 698 HSA/kex2 0 F34.HSA of mature HSA and downstream of HSA/Itex2 leader sequence.
26 1757 pSAC35:IL2.A21- Mature human 11-2 with a single amino pSAC35 242 26 458 699 700 HSA/kex2 T153.145C/S.HSA acid mutation (C to S at position 145) cloned downstream of the HSA/ICEX2 leader and u.stream of mature H&A.
. .
27 1758 pSAC35:HSA.112.A21- Mature human 1L-2 with a single amino pSAC35 243 27 459 701 702 'HSA/kex2 T153.145C/S acid mutation (C to S at position 145) cloned downstream of HSA with HSA/kex2 leader sequence.
= 28 1772 pSAC:EPOco.A28- Amino acids A28-D192 of EPO variant pSAC35 244 28 460 703 704 liSA/Iceic2 D192.HSA (where glycine at amino acid 140 has been replaced with an arginine) fused upstream of mature USA and downstream of HSAikex2 leader sequence. DNA
encoding EPO has been codon optimized.

Table 2 , _______________________________________________________________________________ _______________________________________ Fusion Construct Construct Name , Description Expression SEQ SEQ SEQ SEQ SEQ , Leader No. ID
Vector ID ID ID ID 1 ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
!
_______________________________________________________________________________ __ 29 1774 pSAC:HSA.E'POco.A28 Amino acids A28-D192 of EPO
variant pSAC35 245 29 461 705 706 HSA/kex2 , -D192. (where glycine at amino acid 140 has been replaced with an arginine) fused , downstream of HSA with HSA/kex2 , o , leader sequence. DNA encoding EPO has ' P
. * been codon o Itimized.
, o rs.) - . . . 30 1777 pSAC35:TNFR2.1,23- Mature TNFR2 fused downstream of the pSAC35 246 30 462 707 708 HSAikex2 co 0.
1-`
D257.HSA HSA/kex2 signal and upstream of mature 0 .
ko -4... EISA. =
-.I

. ..... .,.-.. /
ccro 31 1778 pSAC35:IF'Nf3.M22-Residues M22-N187 of full-length TFNb pSAC35 247 31 463 709 710 HSA/kex2 "
o N187:HSA (shown as M1 to N166 of SEQ ID
1-`
LA) , NO:463) fused upstream of mature USA
¨ - = = - 1-`
= .
and downstream of HSA/kex2 leader . 1 rs.) sequence.
ko , 32 1779 pSAC35:HSAIFN1-3.M2 Residues M22-N187 of full-length IFNb pSAC35 248 32 464 HSA/kex2 2-N187 (shown as MI to N166 of SEQ ID
, ,= NO:464) fused downstream of USA
with HSA/kex2 leader sequence.
....
33= 1781 pSAC:EPOcoA28- Amino acids A28-D192 of EPO
variant pSAC35 249 33 465 711 ' 712 HSA/kex2 D192.HSA (where glycine at amino acid 140 has been 51N/S,65N/S,110N/s replaced with an arginine) fused upstream .
/ of mature USA and downstream of 1- HSA/kex2 leader sequence.
Glycosylation , sites at amino acid 51, 65, 110 are .
. . mutated from N to S residue. DNA
.
-encoding EPO has been codon optimized.
i _ , ....
, Table 2 -_______________________________________________________________________________ _____________________________________ Fusion Construct I Construct Name Description Expression SEQ I SEQ SEQ SEQ SEQ Leader No. ID Vector , ID ID ' ID ID ID Sequence NO:Y NO: = NO:Z NO: NO:II
X
A ' 34 1783 pSAC:HSA.EPOcoA28-Amino acids A28-D192 of EPO variant pSAC35 250 34 466 713 714 HSA/Icex2 D192.51N/S,65N/S,110 (where giycine at amino acid 140 has been , N/s replaced with an arginine) fused .
downstream of HSA with HSA/kex2 ' leader sequence. Glycosylation sites at o amino acids 51, 65, 110 are mutated from . 0 N to S residue. DNA encoding EPO has N' , been codon optimized.
0.
1-, , . _______ .

pSAC35:HSA.TNFR2.L = Mature TNER2 fused downstream of FL pSAC35 251 35 ko cr, 23-D257 HSA.
...3 pSAC35:KGF2 28.A63 Amino acids A63 to S208 of KGF2 fused pSAC35 , 252 36468 1 717 718 HSA/kex2 0 .
1-, -3208:HSA upstream of mature HSA and downstream = ' õ ... w =
. - . 1-, . of __ the HSA/kex2 signal peptide.
, .
_______________________________________________________________________________ ____________________________________________ 1 pSAC35:HSA.KGF2{D Amino acids A63 to S208 of KGF2 fused pSAC35 253 37 469 719 720 HSA 1.) ko }28.A63-S208 downstream of HSA.
.
.

pSAC35:HSA.TR2.P37- Amino acids P37 to A192 of TR2 fused pSAC35 254 38 470 721 722 HSA,11cex2 A192 downstream of HSA vvith-HSA/kex2 ________________________________________ leader sequence.
. ..

pSAC35:HSA,EPO.A28 Amino acids A28-D192 of EPO variant pSAC35 255 39 471 HSA/kex2 I
-D192 . (where glycine at amino acid 140 has been (N51A,N65A,N110A) replaced with an arginine; see, for example, SEQ ID N0:499) fused downstream of HSA with HSA/kex2 leader sequence. Glycosylation sites at amino acids 51, 65, 110 are mutated from .
N to A residue.
-Table 2 Fusion Construct Construct Name Description j-Expression SEQ SEQ SEQ SEQ' SEQ Leader No. , ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
40 1794 pSAC35:HSA.EPO.A28 Amino acids A28-D192 of the EPO
pSAC35 256 40 472 HSA/kex2 -D192 variant (where glycine at amino acid 140 has been replaced with an arginine; see, , for example, SEQ ID NO:499) fused downstream of HSA with HSA/kex2 leader sequence.

41 1809 pSAC35.1VIDC.G25- Amino acids P26 to Q93 of MDC with an pSAC35 257 41 473 723 724 HSA/kex2 cc, Q93.HSA N-terminal methiimine, fused downstream 0.
1-, of the HSA/kex2 leader and upstream of ko ...3 c) , mature HSA.
N., 42 1812 pSAC35:IL2.A21- Amino acids Al to T153 of IL-2 fused pSAC35 258 42 474 725 726 HSA/kex2 1-, T153.HSA downstream of the HSA/kex2 leader and w .
.
=
= upstream of mature HSA. ' 1 1-, 1-, .i 43 1813 pSAC35:HSA.II2.A21- Amino acids A21 to T153 of IL-2 fused pSAC35 259 43 475 727 728 HSAikex2 ' N) ko T153 downstream of HSA with HSA/kex2 , leader sequence.
i, , 44 1821 pSAC35:scFv116A01.H BLyS antibody fused upstream of mature pSAC35 260 44 476 729 730 Modified SA HSA which lacks the first 8 amino acids HSA/kex2, ! and downstream from the HSA/kex2 lacking the , . signal sequence which lacks the last two last two amino acids.
amino .
acids 45 1830 pSAC35:HSA.ICEX2.H , Amino acids L19-Q300 of HAGDG59 *
pSAC35 261 45 477 731 732 HSA/kex2 A0D059.L19-Q300 fused downstream of the HSA/kex2 signal, mature HSA and KEX2 cleavage .
¨
, Table 2 _ - , Fusion ' Construct Construct Name Description i Expression SEQ SEQ' SEQ ' SEQ SEQ Leader No. ID , Vector ID ID ID ID ID I Sequence i NO:µ' NO: NO:Z ' NO: NO:B
, X A
46 1831 pSAC35:HAGDG59.L1 HSAIkex2 signal peptide followed by pSAC35 262 46 478 733 734 HSAikex2 . 9-Q300.HSA \
amino acids L19-Q300 of HAGDG59 ' , , I followed by mature HSA.
47 1833 ( pSAC35:humancalcitoni Human Calcitonin (amino acids C98-, pSAC35 263 47 479 735 736 HSA/kex2 n.C1-G33:HSA GI30 of SEQ ID NO:479) fused upstream o P
of mature HSA and downstream of , HSA/kex2 leader sequence.
_ _______________________________________________________________________________ ________________________________________ co 48 1834 pSAC35:HSA.hurnanca1 Human Calcitonin (amino acids C98-IpSAC35 264 48 480 737 738 HSA p=
I
I-`
citonin. C I -G33 G130 of SEQ ID NO:480) fused ko µ....downsream of FL HSA.
___________________________________ .. t 49 1835 pSAC35:sahnoncaleitoni Salmon Calcitonin amino acids C1-G33 pSAC35 265 49 481 739 740 HSA/kex2 0 I-`
n.C1-G3311-1SA . fitsed upstream of mature HSA and I
= downstream of HSA/kex2 leader i - ' = = . I-`
, sequence.
50 1836 pSAC35:HSA.sahnoncal Salmon Calcitonin amino acids C1-033 pSAC35 266 50 482 741 742 HSA ko citonin.C1-G33 fused downstream of HSA.
....
51 1853 pSAC35:PTH(1- Amino acids 1 IpSAC35 267 51 483 743 1-- 744 HSA/kex2 34)N26.HSA upstream of mature HSA and downstream , of HSA/kex2 leader sequence. Amino acid K26 of PTH mutated to N26.
, .
52 1854 A pSAC35:1ISA.PTH(1- Amino acids 1 to 34 of PTH fused pSAC35 268 ,52 484 745 746 HSA
34)N26 downstream of HSA. Amino acid K26 of , PTH mutated to N26.
.
53 1862 pSAC35:HSA.GriREQ Amino acids Q24-033 of human pSAC35 269 53 485 , 747 748 HSAJkex2 24-G33 gonadotropin releasing hormone fused < < downstream of HSA with HSA/kex2 leader sequence, ...

Table .2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID
ID ID ID Sequence NO:Y NO: NO:2 NO: NO:B
X
A
= 54 1863 pSAC35:GnRHQ24- Amino acids Q24-033 of human pSAC35 270 54 486 749 750 HSA/kex2 G33.HSA gonad otropin releasing hormone fused upstream of mature HSA and downstream _________________________________ of HSAAcex2 leader sequence.
55 1866 pSAC35:teprotide.HSA Teprotide fused upstream of mature HSA.
pSAC35 271 55 487 751 752 56 1867 pSAC35:11SA.teprotide. Teprotide fused downstream of FL HSA.
pSAC35 272 56 488 753 754 HSA
r 57 1889 pC4:HSA.PTH.S1-F34 PTH(1-34) fused downstream of HSA. pC4 58 1891 pEE12:HSA.sTR6 Soluble mature TR6 fused downstream of pEE12.1 ESA.

59 1892 pEE12:sTR6.1-1SA - Synthetic full length TR6 fused tpstreana pEEI2.1 275 59 491 759 760 TR6 . of mature HSA.
60 1906 pC4:PTH.S1-F34.IISA Amino acids Si to F34 of PTH fused ' pC4 ' 276 60 492 761. 762 MPIF
(junctioned) upstream of mature HSA and downstream of MPIF leader sequence. There are two cloning junction amino acids (T,S) between PTII and ESA.

61 1907 pC4:HSA.PTH.S I -F34 Amino acids SI to F34 fused downstream pC4 (junetioned) of FL HSA. The last C-terminal amino acid (L) residue is missing for HSA in the cloningiunction between HSA and PTH.
62 1912 pC4:sTR6.HSA Synthetic full length TR6 fused upstream pC4, 62 494 765 766 Native TR6 of mature HSA.
leader 63 1913 pC4:HSA.synTR6.V30- Amino acids V30 to H300 of synthetic pC4 H300 (seamless) TR6 (shown as Vi to H271 of SEQ ID
j. NO:495) fused downstream of full-length HSA.

Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ I SEQ SEQ Leader No. ID Vector ID ID
ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
64 1914 pC4:PTH.S1-F34.HSA Amino acids Si to F34 of PTH fused pC4 (seamless) downstream of MPIF leader sequence and upstream of mature HSA.
65 1916 pC4:HSA.KGF2D28.A6 Amino acids A63 to 8208 of full length pC4 3-S208 'KGF2 fused downstream of HSA.
66 1917 pC4kGF2D28.A63- Amino acids A63 to 8208 of KGF2 fused pC4 282 66 498 EMI HSA/Icex2 ______________ S208:HSA upstream of mature HSA.*
67 1925 pcDNA3.EPO Ml- Amino acids M1 to D192 of EPO variant pcDNA3 283 67 499 776 Native =D192.HSA
(where glycine at amino acid 140 has been EPO leader replaced with an-arginine) fused upstream.
peptide of HSA. D192 of EPO arid DI of mature HSA are the same amino acids in this = - =
=
construct.
68 1926 pcDNA3:SPHSA.EPO Amino acids A28 to D192 of EPO variant pcDNA3 A28-D192 (where glycine at amino acid 140 has been replaced with an arginine) fused upstream of mature HSA and downstream of the M-91F leader . e *tide.
69 1932 pEE12.1:HSA.PTH.S1- Amino acids Ito 34 of PTH fused pEE12.1 285 69 501 779 780 HSA
F34 downstream of full len: h HSA.
70 1933 pSAC35:HCC-1.T20- Amino acids T20 to N93 of HCC-1 fused pSAC35 286 70 502 781 782 HSAikex2 N93 :HSA upstream of mature HSA and downstream of the HSA/lcex2 leader sequence.-Table 2 ' Fusion Construct Construct Name Description Expression 1 SEQ I. SEQ SEQ SEQ SEQ Leader No. ID Vector ID ' ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
.7:.
A
71 1934 pSAC35:HCC-Amino acids T20 to N93 of HCC-I fused pSAC35 287 71 503 783 784 HSA/kex2 1 C.O.T2O-N93:11SA upstream of mature HSA and downstream ' of the HSA/kex2 leader sequence. DNA
sequence is codon optimized for yeast expression.
,.
72 1938 pEE12.1:PTH.S1- Amino acids SI to F34 of PTH fused pEE12.1 288 72 504 785 786 MP1F 0 1..) F34.HSA upstream of mature HSA and downstream co 0.
of MP1F leader sequence.
.

73 1941 pC4:HSA1PTH84 PTH fused downstream of full length pC4 289 73 505 787 788 HSA
ko ...3 (junctioned) HSA. The last amino acid of HSA (Leu) .
1..) bcs been deleted.
_______________________________________________________________________________ _________________________________ , _____________________ ,.., w - 74 ' 1947 pSAC35:d8HCC-1.G28- Amino acids G28 to N93 of 11CC-1 fused pSAC35 290 74 506 789 790 HSA/kex2 1 1-, .
. . ..
N93:HSA i upstream. of mature HSA
andclownstrearn 0 1-, 1..) of HSA/kex2 leader sequence.
ko ....... , 75 , 1948 pSAC35:d8HCC- Amino acids G28 to N93 of HCC4 fused pSAC35 291 75 507 791 792 IISA/kex2 1C.O.G28-N93:HSA upstream of mature HSA and downstream of HSAJkex2 leader sequence. DNA
sequence is codon optimized for yeast ' , _ expression.
76 1949 ' pC4:PTH.S1-Q84THSA P'TH fused downstream of the MPIP' pC4 -(junctioned) leader sequence and upstream of mature , HSA. There are two additional amino acids between PTH84 and HSA as a result , of the cloning site.
, 77 1952 pcDNA3.1:IL2.HSA Full length human IL-2, having a Cysteine pCDNA3,1 293 77 509 795 796 Native 11,-2 , to Serine mutation at amino acid 145, leader , fused upstream of mature HSA. i -e .
, Table 2 , Fusion . Construct Construct Name I Description Expression SEQ SEQ SEQ SEQ SEQ I Leader No. ID Vector ID ID
ID ID ID Sequence NO:Y NO: NO:Z NO: ' NO:B .
_ X
A . , , 78 1954 pC4:1L2.HSA Full length human 1L-2, having a Cysteine pC4 294 78 ' 510 797 798 Native 1L-2 . to Serine mutation at amino acid 145, leader , fused upstream of mature HSA.
./-_______________________________________________________________________________ _____________________________ , 79 1955 pSAC35:t9HCC-1.028- Amino acids G28 to N93 of HCC-1 fused pSAC35 295 79 511 799 800 HSA/kex2 N93:spcHSA upstream of a 16 amino acid spacer and , mature HSA and downstream of HSATIcex2 leader sequence.
, . ____________ , N.) , 80 1956 pSAC35:HSA.scFv116 Single chain BLyS antibody fused pSAC35 296 80 512 801 802 . HS.Afleex.2 ' 0.
1-, A01 . downstream of BSA with HSA/Icex2 to -..) ...3 leader sequence. This construct alSo , , contains a His tag at the 3' end..
, 81 1966 pC4:EPO.M1- Amino acids M1 to 1?192 of EPO variant pC4 ..
297 ' 81 511 .. . : ,.. . Native . w ID19211SK - - (where glycine at arnino.acid 140 has been EPO leader 1-, ' replaced with an arginine) fused upstream .
peptide N., to Construct is also named.of mature HSA.
pC4:EPOM1- .., .
D192.HSA
-, 82 1969 pC4:MPIFsp.HSA.EPO. ' Amino acids A28 to D192 of EPO variant pC4 298 82 514 . MP1F
A28-D192 (where glycine at amino acid 140 has been replaced with an arginine) fused , downstream of IN,TP1F leader sequence and ,.
, upstream of mature HSA.

_______________________________________________________________________________ ________________________________ , 83 1980 ' pC4:EPO.A28-D192. Amino acids A28 to D192 of EPO variant ' pC4 299 83 515 803 804 HSA
HSA (where glycine at amino acid 140 has been replaced with an arginine) fused downstream of the HSA leader peptide :
"
... , and upstream of mature HSA, S.
4 _______________________ , Table 2 ' _______________________________________________________________________________ ________________________________ Fusion Construct Construct Name - Description Expression SEQ I SEQ SEQ SEQ I SEQ Leader I
i . No. ID Vector ID ID
ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
, __________________________________ 84 1981 pC4.HSA-EPO.A28- Amino acids A28 to D192 of EPO variant pC4 300 84 516 805 806 HSA
D192. (where glycine at amino acid 140 has been = replaced with an arginine) fused downstream of the full length HSA.
.
o pSAC35:activeAC2inhi Active inhibitor of ACE2 (DX512) fused pSAC35 301 85 517 807 808 HSA/kex2 bitor:HSA upstream of mature HSA and downstream 1.., f 1 of HSA/lcex2 leader sequence.

0.
86 1994 pEE12.1.HSA- Amino acids A28 to D192 of EPO variant pEE12.1 302 86 518 HSA

ko EPO.A28-D192.
(where glycine at amino acid 140 has been õ...3 -...1 1.., aN replaced with an arginine) fused ___________________________________ downstream of full length HSA.
_ , w , 87 1995 pEE12.1:EPO.A28-Amino acids A28 to D192 of EPO variant pEE12.1 303 ' 87 519 USA =

D192. HSA (where glycine at amino acid 140 has been 1.., replaced with an arginine) fused ' ko downstream of the HSA leader peptide k _ and upstream of mature }ISA. , , 1 _ , ., 88 =1996 pEE12.1:MPIFsp.HSA. ¨Amino acids A28 to D192 of EPO variant pEE12.1 304 88 520 MPiF
EPO.A28-D192 (where glycine at amino acid 140 has been replaced with an arginine) fused downstream of MPIF leader sequence and upstream of mature USA.
_ .
89 1997 pEE12.1:EPO Ml- Amino acids M1 to D192 of EPO variant pEE12.1 305 89 521 Native D192.HSA (where glycine at amino acid 140 has been EPO leader replaced with an arginine) fused upstream I of mature HSA, _J
=
. ' ' Table 2 , , ______ Fusion Construct Construct Name Description 1 Expression SEQ SEQ SEQ i SEQ SEQ Leader i ' No. ' ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
.
X A
90 1998 pC4:CKBI.G28- Amino acids G28 to N93 of CkBetal IpC4 306 90 522 809 810 HSA
N93.HSA fused upstream of mature HSA and downstream of the HSA leader sequence.
. 91 2000 pSAC35:HSA:activeAC Active inhibitor of ACE2 (DX512) fused pSAC35 307 91 523 811 - 812 HSA
2inhibitor downstream of HSA.

i 1 92 2001 pSAC35:inactiveAC2in Inactive inhibitor of ACE2 (DX510) pSAC35 - 308 92 524 813 r 814 HS AfIcex2-' 0 hibitorlISA fused upstream of mature HSA and N' 0.
downstream of IISA/kex2 leader ' .. sea_uence.
L ____________________________ ko A
--.1 93 2002 pSAC35:HSA.inactiveA Inactive inhibitor of ACE2 (DX510) pSAC35 309 93 525 815 816 HSA 1..) C2inhibitor fused downstream of HSA.

, _______________________________________________________________________________ ____________________________________________ 1-, 94 2011 pC4:IFNb-HSA Full length IENb fused upstream of pC4 310 94 ' 526 817 818 Native w , = 1mature HSA.
IFN13 1-, .
= , Fr .
=
leader 1..) -- ko 95 2013 pC4:HSA-IFNb.1V122- Amino acids M22 to N187 of EENb pC4 311 95 527 HSA
: N187 (fragment shown as amino acids M1 to i . N166 of SEQ ID NO:527) fused 1 r downstream. of HSA.
96 2016 pC4:TR1.M1- Amino acids M1 to L401 of TRi fused pC4 312 96 , 528 819 820 Native TR1 , L401.HSA , upstream of mature HSA. Native TR1 . signal sequence used. A Kozak sequence , was added.
97 2017 pC4:HSA.TR1.E22-Amino acids E22 to L401 of TR1 fuied pC4 ' -` 313 97 529 821 - 822 s HSA
L401 downstream of HSA.
-98 2021 pC4:PTH.S1-Q84/HSA Arnino acids 1-84 of PTH
fused upstream pC4 314 98 530 823 824 HSA
(seamless) of mature HSA and downstream of native ,...., , HSA leader sequence. , ..........
L ______ Table 2 _______________________________________________________________________________ ____________________________________ ¨
Fusion Construct Construct Name Description Expression SEQ ' SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
, _______________________________________________________________________________ X A, ___________ 99 2022 pEE12.1:PTH.S1- Amino acids 1-84 of PTH fused upstream pEE12.1 315 99 531 ------- HSA
Q84 RSA of mature HSA and downstream of native ' HSA leader sequence.
r 100 2023 pSAC35.PTH.S1- ¨Amino acids 1-84 of PTH fused upstream pSAC35 ¨ 316 100 532 825 826 HS Afkex2 Q84.HSA of mature HSA and downstream of c:
P
i HSA/lcex2 leader sequence..
o rs.) 101 P 2025 pSAC35:teprotide.space Teprotide fused upstream of a linker and pSAC35 317 101 533 827 828 co 0.
r.HSA , mature HSA.
1-`
_______________________________________________________________________________ ____________________________________ , 0 . 102 2026 pSAC35:1-ISA.spacer.tep Teprotide fused downstream of HSA and pSAC35 318 102 534 829 830 HSA kr) -.1 ¨a co rotide a linker.
rs.) _ 103 2030 pSAC35.ycolL-2.A21- Amino acids A21 to T153 of IL-2 fused pSAC35. 319 103 . 535 831 832 HSA/ltex2 l A) T153.HSA upstream of mature HSA and downstream ' 1-`

1-`
of HSA/kex2 'leader sequence. DNA
. . 1 rs.) .. encoding. IL-2 has been codon optimized. , kr) A
104 2031 pSAC35.HSA.ycolL- Amino acids A21 to T153 of 1L-2 fused pSAC35 320 104 536 I 833 834 HSAJkex2 2.A21-T153 downstream of HSA with the HSA/lcex2 leader sequence. DNA encoding 11-2 has been codon optimized t, , . , 105 2047 pC4HSA:SP.EPO A28- Amino acids A28 to D192 of EPO
variant pSAC35 321 105 537 1 835 836 IVIP1F
D192 .HSA (where glycine at amino acid 140 has been replaced with an arginine) fused upstream of mature HSA and downstream of1v1PIE
¨ leader p6pticle.
106 2053 pEE12:IFNb-HSA Full length IENb fused upstream of pEE12.1 322 106 538 Native mature HSA.
, IFNb also named leader pEE12.1:1FN13-.11SA _ __.. ' j _________ _ , Table 2 , _______________________________________________________________________________ ______________________________ .....
' Fusion Construct Construct Name Description Expression SEQ 1 SEQ I SEQ SEQ SEQ Leader No. ID Vector ID ID j ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
, 107 2054 pEE12:HSA-IFNb Mature IFNI) fused downstream of HSA. , pEE12.1 323 107 539 HSA
108 2066 pC4:GM-CSF.M1- , Amino acids 1141 to E144 of GM-CSF
pC4 324 108 - 540 ' 837 838 Native E144.HSA , fused upstream of mature HSA.
. GM-CSF
109 2067 pC4:HSA.GM- Amino acids A18 to E144 of GM-CSF
pC4 325 109 541 839 840 HSA
CSF.A18-E144 fused downstream of HSA.
o _ ¨
.
110 2085 pEE12.1:TR1.M1- Amino acids M1 to L401 of TR1 fused pEE12.1 326 110 542 , Native TR-_________________ L401.HSA upstream of mature HSA.
1 N.) 111 2086 pEE12.1:HSA.TR1.E22- Amino acids E22 to L401 (fragment pEE12.1 327 111 543 HSA 0.
1-.
L401 shown as amino acids El to L380 of SEQ

ko -.1 -.3 4) ID NO:543) of TR1 fused downstream of ' N., HSA.
, 0 1-, 112 2095 pC4:HSA-BLyS.A134 Amino acids A134 to L2b5 of BLyS fused pC4 328 112 544 841 842 HSA w - do,Nsmstream of HSA. .
it _ 113 2096 pC4:sp.BLyS.A134- Amino acids A134 to L285 of BLyS
pC4 329 113 545 843 844 Native N., ko L285.HSA (fragment shown as amino acids Al to C1(138 L152 of SEQ ID NO:545) fused upstream of mature HSA and downstream of the CKb8 signal peptide.
114 2101 pcDNA3:SP.Ck7 Q22- N-terminal Methionine fused to amino pcDNA3 330 114 546 845 846 MPH' A89.HSA. acids Q22 to A89 of C1437 fused upstream of mature HSA and downstream of MPIF
sivalpeptide.
D192.HSA (where glycine at amino acid 140 has been replaced with an arginine) fused upstream of mature HSA and downstream of MPIF
leaderipeptide.
.
' Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ 1 Leader ' No. ID
Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
' i X A
116 2129 pC4:TR2.M1-Amino acids M1-A192 of TR2 fused pC4 332 116 548 847 848 Native TR2 A192.HSA , upstream of HSA.
, 117 2137 pSAC35.MDC.G25- Amino acids 025 to Q93 of MDC
fused pSAC35 333 117 549 849 850 HSA/lcex2 Q93.HSA. upstream of mature HSA and downstream (-) of HSA/Icex2- leader sequence.
118 2141 HSA-CK-Beta4 Full length CK-beta4 fused downstream pSAC35 334 118 550 851 852 HSA 0 1.) of ESA.
co I. 1190.
2146 pC4:Leptin HSA 1Full length Leptin fused upstream of pC4 335 119 551 853 854 Native I mature HSA.
leptin ko ...3 co .
c) 120 2181 pC4:HSAIL1Ra(R8- ' Amino acids R8 to E159 of IL1Ra (plus pC4 336 120 552 855 856 HSA 1.) E159) an added methionine at N-terminus) fused 1-, .
w _. .
k downstream of HSA.
4, 121 2182 pC4:MPIFsp(M1- i Amino acids R8 to E159 of IL1Ra (Plus pC4 ' = 337 - 121 553 857 858 MPIF
, 1.) A21),ILIRa(R8- an added methionine at N-terminus) fused ko E159) HSA downstream of the MP7 leader sequence ; I
and upstream of mature HSA. ' .
122 2183 pSAC35ESA.IL1Ra(R8 Amino acids k8 to E159 of1L1Ra (plus pSAC35 338 122 554 859 860 HSA
, -El 59) an added methionine at N-terminus) fused downstream of HSA.
i 123 2184 ' pC4:HSA.Leptin.V22- - Amino acids V22 to C167 of Leptin fused pC4 339 123 555 861 862 HSA
-- ____________________ C166 downstream of HSA.
..---...-47.
124 1 2185 pSAC35:IL1Ra(R8- Amino acids R8 to E159 of IL1Ra (plus pSAC35 340 124 556 863 864 HSA/kex2 E159).HSA an added methionine at N-terminus) fused upstream of mature HSA and downstream , of HSAJIcex2 leader sequence Table 2 ______________________________________________________________________ ...
. _ Fusion Construct I Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
, 125 2186 pSAC35:Leptin.V22-Amino acids V22 to C167 of Leptin fused pSAC35 ' 341 125 557 865 866 IISAikex2 IC166.HSA upstream of mature HSA and downstream I of HSA/Icex2 leader sequence. µ

pSAC35:HSA.Leptin.V Amino acids V22 to C167 of Leptin pSAC35 342 126 558 867 868 HSA/kex2 22-C166 fused downstream of HSA with o HSA/kex2 leader sequence.
, 127 2226 pcDNA3(+):TREM- Amino acids A21 to P202 of TREM-1 pCDNA3.1' ' 343 127 559 869 870 MPIF

1(21-202)-HSA fused upstream of mature HSA and 0.
1-, downstream of the NTT leader sequence, ke -128 2230 pC4:TREM-1.M1- , Amino acids M1 to P202 of TREM-I pC4 - 344 128 ' 560 871 872 Native ...3 p202,HSA fused upstream of mature HSA.
N., , 129 2240 pC4:SP.Ck7 Q22- N-terminal Methionine fused to amino pC4 345 129 561 873 874 MPIF
w A89,HSA. acids Q22 tp A89 of Cki37 fused upstream -.

of mattie HSA and downstream of the ke MPIF leader sequence. Contains a linker ' f sequence between Ck137 and HSA.
I _______ pC4:HSA.Ck7metQ22-- N-terminal Methionine fused to amino pC4 346 130 562 875 876 HSAAcex2 A89. acids Q22 to A89 of Chemolcine beta 7 , (Ckbeta 7 or CK7) fused downstream of ' FISA with HSAikex2 leader sequence.
, Contains a linker sequence between CkB7 and BSA. i __________________________________ ._.
____ -131 , 2244 pC4.HCNCA73.HSA HCNCA73 fused upstream of mature pC4 347 131 563 877 HSA. . ¨
¨

Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ
SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
, . ) X, A
132 2245 pScNIISA:CK7.Q22- Amino acids Q22 to A89 of Ck137 fused pSeNHSA 348 132 564 879 880 HSA/kex2 ' A89 downstream of HSA with HSA/kex2 leader sequence. Contains a linker , sequence between Cki37 and HSA.
133 2246 pScCHSA.CK7metQ22- f-N-terminal Methionine fused to amino pScCHSA 349 133 565 881 882 HSA/kex2 P
A89 acids Q22 to A89 of Ck137 fused upstream rs.) of mature HSA and downstream of , 0.
HSAikeic2 leader sequence.
, 134 2247 pSAV:C1(7metQ22- N-terminal Methionine fused to albino pSAC35 350 S 134 566 883 884 HSA/1ex2 kr) -.I

A89.HSA. acids Q22 to A89 of C1437 fused upstream rs.) of mature HSA and downstream of LA) HSA/kex2 leader sequence._ , 135 2248 pSAC35:HSA.C1C7inet N-terminal Methionine 'fused to amino , pSAC35 351 135 567 8$45 886 HSA/kex2 Q22-A89. ' acids Q22 to A89 of C1437 fused rs.) ko downstream of HSA with HSA/kex2 , leader sequence. Contains a linker ' sequence between Ckf37 and HSA.
136 2249 pSAC35:IFNa2-HSA Mature lTha2 fused upstream of mature pSAC35 352 136 568 887 888 HSA/kex2 HSA and downstream of HSA/kex2 leader also named: sequence. , ..
pSAC23:1FNct2-HSA
137 2250 pSAC35:HSA.INSULIN Mantic Insulinwherein the C-peptide is ¨pSAC35 353 137 569 889 890 HSA
' (GYG) replaced by the C-domain of IGF-1 fused , downstream of HSA. DNA encoding also named: Insulin was codon optimized.
pSAC35.HSA.INSUI1N , G(GYG).F1-N62 i ..
' Table 2 _ Fusion Construct I Construct Name Description Expression SEQ SEQ ' SEQ SEQ I SEQ Leader . .
No. ID
Vector ID ID ID ID ID Sequence ' NO:Y NO: NO:Z NO: NO:B
X
A

, pScCHSA:VEGF2,T103- Amino acids T103 to R227 of VEGF2 - pSeCIISA
354 138 - 570 891 892 HSA/kex2 -R227. fused upstream of mature HSA and , downstream of HSA/kex2 leader i sequence.
_ 139 2252 pScNHSA:VEGF2.T103 Amino acids T103 to R227 of VEGF2 - pSeNHSA - 355 139 571 893 894 HSA/kex2 -R227. fused downstream of HSA with . 0 "
HSAfkex2 leader sequence.
co , 140 2255 pSAC35:INSULIN(G Mature Insulin wherein the C-peptide is pSAC35 356 140 572 / .895 896 HSAIkex2 0.
1-, YG).HSA . replaced by the C-domain of IGF-1 fus$ . ko ...3 4i, 00 . - (...a upstream of mature HSA and downstream also named = of HSA/kex2 leader. DNA encoding 1-, pSAC35.INSULING(G Insulin was codon gptimized. .
' = w ....
, , YG)21-N62.HSA .4 . ,4 . I-`
, -141 , 2256 pSAC35: VEGF2.T103- - Amino acids T103 to R227 of VEGF2 pSAC35 357 141 573 897 898 HSA/kex2 ko R227.HSA fused upstream of mature HSA and ,., , downstream of HSA/kex2 leader , .
,. sequence.
142 2257 pSAC35:HSA.VEGF2.T Amino acids t103 to R227 of VEGF2 - pSAC35 358 142 574 ' 899 900 HSAl1cex2 103-R227 fused downstream of HSA with ,IISA/kex2 leader sequence. :
143 2271 pEE12.1:HCHNF25M1- Amino acids MI to R104 of HCHNF25 ' pEE12.1 359 143 575 Native R104.HSA fused upstream of mature HSA.

, Table 2 ______________________________________________________ _ _____________________________ Fusion Construct Construct Name Description Expression SEQ SEQ i SEQ SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
144 2276 pSAC35:HSA.INSITLIN Mature Insulin wherein the C-peptide is pSAC35 360 ( 144 ' 576 901 902 HSA
, . (OGG) replaced by a Synthetic linker fused downstream of HSA. DNA encoding /
also named: Insulin was codon optimized.

pSAC35.HSA.INSUUN
co G(GGG).F1-N58 "

k - ¨ ____________ 145 2278 pSAC35:insulin(000). Mature Insulin wherein the C-pepfide is pSAC35 361 145 577 903 904 HSA/kex2 0.
1-, HSA replaced by a synthetic linker fused co ko ..3 downstream of WA/kex21eader and ..-N., ooco 4.= upstream of nAture USA. DNA encoding w , Insulin was codon optimized.

146 _ 2280 pC4:HCHNF25.HSA HCIINF25 fused upstream of mature pC4 362 146 578 905 906 Native , 1 ,-RSA.

p- #*,=-. ko 147 2283 pScCHSA:EPOcoA28- Amino acids A28 to D192 of EPO variant pSeCTISA 363 147 579 907 908 HSA/kex2 D192,51N/Q, 65N/Q, (where glycine at amino acid 140 has been 110N/Q EPO replaced with an arginine) are fused upstream of mature HSA and downstream of HSA/Icex2 leader sequence.
.
. ; Glycosylation sites at amino acids 51, 65 , and 110 are mutated from N to Q residue.
DNA =coding EPO is codon pptimixed.
_ _ -Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ 1 SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: , NO:Z NO: NO:B
X
A
=
148 2284 pScNHSA:EPOcoA28- Amino acids A28 to D192 of EPO variant pScNHSA 364 148 580 909 910 HSA/kex2 = D192.51N/Q, 65N/Q, (where glycine at amino acid 140 has been =
110N/Q EPO replaced with an arginine) fused = downstream of mature HSA and HSA/kex2 leader sequence. Glycosylation o sites at amino acids 51, 65 and 110 are ..
e:.
mutated from N to Q residue. DNA
co encoding EPO is codon optimized.
0.
1-, 149 2287 pSAC35:EPOcoA28- Amino acids A28 to 0192 of EPO
variant pSAC35 365 149 581 911 912 HSA/kex2 ko oo D192.51N/Q,65N/Q,110 (where glycine at amino acid 140 has been ...3 kf.
N/Q.HSA. . replaced with an arginine) fused upstream 1-, =
of mature HSA and downstream of w . . .
=-HSAikex2 leaaer sequence. Glycosylation . ..
1-, sites at amino acid 51, 65 and 110 are , N., =
mutated from N to Q residue. DNA-ko ______________________________________ encoding EPO is codon optimized.
150 2289 pSAC35:HSA.EPOcoA Amino acids A28 to 0192 of EPO valiant pSAC35 366 150 582 913 914 HSA/kex2 = 28- (where glycine at amino acid 140 has been ' D192.51N/Q,65N/Q,110 replaced with an arginine) fused.
.IN/Q. downstream of mature HSA and HSA/kex2 leader sequence. Glycosylation ' , . sites at amino acid 51, 65 and 110 are `
` mutated from N to Q residue. DNA
..

encoding EPO is codon optimized.

, .t Table 2 Fusion Construct Construct Name Description Expression SEQ I SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID ID I ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
151 2294 pC4:EPO.R140G.HSA Amino acids MI-D192 of EPO fused pC4 367 151 587 915 916 Native upstream of mature HSA. The EPO
EPO
also named sequence included in construct 1997 was pC4.EPO.R1406.HSA used to generate this construct, mutating arginine at EPO amino acid 140 to glycine. This mutated sequence matches the wildtype EPO sequence.
152 2295 pSAC35:humanresistin. Amino acids K19 to P108 of Resistin pSAC35 368 152 584 917 918 HS A/kex2 K19-P108:HSA fused upstream of mature HSA and = =
=
oo downstream of HSA/kex2 leader sequence. =

1-=
153 2296 pSAC35:HSA:humanres Amino acids K19 to P108 of Resistin pSAC35 , 369 153 585 919 1 920 HSA
1-=
= istin.K19-P108 fused downstream of FISA. 1-=
154 2297 pSAC35:humanresistin. Amino acids K19 to P108 of ResistinPS AC35 370 154 586 921 922 HSAJkex2 K19-P108.stop:HSA fused upstream of mature HSA and downstream of HSA/kex2 leader sequence. Includes two stops at 3 end for termination of translation before the HSA.
155 2298 pEE12.1:EPO.R1400.H Amino acids M1 to D192 of EPO fused pEE12.1 371 155 587 923 924 Native SA Upstream of mature HSA. The EPO
EPO
sequence included in construct 1997 was used to generate this construct, mutating arginine at EPO amino acid 140 to glycine. This mutated sequence matches } the wildtype EPO sequence.
=

, .
Table 2 ..
_ _______________________________________________________________________________ ______________________________________ Fusion Construct Construct Name Description Expression SEQ SEQ SEQ I SEQ SEQ
Leader No. ID Vector ID
ID ID i ID ID Sequence NO:Y NO: NO:Z, I NO: NO:B
X
A
_ _______________________________________________________________________________ ______________________________________ , .

pC4:humanresistin.1141- Amino acids M1 to P108 of Resistin fused pC4 372 156 588 925 926 Native P108:HSA upstream of mature HSA.
...
resistin.

pEE12.1:hunciamesistin. Amino acids MI to P108 of Resistin fused pEE12.1 373 157 , 589 927 Native M1-P108:HSA upstream of mature RSA, resistm , 158 2310 pc4:EPOco.M1- Amino acids M1 to D192 of EPO variant pC4 374 158 590 928 929 Native o , D192 .HSA fused upstream of mature USA. DNA

N., encoding EPO is codon optimized. The co 0.
EPO sequence included in construct 1997 was used to generate this construct, =ko ..3 , --..1 mutating arginine at EPO amino acid 140 - . , to glycine. This mutated sequence . 0 1-, . matches the wildtype EPO sequence.
; w J ..I
. 159 2311 pC4:EPO.M1- Arnith acids MI
to D192 of EPO fized pC4 - 375 159 591 930 931 Native G27.EPOco.A28- upstream of mature USA. DNA
encoding EPO
ko D192.HSA only EPO portion is codon optimized. r The EPO sequence included in construct 1997 was used to generate this construct, , , mutating arginine at EPO amino acid 140 to giycine. This mutated sequence .
matches the wiIdtype EPO sequence.
160 2320 pC4:HCIINF25M1- Amino acids MI to R104 of HCHNF25 pC4 376 160 592 932 933 Native R104.HSA fused upstream of mature USA.HCHNF25 --, * ______________________ .õ
161 2325 pC4.E1"6:MI- Amino acids MI to D192 of EPO fused 'pC4 377 161 593 ' Native D192.HSA.Codon opt. upstream of mature USA. DNA encoding EPO
EPO is codon optimiz ;ed, _ , .
.

, , Table 2 -_______________________________________________________________________________ ________________________________ Fusion Construct Construct Name Description Expression SEQ SEQ SEQ 1 SEQ SEQ i Leader I
No. ID Vector ID ID ID ID ID 1 Sequence NO:Y NO: NO:Z NO: NO:B ;
i X A
,-162 2326 pEE12.1.EPO:M1- Amino acids M1 to D192 of EPO fused pEE12.1 378 162 594 Native D192.HSA.Codon opt. upstream of mature HSA. DNA encoding EPO
i EPO is codon optimized.
_______ , _______________________________________________________________________________ ______________________ i 163 , 2328 pC4:HLDOU18.K23- lAmino acids K23 to R429 of IILDOU18 pC4 379 163 595 934 R429.HSA fused upstream of mature HSA and o downstream of native HSA leader N., sequence.
0.
164 2330 CK-13eta4-HSA Full length okbeta4 fused upstream of. pSAC35 380 164 596 936 937 Native , mature NSA.
CK134 ko -.3 go 165 2335 pC4:MPIFsp.ek{b}4D3 Amino' acids D31 to M96 of Ckbeta4 pC4 381 165 597- 938 939 1 MPIF ' tv 1-M96.HSA fused upstream of mature HSA and 1-, w downstream of MP 1F leader sequence. , o 1-, pC4:MPIFsp.ck{b}:403- Amino acids 035 to M.46 of Ckbeta4 pC4 382 166 5-M96.HSA fused upstream of mature HSA and ko , downstream of MAW leader sequence.

pC4MPIFsp.ck{b}4G4 Amino acids G48 to M96 of Ckbeta4 pC4 383 167 599 942 943 : MPIF
8-M96.11SA , fused upstream of mature HSA and 4- downstream of MPIF leader sequence.

pC4:MPLFsp.ck{b)4A6 'Amino acids A62 to M96 of Ckbeta4 pC4 384 168 4.
2-M96.HSA fused upstream of mature HSA and downstream of MPIF leader sequence.
' 23-R429 , ,. fused dovmstream of HSA.
170 2343 pSAC35.INV-Mature Interferon alpha2 fused upstream - pSAC35 386- 170 602 948 949 invertase IFNA2.HSA of mature HSA and downstream of , invertase sipal peptide.
___________________________________________________ ' Table 2 .
...
, _______________________________________________________________________________ _________________ Fusion Construct [Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:D
X
A
f 171 2344 pC4.SpIg.EPO:A28- Amino acids A28 to D192 of EPO fused pC4 387 171 603 950 951 Mouse 4 D192.HSA.Codon opt. upstream of mature HSA and downstream ' leader of mouse Ig leader sequence. DNA
encoding EPO is codon _Rptimiz,ed.
172 2348 pC4:11/111:Fsp.ck{b}4G5 Amino acids 057 to M96 of Ckbeta4 pC4 388 172 604 952 953 MPEF 0 7-M96.HSA fused upstream of mature HSA and downstream of MPIF leader se. uence.
N.) co 173 2350 pC4:MPLEsp.HLDOU18 Amino acids S320 to R429 of HLDOU18 pC4 389 173 605 954 955 MPIF 0.

(S320-R429).HSA fused upstream of mature HSA and ko co--.1 %.o downstream of MPIF leader sequence. -= . , N.) 174 2351 p04:HSA.HLD01:18(S Amino acids S320 to R429 of HLDOU18 pC4 = 390 174 606 ' 956 957 HSA 0 32&-R429 fused downstream of HSA.
L..) 175 2355 pSAC35:MATalpha.d8c Amino acids G28 to N93 of Ckbetal : pSAC35 391 607 958 959 MFo.-1 I- `

kbetal.G28-N93:HSA fused upstream of mature HSA arid N.) ko downstream of the yeast mating factor ¨ alpha leader sequence.
, 176 2359 pEE12:HLDOU18.K23- Amino acids K23 to R429 of HLDOU18 pEE12.1 392 176 608 HSA
R429.HSA fused upstream of mature NSA and ' downstream of native NSA leader sequence.
177 2361 pC4:11RDFD27:HSA HRDFD27 fused upstream of mature pC4 393 177 609 960 961 Native HSA.

178 2362 pEE12:HSA.HLDOU18 Amino acids K23 to R429 of HLDOU18 pEE12.1 394 178 610 1111111111=
K23-R429 fused downstream of HSA.

Table 2 Fusion Construct Construct Name Description I Expression SEQ SEQ SEQ SEQ SEQ
Leader No. ID
Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z1 NO: NO:B
X
A.
179 2363 pC4GCSF.HSA.EPO.A Amino acids M1 to P204 of GCSF
fused pC4 395 179 611 Native 28-D192 upstream of mature HSA which is fused GCSF
upstream of amino acids A28 to D192 of EPO variant (where amino acid 140 of =
EPO is mutated from glycine to arginine.) 180 2365 pEE12.1.HCNCA73HS HCNCA73 is fused upstream of mature pEE12.1 396 180 612 962.....963 Native 0 A HSA.

= 181 2366 pSAC35.MAF-Mature 1FNa2 fused upstream of mature PSAC35 397 181 613 964 965 M.1-'0.-1 LFNa2.HSA HSA and downstream of yeast mating = No =
factor all ha leader sequence. =

182 2367 pEE12.MPIFsp.HLDOU Amino acids S320 to R429 of HLDOU18 pEE12.1 ¨898 182 614 966 967 MPIF
18.S320-R429.HSA fused upstream of mature HSA and . . downstream of IVTPIF leader sesuence.
183 2369 pC4:HLDOU18.HSA Amino acids M1 to R429 of HLDOU18 pC4 399 183 615 968 969 Native N.) fused upstream of mature HSA.

184 2370 pEE12:HLDO1J18.HSA Amino acids M1 to R429 of HLDOU18 pEE12.1 400 184 616 Ina Native fused u = stream of mature HSA.

185 2373 pC4.GCSF.HSA.EPO.A Amino acids M1 to P204 of GCSF
is pC4 401 185 617 Native 28-D192.R140G fused upstream of mature HSA
which is GCSF
fused upstream of amino acids A28 to D192 of EPO, wherein amino acid 140 is glycine. The EPO sequence included in construct 1997 was used to generate this construct, mutating arginine at EPO amino acid 140 to glycine. This mutated sequence matches the wildtype EPO
I sequence.

s.
=
=
Table 2 Fusion Construct Construct Name Description Expression SEQ ' SEQ SEQ SEQ 1 SEQ Leader No. ID
Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B , _______________________________________________________________________________ __________ X A
186 2381 pC4:HSA--1FNa2(C17- Amino acids C17 to E181 of 1FNa2 - pC4 402 186 618 970 971 HSA
E181) (fragment shown as amino acids Cl to E165 of SEQ ID NO:618) fused downstream of HSA.
_______________________________________________________________________________ ___ ¨ _____________ 187 2382 pC4:1FNa2-HSA TYNa2 fused upstream of mature HSA. pC4 403 187 619 972 973 Native ci ' IFNa.2 , leader , 1..) 188 2387 pC4:EPO(0140)-HSA- - Amino acids M1-D192 ofEPO
fused pC4 - - 404 188 620 Native 0.
= GCSF.T31-P204 upstream of mature HSA which is fused , ,EPO 0 ko .c.
...3 , upstream of amino acids T31 to P204 of GCSF.
1..) , 189 2407 pC4:HWHGZ51.M1-Ammo acids to N323 of HWHGZ51 pC4 405 189 621 974 975 Native 1-w .

N323.HSA fused upstream of mature HSA. t . , HWHGZ5 1-, ,, 1..) 190 2408 pEE12.1:HWHOZ51.M Amino acids M1 to N323 of HWHGZ51 pEE12.1 406 190 622 976 977 ' Native ko 1-N323.HSA fused upstream of mature HSA.

, 191 2410 ,pSAC35INTV:1FNa-HSA Mature 1FNa2 fused downstreamof the pSAC35 ' 407 191 623 978 979 , invertase invertase signal peptide and upstream of i mature HSA.
___________________ ¨
192 2412 pSAC35:de1l(EX.d8ckb Amino acid.s G28 to N93 of Ckbetal pSAC35 - 408 192 624 980 981 HSA minus etal.G28-N93:HSA fused downstream of the HSA signal the NEX
sequence (with the KEX site deleted - last' site ' 6 amino acids of the leader) and upstream of mature HSA.

Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID .
Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:13 =
X A
_ 193 2414 pC4.EPO:M1- Amino acids M1 to D192 of FPO fused pC4 409 193 625 982 983 Native D192copt.HSA.GCSF.T upstream of mature HSA which is fused EPO
31-P204 upstream of amino acids T31 to P204 of GCSF. DNA encoding EPO has been , , o also named: codon optimized.
=
pC4.EFO:M1- 0 1..) D192copt.HAS.GCSF.T , co 0.

1-, , ---194 2428 = pN.4:PTH.S1-Q84/HSA. Amino acids Si to Q84 of PTH fiised pN4 410 194 626 HSA ko .4 k.a f 4 ) . sob . I ,...
N.) 1 ==

= I) upstream of mature HSA and downstream 1.) I .
,. of the native HSA leader sequence.
, . . =1-, w 195 2441 pEEIIEPO:M1- . Arnino.acids M1 to D192 of EFO fused pEE12.1 409 196 628 1 EPO leader 1-,1 . ..!... . ..
D192copt.HSA.GCSF.T upstream of mature HSA which is fused Fr 31-P204 upstream of amino acids T31 to P204 of 1..) ko GCSF. DNA encoding EPO has been , , ' also named: codon optimized.
pEE12.EPO:M1-D192copt.HAS.GCSF.T ' , 196 2447 pC4:HSA.humancalcito Amino acids C98 to G130 of SEQ ID ' pC4 413 197 629 986 987 HSA ' nin..C1-G33 =NO:629 fused downstream of HSA. .
., .---197 2448 pSAC35:GLP-1(7- Amino acids H98 to R127 of pSAC35 414 198 630 988 989 HSA/kex2 i' 36).HSA preproglucagon (SEQ ID NO:630) (hereinafter this specific domain will be referred to as "GLP-1(7-36)") is fused upstream of mature HSA and downstream of IISA/kex2 leader sequence.
_ _ Table 2 .
' Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ ' Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
I
X
A
...i.

pSAC35:INV.d8CKB1. Amino acids 028 to N93 of Ckbetal pSAC35 415 199 631 990 991 Invertase 028-N93:HSA fused downstream of the invertase signal , õpeptide and upstream of mature HSA.
1 ______ -pSAC35:HSA.GLP-1(7- GLP-1(7-36) is fused downstream of pSAC35 416 200 1. 632 992 993 HSA/kex2 36) mature IBA and HSA/kex2 leader ! =-+- i sequence.

200 2456 pSAC35:GLP-1(7- Amino acids 1198 to IZ.127 of pSAC35 417 201 633 994 995 HSA/kex2 "

36(A80)).HSA Preproglucagon (SEQ ID NO:633)(also 0.
1-, ., referred to as "GLP-1(7-36)") is mutated . 0 ko tcs .4 (.).) at amino acid 99 of SEQ ID NO:633 to =
replace the alanine with a glycine. This , 1-, , particular GLP-1 mutant will be w hereinafter referred to as "GLP-I (7-1-, 1. 36(A8G))" and corresponds to the ,.., sequence shown in SEQ ID NO:1808.
, ko , GLP-1(7-36(A8G)) is fused upstream of mature HSA and downstream of HSAikex2 leader sequence.
,.. ____ pSAC35:HSA.GLP-I (7- GLP-1(7-36(A8G)) (SEQ ID NO:1808) is pSAC35 418 202 634 996 997 HSAlkex2 36(A8G)) fused downstream of mature USA and ___________________________________ IISA/Icex2 leader sequence.
' *

pSAC35:11SA.exendin. Amino acids 1148 to S86 of Extendin pSAC35 419 H48-S86 fused downstream of full length USA.

pSAC35:Exendin.H48- Amino acids 1148 to S86 of Extendin pSAC35 420 204 636 HSAJkex2 S86.HSA fused upstream of mature USA and downstream of HSA/kex2 leader 'sequence. , Table 2 _ _______________________________________________________________________________ ________________________________ Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
A
204 2473 pC4.111,DOU18:HSA:S I Ml-R319 of HLDOU18 (containing the pC4 421 205 637 998 999 Native 320-R429 furin site RRKR) followed by residues ' `LE' followed by mature HSA followed = by 'LE' and amino acids S320 through ' R429 of HGDOU18 (fragment shown as ci = SEQ 1D NO:637).
205 2474 pSAC35.MDC.P26- Amino acids P26 to Q93 of MDC fused pSAC35 422 206 638 1000 1001 HSA/kex2 co 0.
Q93.HSA downstream of the HSA/kex2 leader and e:.
upstream of mature HSA.
ke ...3 t 206 2475 pSAC35.MDC.11426- Amino acids Y27 to Q93 of MDC with an pSAC35 423 207 639 1002 1003 HSA/kex2 1..) e:.
Q93.HSA N-terminal methionine, fused downstream 1-, .
w , of the HSA/kex2 leader and upstream of . 1-,1 1-, mature HSA. _ i µ
k ) IV
207 . 2476 pSAC35.MDC.Y27- Amino acids Y27 to Q93 of MDC fused pSAC35 424 208 640 1004 1005 HSA/kex2 ke Q93.HSA downstream of the HSA/kex2 leader and , upstream of mature HSA.
208 2477 pSAa35.MDC.M27- Amino acids 628 to Q93 of MDC with an pSAC35 425 209 641 1006 1007 HSA/kex2 Q93.HSA N-terminal methionine, fused downstream , of the HSA/kex2 leader and upstream of mature HSA.
_ _ 209 2489 ' pSAC35:11SA.C17.A20- Amino acids A20 to R136 of C17 fused pSAC35 426 = 210 642 1008 1009 HSA/kex2 R136 downstream of mature HSA with HSA/kex2 leader sequence.
t _______________________________________________________________________________ ________________________ -210 2490 pSAC35:C17.A20- Amino acids A20 to R136 of C17 fused pSAC35 427 211 643 1010 1011 HSA/kex2 = R136.HSA downstream of the HSA/kex2 leader and upstream of mature HSA.
-, Table 2 r _______________________________________________________________________________ ________________________________ Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID = Vector ID
ID ID ID ID Sequence NO:Y NO: NO:2 NO: NO:B
___________________________________________________________________________ .
X A
211 2492 pC4.IFNb(deltaM22).H Mutant full length INFbeta fused pC4 i 428 644 Native SA -upstream of mature HSA. First residue of native, mature IFNbeta (M22) has been leader deleted.
212 2498 pC4:HSA.KGF2D60.G9 Amino acids G96 to S208 of KGF-2 fused pC4 429 213 645 1012 1013 HSA 0 6-S208 downstream of HSA.
213 2499 pC4:KGF2D60.096- Amino acids 096 to S208 of KGF2 fused pC4 430 214 646 1014 11= c) "
op 0.
01¨

S208:HSA upstream of mature HSA and downstream of the HSA signal peptide.
ko ...3 5, 214 2501 pSAC35:scEvI006D08. BLyS antibody fused upstream of mature pSAC35. 431 215 647 1016 1017 HSA/kex2 HSA HSA and downstream of HSA/kex2 signal E0 1-, peptide. ' i = w , 215 2502 pSAC35:seFvI050B11. BLyS antibody fused upstream of mature pSAC35 432 216 648 1018 1019 HSA/kex2 1-, , HSA HSA and downstream of HSA/kex2 leader N., ko sequence.
, 216 2513 pC4:HSA.salmoncalcito Cl through 033 of salmon calcitonin pC4 1513 1345 1681 1854 1855 HSA
nin.C1-033 fused downstream of full len:. h HSA.
217 2515 pC4:HDPBQ71.M1- M1 through N565 of HDPBQ71 fused pC4 1514 1346 1682 1856 1857 Native N565.HSA upstream of mature EISA
HDPBQ71 ' 218 2529 pC4:TR1.M1- Amino acids M1 to K194 of TR1 pC4 1223 1208 1238 1253 1254 Native TR1 K194 .HSA (including native signal sequence) fused uostream of mature }ISA.
219 ' 2530 pC4:TR1.M1- Amino acids M1 to Q193 of ilt1 pC4 1209 1239 1256 Native TR1 Q193.HSA (including native signal sequence) fused u.stream of mature HSA.

., -.
Table 2 Fusion Construct Construct Name [Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: = NO:Z NO: NO:B
, _ X
A
, ¨
, 220 2531 -pC4:TR1.M1- Amino acids MI to E203 of TR1 pC4 1225 1210 1240 1257 1258 Native TR1 E203.HSA (including native signal sequence) fused upstream of mature HSA.
, , A
221 2532 pC4:TR1.M1- .Amino acids M1 to Q339 of TR1 pC4 1226 1211 1 1241 1259 1260 Native TR1 Q339.HSA (including native signal sequence) fused , upstream of mature HSA.

, 222 2545 pEE12.1:HDPBQ71.MI MI through N565 of HDPBQ71 fused pEE12.1 1515 1347 ' 1683 Native co 0.
-N565.HSA upstream of mature HSA
___________________________________________________ HDPEQ71 _ __ ...

223 2552 pSAC35:KGF2delta33.S Atnino acids SO through S208 of KGF2 pSoA 1516 ' 1348 1684 1858 1859 ' HSA/kex2 ko --.1 \ 0 IV
C71 69-S208.HSA fused upstream of HSA.
, _______ A

224 2553 psAc351BA.KOF2delt HSA/kex2 signal peptide followed by I
pSeNHSA 1517 1349 1685 1860 = 1861 HSA/kex2 a33.S69-S208 HSA peptide followed by amino acids w S69 to S208 of ICGF2.

225 2555 pEE12.1:TR1.M1- Amino acids MI to Q193 of TRI
pEE12.1 1227 1212 1242 Native TR1 ko Q193.HSA (including native signal sequence) fused upstream of mature HSA.
, 226 2556 pEE12.1:TR1.M1- iAmioacidsMltoKl94ofTRl pEE12.1 1228 1213 1243 Native TR1 , K194.HSA (including native signal sequence) fused \
upstream of mature HSA.
227 2557 pEE12.1:TR1.M1- Amino acids M1 to E203 of Tlil - pEEI2.1 1229 1214' 1244 ' 1 Native TRI
E203 ,HSA (including native signal sequence) fused ' upstream of mature HSA.
,.. _ 228 2558 pEE12.1:TRI.M1- Amino acids M1 to Q339 of TR1 ' pEE12.1 1230 1215 ' 1245 Native TRI
Q339.HSA (including native signal sequence) fused , upstream of mature HSA.
At.
.....----.....-...
A
. .

Table 2 _ _______________________________________________________________________________ _____________________________________ ' Fusion Construct Construct Name Description Expression SEQ i SEQ SEQ SEQ SEQ Leader No. ID
Vector ID ID ID ID ID Sequence = NO:Y NO: NO:Z NO: NO:Bt i X A , 229 , 2571 pC4.0SCAR.R232.11SA M1-R232 of OSCAR fused upstream of pC4 1518 1350 1686 11862 - 1863 Native ., : mature RSA.
OSCAR
:
receptor leader t 230 2580 , pC4.IFNb(deltaM22,C3 IFNb fused upstream of mature HSA. The pC4 1519 1351 1687 Native (-) = 8S),IISA
IFNb used in this fusion lacks the first IFNil .

, residue of the mature form of JENb, which . 1..) corresponds to M22 of SEQ ID NO:1687. , 0.
i-t Also amino acid 38 of SEQ IDNO:1687 , 0 . .
ko %.0 ...3 has been mutated from Cys tf...Ser.'. Ai -= 231 2584 pC4:MPIFsp.KGF2delta MPIF signal sequence followed by A63 pC4 1520 1352 1688 1864 1865 MPIF 1..) i-t 28.A63-S20811SA through S208 of KGF2 followed by w = mature HSA.
i-t , 232 2603 - pC4:1-ISA(A14)- , Modified IBA A14 leader fused upstream pC4 -L521 1353 1689 Modified ' 1..) : ko EPO(A28-D192.0140)_ of mature USA which is fused upstream , HSA (A14) of A28 through D192 of EPO. Amino _acid 140 of EPO is a'(3'.

233 2604 pC4:1-ISA(S14)- Modified HSA S14 leader fused upstream pC4 1522 1354 1690 Modified = i EPO(A28-D192.G140) ' of mature HSA which is fused upstream RSA (S14) of A28 through D192 of EPO. Amino acid 140 of EPO is a'(3'.
..) 234 ' 2605 pC4:HSA(G14)- Modified HSA G14 leader fused upstream pC4 1523 1355 1691 Modified EPO(A28-13192.0140) of mature HSA which is fused upstream HSA (G14) of A28 through D192 of EPO. Amino =
I ' ' acid 140 of EPO is a 0.
, 1. 4 _____ ,,,õ

. \
Table 2 ' _ _______________________________________________________________________________ ____________________ Fusion Construct Construct Name -Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID
Vector ID ID ID ID ID I Sequence NO:Y NO: NO:Z NO: NO:B
X
A
- .
235 2606 pC4:HSA#64.KGF2D28 A63 through S208 of KGF2 fused pC4 ' 1524 1356 1692 1866 1867 Modified .A63-S208 : downstream of mature HSA and the NSA #64 =
.modified #64 leader sequence. .
236 2607 pC4:HSA#65.K0F2D28 A63 through S208. of KGF2 downstream p04 1525 1357 1693 1868 1869 Modified .A63-S208 of mature HSA and the modified #65 HSA #65 o leader sequence.

, - =
. 1..) 237 2608 pC4:HSA#66.K0F2D28 A63 through S208 of KGF2 fused p04 1526 ' 1358 1694 1870 1871 Modified 0 0.
.A63-S208 downstream of mature HSA and the = . . HSA #66 ko modified #66 leadersequence.
-.3 , -, .= _ 238 2623 pC4:(AGVSG,14- A modified HSA Al4 leader followed by pC4 . 1527 1359 1695 = Modified 1..) 18)HSAELDOU18.K2 mature HSA and amiro acids K23 S. HSA (A14) w 1-, 3-R429 through R429 of 1-3LDOU18.
leader , .
, 239 .. 2624 pC,4:(SGVSG,14-Modified HSA S14 leader followed by pC4 1528 1360 1696 Modified - 18)HSA.FILD01.118.K2 mature HSA and amino acids K23 t i , HSA (S141) - :)=) 3-R429 R429 of HLDOU18.
1 leader ¨ _______________________________________ J
>
240 2625 , pC4:(GGVSG,14- A
modified HSA G14 leader sequence p04 ' ¨ 1529 1361 1697 Modified 18)HSA.HLDOU18.K2 followed by mature HSA and amino acids HSA (014) 3-R429 K23 through R429 of HL.DOU18.
I leader 241 2630 pC4:HSA.KGF2D28.A6 'Amino acids A63 to S208 of KGF-2 p04 1530 1362 1698 1872 1873 HSA
3-S208#2 fused to the C-terminus of HSA.
-I
_______________________________________________________________________________ ______________________________________ 242 2631 pEE12.1:(AGVSG,14-A modified HSA A14 leader sequence = pEE12.1 1531 1363 1699 Modified 18)HSA.HLDOU18.K2 followed by mature HSA and amino acids = HSA (A14) 3-R429 K23 through R429 of HLDOU18 leader.
. _____________________________________________________________________ 4 'pEE12.1:(SGVSG,14- Modified HSA S14 leader followed by = pEE12.1 1532 1364 1700 Modified ' 1.8)11SA.HLDOU18.K2 mature HSA and amino acids K23 to HSA (S14) -----..-3 3-R429 R429 of HLDOU18.=
leader .a , =

Table 2 = -, _ Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ 1 SEQ Leader No. ID . Vector ID ID ID ID 1 ID Sequence NO:Y NO: NO:Z , NO: NO:B
X
A.
, .
244 2633 pEE12.1:(GGVS0,14- A modified HSA 014 leader sequence pEE12.1 1533 1365 1701 Modified 1 i 18)HSA.HLDOU18.K2 followed by mature HSA and amino acids HSA (014) 3-R429 K23 through R429 of HLD01318. 1 leader , 245 2637 pSAC35:HSA.GCSF.T3 HSA/kex2 leader fused upstream of pScNHSA 1534 1366 1702 -4' 1874 1875 HSAlkex2 1-P207 mature NSA followed by T31 through , P207 of GCSF (SEQ ID NO:1702). ._ 246 2638 pPPC007:116A01.HSA scFv Il I6A01 with C-terminal HSA
pPPC007 1 1535 1367 1703 1876 1877 scFvI006A 1..) fusion, where the mature form of HSA01 0.
1-, .

= , , lacks the first 8 amino acids. , ko ..3 T 247 2647 pSAC35:T7,11SA.
The T7 peptide (SEO ID NO: 1704) of ' pScCHSA 1536 1368 1704 1878 1879 HSAficex2 1..) ..

Tumstatin was fused with a C-terminal 1-, w HSA and N terminal HSA/Icex.2 leader.
, 1 248 2648 pSAC35:T8.HSA The Tg peptide (SEQ IQ NO: 1705) of pScCHSA-. 1537 , 1369 1705 1880 1881 TISA/kex2 1..) - Tumstatin is fused upstream to mature ko HSA and downstream from .
,.. HSA/Icex2.
249 2649 pSAC35:HSA.T7 The T7 peptide (SEQ ID NO: 1706) of pScNHSA 1538 1370 1706 1882 .1883 HSA/Icex2 'f Turnstatin was fused with a N-terminal , _., , HSA/Icex2 siaual sequence. ,, _ 250 2650 p-SAC35:HSA.T8 The T8 peptide (SEQ ID NO: 1767) of pScl\THSA 1539 1371 1707 1884 1885 HSA/Icex2 Tumstatin is fused downstream to HS Aikex2 signal sequence and mature _ H$A.

. .
Table 2 _ _______________________________________________________________________________ ________________________________________ Fusion Construct Construct Name ' Description Expression SEQ SEQ 1 SEQ 1 SEQ ' SEQ Leader No. ID
Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
f X
A
_ _______________________________________________________________________________ _______________________________________ .,.1 251 2656 pSac35:Insulin(KR.GG Synthetic gene coding for a single-chain - pScCHSA . 1540 -1372 1708 1886 1887 HSAilex2 G.KR).HSA insulin with HSA at C-terminus.
Contains a modified loop for processing resulting in ' correctly disulfide bonded insulin coupled to HSA.
o 252 2667 pSAC35:HSA.T1249 T1249 fused downstream of full length - pSAC35 1178 ' 1179 1180 1181 1182 HSA 0 1..) HSA
co 0.
253 2668 pSac35:HSA.Insulin(KR Synthetic gene coding for insulin with FL pScNHSA 1541 1373 1709 1888 1889 HSA

= = .GGGI(R) _ HSA at N-terminus. Contains a modified = ko -.3 , . =
S=loop for processing resulting in correctly 1..) .
i - disulfide bonded insulin coupled to HSA. 1-, w 254 2669 _ ISac35:Insulin(GGG.K Synthetic gene coding for a single-chain pScCHSA 1542 1374 1710 1890 1891 HSA/kex2 1 1 1-, , = K).HSA insulin with HSA at C-terminus.
Contains 1-, , =a modified loop.
S... 1..) ko , _ 255 2670 pSAC35:T1249.1ISA T1249 fused downstream of :HSAdekeI2 ¨ pSAC35 1183 1179 1180 1184 1185 HSA/Icex2 ),eader and upstream of mature HSA.
256 2671 pSac35:1=ISA.Insulin(GG Synthetic gene coding for a single-chain pSeNHSA 1543 1375 1711 1892 1893 USA
G.KX) insulin with USA at N-terminus.
Contains a modified loop for rater stability 257 2672 pSAC35:HSA.T20 Amino terminus of T20 (codon optimized) pSAC35 1186 1187 1188 1189 1190 USA
fused dovvnstream of fidl length HSA
______________ ...-258 2673 pSAC35:T20.HSA 'Amino terminus of T20 (codon optimized) pSAC35 1191 1187 1188 1192 1193 HSAikex2 , fused downstream of HSARcex2 leader and upstream of mature HSA.
259 2700 'pSAC35:HSA.GCSF.T3 ¨C-terminal deletion of GCSF
fused pSAC35 1544 1376 1712 1894 1 1895 MI
1-R199 downstream of mature HSA.

Table 2 Fusion Construct Construct Name Description 1Expression SEQ SEQ SEQ SEQ SEQ Leader No, ID = Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
260 2701 pSAC35:HSA.GCSF.T3 C-terminal deletion of GCSF fused pScNHSA 1545 1377 1713 1896 1897 HSA/kex2 1-H200 downstream of mature HSA.
261 2702 pSAC35:HSA.GCSF.T3 HSA/kex2 leader followed by mature pSAC35 1194 1195 1196 1197 1198 HSA/kex2 1-L201 HSA and amino acids T31-L201 of GCSF
(corresponding to amino acids Ti to L171 of SEQ ID NO1196).

262 2703 pSAC35:HSA.GCSF.A3 HSA/kex2 leader followed by mature pSeNHSA 1546 1378 1714 1898 1899 HSAl1ex2 co 6-P204 USA and amino acids A36-P204 of GCSF, S. 263 2714 pC4:HSASP.PTH34(2)/ PTH34 double-tandem repeats fused pC4 1199 1200 1201 1202 1 1203 HSA leader HSA downstream of HSA leader (with the KEX
minus Kex 0 site deleted ¨ last 6 amino acids of the site leader) and upstream of mature IBA.
. ' 264 2724 pSAC35.sCNTF."HSA HSA/Kex2 fused to CNTF, and then pSAC35 1547 1379 1715 1900 1901 .HSA/kex2 :)=) fused to mature HSA.
265 2725 pSAC35:1-ISA.sCNTF HSA/Kex2 fused to mature HSA and pSAC35 1548 1380 1716 1902 1903 HSA/kex2 then to CNTF
266 2726 pSac35.1NV.GYGinsuli Synthetic gene coding for a single-chain pSAC35 1549 1381 1717 1904 1905 Invertase n.HSA insulin with HSA at C-terminus. The signal peptide of invertase is used for this construct.
267 2727 pSac35.ENTV.GYGinsuli Synthetic gene coding for a single-chain pSAC35 1550 1382 1718 1906 1907 invertase n(delF1).HSA insulin with HSA at C-terminus.
Construct uses the invertase signal peptide and is lacking the first amino acid (F) of 'nature human insulin.

Table 2 _ 'Fusion Construct Construct Name Description Expression SEQ SEQ ' SEQ SEQ SEQ 1 Leader No. ID ' Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X A
HSA OSCAR fused upstream of mature HSA.
OSCAR
leader HSA codon optimized insulin with HSA at C-terminus.
o , ______ 1270 2789 pSAC35:Insulin(GGG). Synthetic gene coding for a single-chain pSAC35 1553 1385 1721 ' 1912 ( 1913 invertase 0 N., ______________________ HSA (codon optimized) insulin with HSA at C-terminus.
co . 0.

..3 ,--.
..
c:, 1 downstream from HSA signal peptide - site i,..) (with the KEX site deleted ¨ last 6 amino , .
w acids of the leader) ' 1-, , . =
IENb.M22-N187 C-terminus of HSA, which contains an FigA (A14) ke modified signal peptide, designed to , improve processing and homogeneity.
.. _________________ IFNb.M22-N187 C-terminus of HSA, which contains a HSA (S14) modified signal peptide, designed to , improveyrocessinj and homogeneity. .
IFNb.M22-N187 C-terminus of I-ISA, which contains an : HSA, (014) ________________________________________ modified signal peptide.
, 275 2798 pSAC35:Somatostatin(S A 14 amino acid peptide of Somatostatin pScCHSA 1558 1390 1726 1914 1915 HSAfIcex2 14).HSA fused downstream of HSA/Icex2 leader , ,, and uystream of mature HSA.
.

Table 2 .
Fusion I Construct ' Construct Name ' Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A ' =
276 2802 =pSAC35:GLP-1(7- GLP-1(7-36(A80)) (SEQ BD NO:1808) is pScNHSA 1559 1391 1727 , HSAikex2 36(A80)),T2.HSA fused downstream from the HSA/kex2 leader sequence and upstream from the =
intervening peptide-2 of proglucagon ' , peptide and upstream from mature HSA, =0 I P
277 2803 pSAC35:GLP-1(7- = ' GLP-1(7-36(A8G)) (SEQ ID NO:1808) is pScCHSA 1231 1216 1246 1261 1262 HSA/kex2 0 36(A8G))x2.HSA tandemly repeated and fused downstream "

i of the HSAlkex2 signal sequence, and p=
1-`
.=

upstream of mature HSA.
ko .
-.1 278 2804 pSAC35:coGLP-1(7- GLP-1(7-36(A8O)) (SEQ 1D NO:1808) is pScCHSA 1232 1217 1247 1263 1264 HSA/kex2 N.) J.) =

36(A8G))x2.HSA tandemly repeated and fused downstream l A) =
of the HSAikex2 signal sequence, and 1 1-`
.,, upstream of mature HS.A.
1-`

279 2806 pC4:HSA#65.salmoncal Modified NSA leader #65 followed by pC4 1560 1392 1728 1916 1917 I
Modified "
ko citonin.C1-G33 mature NSA and C1-033 of salmon NSA #65 ca1citonin.
_ 280 2821 pSac35.delKex2.Insulin(- Synthetic gene coding for a single-chain pScCHSA 1561 1393 1729 Modified , GYG).HSA insulin with NSA at C-terminus. The kex2 HSA/kex2, site has been deleted from the HSA/KEX2 lacking the signal peptide.
' , Kex2 site. 4 281 2822 pSac35.alphaMF.Insulin Synthetic gene coding fora single-chain pSAC35 1562 1394 1730 1920 1921 MFo.-1 = (GYG).HSA insulin with HSA at C-terminus. The =signal peptide of alpha mating factor QVIFa.-1) is used for this construct.
=.
282 2825 pSAC35:HSA.Somatost 14 amino acid peptide of Somatostatin pSel\THSA 1563 1395 1731 1922 1923 HSAikex2 atin(S14) was fused downstream of HSA/kex2 , =leader and mature HSA.
¨ ,, ¨ ______________________ .. , Table 2 _ Fusion I Construct Construct Name Description Expression SEQ SEQ 1 SEQ SEQ SEQ Leader -4 No. ID, , , ' Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: . =NO:B
X
A

¨
2830 pSAC35:S28,HSA 28 amino acids of sornatostatin fused pScCHSA 1564 1396 1732 1924 1925 IISAlkex2 downstream of HSA/kex2 leader and , upstream of mature HSA.
..
284 2831 pSAC35:HSA.S28 28 amino acids of somatostatin fused , pScNIISA. 1565 1397 1733 = 1926 1927 HSA/kex2 downstream of HS A/kex2 leader and i0 1 mature HSA.
, :
, .
_.. ____ 4 0 285 2832 pSAC35:Insulin.HSA. Long-acting insulin peptide fused pScCHSA 1566 1398 1734 , 1928 1929 invertase "
0.
, (yeast codon optimized) ' upstream of mature HSA.
.

286 H 2837 pSAC35:CKB1.K21-K21-N93 of CK.D1 (fragment shown as .pScCHSA 1567 1399 1735 1930 1931 HSA/kex2 ko -.3 .-0 - N93:HSA 1(2 to N74 of SEQ ID NO:1735) fused 1..) .tz..0 downstream of the HSA/kex2 leader and 1-, w upstream of mature HSA.

, - -.
,,-.. . _ . 1-, 287 2838 pSAC35:CK131.T22-T22-N93 of CI031 (fragment shown as T3 pScCHSA 1568 1400 1736 1932 1933 HSA/kex2 N93:HSA to N74 of SEQ ID NO:1736) fused 1..) ko downstream of the HSA/kex2 leader and upstream of mature HSA.
, , 288 2839 pSAC35:CK.131.E23;
E23-N93 of CKB1 (fragment shown as E4 pScCIISA 1569 " 1401 1737 1934- 1935 1 HSA/kex2 N93:HSA to N74 of SEQ ID NO:1737) fused downstream of the HSA/kex2 leader and , upstream of mature HSA.
, -289 2840 pSAC35:C1(131.S24- S24-N93 of CIU31 (fragment shown as S5 ' pScCHSA ' 1570 1402 1738 1936 ' 1937 HSAJkex2 ' N93:HSA to N74 of SEQ ID NO:1738) fused , downstream of the HSA/kex2 leader and . , E
i upstream of mature HSA.
,I , , . , .._ ... ..

=
Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader = No. ID .
Vector . ID ID ID ID ' ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
-_______________________________________________________________________________ _____________________________ , 290 2841 pSAC35:CKB1.825-' S25-N93 of CKB1 (fragment shown as S6 pSeCHSA 1571 1403 1739 ' 1938 1939 HSAikex2 N93:HSA to N74 of SEQ ID NO:1739) fused downstream of the HSA/kex2 leader and upstream of mature IISA.
1 .
291 2842 p5AC35:CKB1.S26- S26-N93 of CKB1 (fragment shown as 57 pSeCHSA ' 1572 1404 1740 1940 1941 ' HSAncex2 N93:HSA , to N74 of SEQ ID NO:1740) fused , downstream of the HSA/kex2 leader and cp 1..) upstream of mature EISA.
1 _,,, co 0.
.
1-, , 292 ' 2843 pSAC35:CKB1.R27- R27-N93 of CKB1 (fragment shown as PSeCIISA 1573 ' 1405 1741 1942 1943 ' HSA/kex2 cp ko ,--, N93:HSA R8 to N74 of SEQ ID NO:1741) fused...3 t..h 1..) downstream of the HSA/kex2 leader and cp 1-, upstream of mature HSA.' w , . _____________________ - 1 = 293 2844 [
pSAC35:CKBLP29- P29-N93 of CKB1 (fragment shown as pSeCHSA 1574 1406 , 1742 1944 1945 HSA/kex2 1-, 1-, .N93:HSA P10 to N74 of SEQ ID NO:1742) fused ' 1..) downstream of the HSA/kex2 leader and = =
ko , . upstream of mature HSA.
r ___________ 294 ) - 1 2845 pSAC35:CKBLY30- Y30-N93 of CKB1 (fragment shown as pSeCHSA 1575 1407 i 1743 1946 H' 1947 - HSAilcex2 = N93:HSA
f Yll to N74 of SEQ ID NO:1743) fused = .
downstream of the HSA/kex2 leader and =
upstream of mature I-ISA.
õ
.
, , -N93.HSA 09 to N74 of SEQ ID NO:1744) fused i downstream of the 11/P1F signal peptide =and upstream of mature HSA. , . -,, 296 2872 ¨ pSAC35:HSA.IFNaA(C ' This construct contains a hybrid form of pSAC35 1309 1310 ' 1311 1312 1313 HSAikex2 = 1-Q91)/ D(L93-E166) IFNaA and 1FNaD
fused downstream of mature }ISA. , , , Table 2 ' Fusion Construct 7 Construct Name Description Expression i SEQ SEQ SEQ SEQ SEQ ' Leader No. ,,. ID , Vector ID ID ID ID ID Sequence , NO:Y NO: NO:Z NO: NO:B
=
, A
297 2873 ! :pSAC35:1ISAJFNaA(C This construct contains a hybrid form of pSAC35 1314 1315 1316 1317 1318 HSA/kex2 1-Q91)/ B(L93-E166) IFNaA and1FNaB fused downstream of ' mature HSA, 298 2874 , pSAC35:HSA.IFNaA(c this construct contains a hybrid form of pSAC35 1319 1320 1321 1322 1323 HSA/kex2 1-Q91)/ F(L93-E166) 1FNaA and 1}'NaF fused downstream of r) mature BSA, cp _______________________________________________________________________________ ___________________________________ , 299 2875 . pSAC35:HSAIFNaA(C This construct contains a hybrid form of ! pSAC35 1324 1325 1326 1327 1328 HSA/kex2 co 0.
:1Q-62)/D(Q64-E166) IFNaA and IFNaD fused downstream of ' , 1-, cp mature USA. ki) ..3 300 , 2876 pSAC35:HSAIFNaA(C This construct contains a hybrid form of pSAC35 ' 1329 1330 1331 1332 1333 HSA/kex2 1..) as ,, cp 1-Q91)/ D(L93-E166); IFNaA and 1FNaD fused downstream of ' 1-, w E13.23K,A113V mature HSA. .
1 _________________________________________ 1-, 301 t 2877 pSAC35:KT.Insulin.EIS , Killer toxin signal peptide fused to pSeCHSA =! 1577 1409 : 1745 1950 1951 Killer toxin , ;A ; synthetic gene coding for a single-chain ki) , 1 insulin with C7terminal HSA
: ...
302 2878 pSAC35:AP.Insu1in.HS 1 Acid phospatase signal peptide fused to pSAC35 1578 1410 1746 ' Acid , ;A synthetic gene coding for a single-chain phosphatas :
, insulin with C-terminal EISA.
e . ,..........,...e...........,w**..Y
' 303 ; 2882 pSac35.alphalvilprepro.1 MFa-1 prepro signal followed by GYG pSAC35 `
1579 1411 1747 , ' 1v1Fct-1 nsulin(GYG).HSA insulin followed by mature HSA.
.
_______________________________________________________________________________ _________________________________ , 304 ,:, 2885 TPSac35.alphalVIFpreproElYeastl\ifFix-1 prepro signal followed by pSAC35 1580 1412 1748 Yeast EA.Insulin(GYG).HSA I GYG insulin follwed by mature HSA,1\4_Fot-1 !
. , 305 2886 ' pSAC35:HSA.GCSF.P4 HSAJkex2 signal peptide followed by pSAC35 1581 1413 1749', 1952 , 1953 HSA/kex2 0-P204 mature HSA followed by GCSE (P40-P204). _ , , Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ I SEQ Leader No. ID Vector ID ID ID ID ID Sequence -NO:Y NO: NO:Z NO: NO:B
X
A
306 2887 pSAC35:IISA.GCSF.P4 HSAikex2 signal peptide followed by pSAC35 1582 1414 1750 1954 1955 FISA/kex2 0-L201 mature HSA followed by GCSE (P40 L201).
307 2888 pSAC35:11SA.GCSF,Q4 HSA/kex2 signal peptide followed by pSA C35 1583 1415 1751 1956 1957 HSA/kex2 1-L201 mature HSA followed by GCSF (Q41-L201).

308 2889 pSAC35:11SA.GCSF.Q4 HSA/kex2 signal peptide followed by pSAC35 1584 1416 1752 1958 1959 IISA/kex2 1-P204 mature HSA followed by GCSF (Q41-P204).
'8 309 2890 pC4.1-ISA,GCSF.T31- HSAikex2 signal peptide followed by pC4 1585 1417 1753 1960 1961 IISA/kex21 P204 mature NSA followed by GCSF (T31-P204).
310 2891 pGAP.alphaMF.Insulin( Synthetic gene -coding for a single-chain pYPGaf 1586 1418 1.754 " 1962 1963 HSAikex2 GYG).HSA insulin with HSA at C-terminus. The signal peptide of liS,Aikex2 is used for this construct.
311 2897 pGAP.Insulin(KR.GGG. Long-acting insulin analog using a pYPGaf 1 1587 1419 1755 1964 1965 HSAikex2 KR).HSA synthetic gene coding for a single-chain insulin with HSA at C-terminus. Contains a modified loop for processing resulting in correctly disulfide bonded insulin coupled to HSA
312 2900 pSAC:GLP-1(7- GLP-1(7-36) is tandernly repeated and pScCHSA 1233 1218 1248 1265 1266 HSA.Ikex2 36)x2 NSA then fused downstream of the HS.Aikex2 signal sequence and upstream of mature = HSA.

Table Z
..
, Fusion Construct Construct Name Description Expression SEQ SEQ SEQ t SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
313 2901 pSAC35:IL22.A18- Amino acids A18-P202 of 11,22 fused pSAC35 1588 1420 1756 1966 1967 HSA/kex2 P202.HSA downstream of HSAticex2 leader and , ulstream of mature HSA.
, 4 314 2902 pSAC35: A 14 amino acid peptide of Somatostatin, , pScCHSA 1589 1421 1757 1968 1969 HSAlkex2 Somatostatin(S14(A- an inhibitor of growth hormone, Io G)).1.1SA synthesized as a C-terminal HSA fusion.

N., Somatostatin has an alanine to glycine co 0.
change at amino acid 1 of SEQ ID
1-, NO:1757.
ko ...3 *"r '8 315 2903 pSAC35:1ISA.A18- Amino acids A18-P202 of1122 fused pSAC35 1590 1422! 1758 1970 1971 HSA
no P202.1L22 downstream of full length NSA.

1-, w 316 2904 pSAC35:GLP-1(9- Amino acids E100 to R127 of pScCHSA
1234 1219 1249 1267 1268 HSAilcex2 1 1-, 36).GLP-1(7-36).HSA preproglucagon (SEQ ID NO:1249) 1-, N., (hereinafter, this particular mutant is ko referred to as GLP-1(9-36)) is fused downstream from the HSA/kex2 signal 1 sequence and upstream from GIP-1(7- I
'<
36), and mature IISA.
317 1 2908 pSAC3511SAIICE1P8 -Mature HSA fused downstream of the pSAC35 ' 1-591 1423 1759 1972 1973 HSAikex2 0 HSAikex2 leader and upstream of HCE1P80.
, 318 2909 pSAC3511SA.HDRM18 Mature HSA fused downstream of the pSAC35 1592 1424 1760 1974 1975 HSA/Icex2 2 HSA/kex2 leader sequence and upstream ____________________________________ of HDRMI82.
, 319 2910 pSAC35:HSA.Reg1V Mature HSA fused downstream of the pSAC35 1593 1425 1761 1976 1977 HSAIkex2 , ILSA/Icex2 leader sequence and upstream of Reg1V. .
, -"
Table 2 _ Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID i Vector ID ID ID ID ID Sequence ' 1 NO:Y NO: NO.Z NO: NO:B
X
A
, 320 r 2915 -pC4:1ISA#65.hurnancalc Modified HSA leader i4.65 followed by.pC4 1594 1426 1762 1978 1 1979 Modified itonin.C1-G33 mature HSA and C98 through G130 of HSA #65 SEQ DD NO: 1762.
321 2930 pC4.1VIPIP.Insulin(GYG Insulin is downstream of an IvIPIF
signal pC4 1595 1427 1763 1980 1981 j ivIPIP
).HSA peptide arid upstream of mature HSA.
, o 322 2931 pe4.HSA.Insiiiin(GY(3) Synthetic 'gene coding for a mature single- pC4 1596 1428 1764 1982 1983 Modified 4) chain insulin fused downstream of the IHSA (A14) 0 N.) modified HSA A14 leader and mature leader 0 0.
1-, , HSA.

323 2942 pSac35.TA57.Insulin(G The TA57 Propeptide fused to a single pSeNHSA - 1597 1 1429 1765 1984 1985 TA57 ....1 YG).HSA chain insulin (GYG), and then mature propeptide 0 1-, HSA.
w 324 2943 pSAC35:PISA.T7.77,T7 Dirtier construct- HSA/kex2 leader pScNI-ISA 1598 1430 1766 1986 1987 HSA/kex2 1-, 4-L98 followed by mature HSA followed by two 1..) ko copies of T7 peptide (SEQ ID NO:1766) of Tunistatin.
____ .........
325 2944 pSACESA.T8.T8.1(69- HSA/kex2 leader followed by mature pScNHSA 1599 1481 1767 1988 1989 HSA/kex2 S95 HSA followed by two copies of T8 peptide (SEQ ID NO: 1767) of Timistatin . .. .
, _ .

Table 2 ., Fusion Construct Construct Name Description ¨Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence .
, NO:Y NO: NO:l NO: NO:B
, X A
,..
326 2945 pSAC35:GLP-1(7- Amino acids H98 to R127 of pScCHSA
1235 1220 1250 1269 1270 HSA/kex2 36(A8S)).GLP4(7- preproglucagon (SEQ ID NO:1250) is .
36).FISA mutated at position 99 from alanine to , serine (hereinafter, this particular mutant is referred to as GLP-1(7-36(A8S)), which . 0 , is fused downstream from the HSAJkex2 1..) signal sequence and upstream from GLP-0.
, 1(7-36), and mature HSA.
.

327 2946 pSAC:T1249(x2).HSA This dimer represents the wild type pScCHSA 1600 1432 1768 1990 1991 HSA/kex2 ko ..3 '8 sequence for T1249. Both (Inners have=
1..) been yeast codon optimized. The second w dimer was optimized to be different from 1-, =
the first (at the wobble position) to ensure good amplification. Construct has the 1..) ko HSA/kex2 leader followed by T1249 , dirner followed by mature HSA.
. , 328 2947 pSACCICb- Invertase signal peptide followed by pSAC35 1601 1433 1769 1992 1993. myertase 188(x2).1-ISA amino acids G28-N93 of full length CK01 (SEQ 1DNO:1769), tandemly repeated, followed by mature HSA.
. , 329 2964 pSAC35:GLP-1(7: GLP-1(7-36) is tindernly repeated as a pSAC35 1236 1221 1251 1271 1272 HSAikex2 36)x2.HSA dirtier and fused downstream from the .
f HSA/Icex2 leader sequence and upstream from mature HSA.
330 2965 pC4:MPIFspP.PTH(1- IVIPIF signal peptide followed by 34 pC4 1602 1434 1770 1994 1995 MP1F
, 34).HSA amino acids of PTH followed by mature HSA.
_______________________________________________________________________________ __________________________________ .
, ., 1, 1 "

Table 2 _______________________________________________________________________________ ____________________________ _ ____ Fusion Construct Construct Name Description Expression SEQ I SEQ SEQ SEQ SEQ I Leader No. ID Vector ID I IP ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A

pEE12:MPIPsp.PTH(1- IVEIF signal peptide followed by 34 PEE12.1 1603 34).1-1SA amino acids of PTH followed by mature HSA.

HSA/kex2 332 2982 pSAC35:GLP-1(7-GLP-I(7-36(A8G)) (SEQ ID NO:1808) is pScCHSA 1237 1222 1252 1273 1274 36(A80).GLP-1(7- fused downstream fTorn the HSA/kex2 ri , 36).HSA sig-nal sequence and upstream from GLP-1(7-36) and rnature EISA.
_______________________________________________________________________________ _________________ 1..) op 333 2983 pC4.HSA.Growth ' Modified (A14) HSA feader followed by pC4 1604 1436 1772 1998 1999 Modified 0.
1-, Hormone.F27-F-217 mature HSA followed by F27 through HSA (A14) 0 ko ,¨
...3 ,¨ F217 of growth hormoile (corresponding ' ,¨
1..) tS amino acids Fl to F191 of SEQ ID
e:, =i NO:1772).
1-, w , , _______________________________________________________________________________ _________________________________ ¨

pSac35.y3SP.TA57PP.1 The TA57 Propeptide fused to a single pSeCHSA 1605 1-, , nsulin(GYG).HSA chain insulin (GYG), and then mature propeptide 1..) ko HSA.

pSAC35:11\111.Insulin.H Inulinase signal peptide is fused pScCHSA 1606 1438 1774 2002 2003 inulinase SA upstream of single chain insulin (GYG) , and HSA.
= .., pSAC35:INV.GLP-1(7- I Invertase signal peptide followed by GLP- pSAC35 1607 1439 1775 2004 2005 invertase 36A80)x2.HSA 1(7-36(A8G)) (SEQ DJ NO:1808) , tandemly repeated as a dimer, followed by , mature HSA.
, 337 3028 pSAC35:1NV,GLP-1(7- , Invertase signal peptide followed by GLP- pSAC35 1608 1440 1776 2006 2007 invertase 36(A8G)).GLP-1(7- 1(7-36(A8G)) (SEQ ID NO:1808), then 36).HSA GLP-1(7-36(A8G)), and then mature HSA.!
-, Table 2 .,_ = Fusion: Construct Construct Name Description Expression SEQ . SEQ SEQ
[SEQ SEQ Leader ' No. . ID Vector ID ID ID = ID ID Sequence NON NO: NO:1 NO: NO:B
. X , . A .. .
, . .. .
338 = ' 3045 pSAC35:DeltaKex,GLP- HSA/kex2 signal sequence, minus the last pSAC35 1609 1440 1776 2008 2009 HSAIkex2 = 1(7-36A80)x2ESA
six amino acids of the leader, is fused to last six =
GLP-1(7-36(A8G)) (SEQ ID NO:1808) =
amino ' which is tandemly repeated as a dimer, acids followed by mature HSA.
(-) . ______________________________________________________________ ._:... .
. . . ... = == = ==_.= = = = = =
339 3046 pSAC35:Delta 'ITSAikex2 signal sequence, minus the last = .pSAC35 1610 1440. 1776 2010 .2011 ....HSA/kei2 0 ' , Kex.GLP-1(7- six amino acids of the leader, is fused to last six "

36A8G).GLP-1(7- ' GLP-1(7-36(A8G)) (SEQ ID NO:1808), . amino . 0.
1-, 36).11SA GLP-1(7-36), and mature HSA.
. . :. acids .; ko ..3 340 3047 pSAC35: HSA.Tum5 Full length HSA fused to the Tum5 pSeNHSA 1611 1443 1779 2012 2013 HSA = in _peptide (SEQ ID NO:1779) of Turnstatin. ., . ..
, ! 341 3048 pSAC35: Tum5.HSA. The Tum5 peptide. (SEQ ID NO:1780) of pScCHSA 1612 1444 . 1780 . 2014 2015 i HSA/kex2 . w 1-, Tumstatin is fused to HSA and. HSA/kex2 1-, leader.
"
l0 1 . . , õ . õ - +- ,.. --,.1' = ..
342 ==i 3049 pC4.HSA.HCE1P80.D9 Amino acids D92 to L229 of HCE1P80 pC4 1613 1445 1781 2016 = 2017 HSA :
.2-L229 are fused downstream of the full length , HSA.
=.
: 343 3050 ' pC4.HSA.HCE1P80.A2 ' Amino acids A20-L229 of HCE1P80 are pC4 . 1614 1446 . 1782 2018 2019 HSA
:0-L229 fused downstream of the full length human RSA
.. , = = === =
. 344 3051 pSAC35.11SA.PICE1P8 Amino acids D92 to L229 of HCE1P80, a pSAC35 1615 1447 1783 = 2020 = 2021 HSA
O.D92-L229 member of the Cl.q family of proteins, are .
., fused downstream of the full length human HSA
- = = ===========,. , 345 3052 ,pSAC35.HSA.HCEIP8 .. Amino acids A20-L229 of HCEI P80 are pSAC35 1616 1448 1784 :=2022 2023 . PISA
.. 0.A20-L229 := fused downstream of the full length , ..., Junnan.HSA
,. , =
. ... ...::: ,:::.. , . = ..
-, .... . .

Table 2 .
Fusion i Construct I Construct Name I Description Expression ' SEQ SEQ SEQ SEQ SEQ Leader = No. ID .
Vector ID ID ID ID ID ' Sequence ' = NO:Y NO: NO:Z NO: NO:8 A
X
, ,,. , 346 3053 pC4.11SARDALV07.k The globular domain of adiponectin pC4 1617 1449 1785 2024' 2025 BSA I
= 101-N244 (amino acids K101-N244) has been inserted downstream of full length human NSA.
, 347 3055 pSAC35.11SA.HDALVO Full length NSA followed by amino acids =pSAC35 1618 1-1450 - 1786 2026 2027 ' }ISA 0 7(GD) K101-N244 of IIDALV07 (OD)/=
, Adiponectin. .
N' _ ..
.
348 3056 pSAC35.11SARDALVO Full length HSA followed by amino acids pSAC35 1619 1451 1787 2028 2029 NSA 0.
1-, i 7.MP Q18 to N244 of IMALV07.
. 0 ko .
...3 349 3066 a pSAC35:CK13- - Inverasgna pep followed te signal tide fod by ; pS c CI-ISA 1620 1452 1788 2030 2031 mvertase 1d8.GLP-1(7-36).11SA amino acids G28-N93 offal! length CKP1 1-, , : (SEQ IDNO:1788), followed by GLP-1(7-w 1-, ' 136), followed by mature NSA, 1 1-, , 350 3069 ! pSAC35:INUGLP-1(7- =' The-inulinase signal sequence is fused to pSAC35 1621 1453 1 1789 ' 2032 2033 inuiinase N' ko =
36(A80))x2.HSA OLP-1(7-36(A80)) (SEQ ID NO:1808), which is tandemly repeated as a dimer and -fused to mature HSA.
, 351 3070 pSAC35aT.GLP-1(7- OLP-1(7-36(A8G)) (SEQ ID NO:1808) is pSAC35 1 1280 1281 - 1282 1283 , 1284 Killer toxin 1 36(A8G))x2.HSA tandernly repeated as a dimer and fused , upstream from mature NSA and downstream from the killer toxin signal ' I sequence.
. I _ 352 3071 pSAC35:11,4AF.OLP-1(7- The yeast mating factor a-1 (hereinafter pSAC35 - 1622 1454 1790 2034 _ 2035 MFa-1 36(A8G))x2.1-1SA MFa-1) signal sequence is fused to itandemly repeated copies of OLP-1(7-36(A80)) (SEQ ID NO:1808), which are , fused to mature NSA.

Table 2 - I ___ -Fusion , I Construct Construct Name Description Expression SEQ SEQ I SEQ 1 SEQ SEQ I Leader 1 No, ID Vector ID ID 1 ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
, X A
' 353 3072 pSAC35:AP,GLP-1(7- The acid phosphatase signal sequence is pSAC35 1623 1455 1791 2036 2037 Acid 36(A8G))x2.1-1SA fused to tandemly repeated copies of phosphatas GLP4(7-36(A8G)) (SEQ ID NO:1808), e ` ______________________________ which are fused to mature USA.
i 354 3085 pSAC35:MAF.GLP-1(7- The yeast mating factor a-1 (hereinafter pSAC35 1624 1456 1792 2038 2039 MFa-1 o 36(A8G)),GLP-1(7- IVIFa-1) signal sequence is fused to OLP-1..) 36).HSA 1(7-36(A8G)) (SEQ ID NO:1808), GLP-co 0.
1(7-36), and mature HSA.
1-, , ¨ 0 -.3 . 36(A80)).GLP4(7- GLP-1(7-36(A8G)) (SEQ ID NO:1808), 1..) 36).HSA GLP-1(7-36), and mature HSA.
1-, .-w 356 3087 pSAC35:AP.GLP-1(7- The acid phosphatase signal sequence is pSAC35 1626 1458 1794 2042 2043 Acid 1 1-, 36(A8G)).GLP-1(7- fused to GLP-1(7-36(A8G)) (SEQ
IDphosphatas , 1..) 36).HSA NO:1808), GLP-1(7-36), and mature e ko ................................... HSA.
.. i ...
sequence. j 358 3105 pSAC35:INV.t9HCC- Invertase signal peptide followed by pSAC35 1628 1460 1796 2046 2047 invertase 1.G28-N93:spc.HSA amino acids G28 to N93 of HCC4 fused upstream of a spacer and mature HSA. _____________________________ 359 3106 pSACHSA.HCBOG68 mature Hti3OG68 fused downstream of pSAC35 1629 1461 1797 HSA/Icex2 mature HSA and the HSAJIcex2 leader sequence. , 360 3108 pSAC35HSA.PYY Mature PYY fused downstream of mature pSAC35 1630 1462 1798 HSAlicex2 IISA and the HSAiltex2 leader.
_ 4 . ____________________________ z Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:1( NO: NO:Z NO: NO:13 X
A
361 3109 pSAC35HSA.PYY3-36 HSA/kex2 leader followed by mature pSAC35 1631 1463 1799 HSA/kex2 HSA and then PYY3-36 (SEQ ID
N01799).
362 3117 pC4:PYY3-36/11SA HSA leader followed by PYY3-36 (SEQ :pC4 1632 :146411800 2048 2049 HSA
ID NO:1800) and mature HSA.
363 3118 pSAC35:PYY3-36/HSA HSA/kex2 leader followed by PYY3-36 pSAC35 1633 1465 1801 2050 2051 HSA/kex2 (SEQ ID NO:1801) and mature HSA.
co 364 3119 pSAC35:BNIVHSA HS4jicex2 leader followed by BNP and pSAC35 1634 1466 1802 I 2052 2053 HSA/kex2 mature HSA.

365 3124 pSAC35:INV.CK331.P2 Invertase signal peptide followed by pSAC35 1635 1467 1803 2054 2055 invertase 9-N93:HSA amino acids 29 to 93 of full length ckbetal fused to N-tenninus of HSA.
366 3125 pSAC35:INV.CK.b- Invertase signal peptide followed by pSAC35 1636 1468 1804 2056 2057 invertase 1.R27-N93:HSA amino acids 27 to 93 of full length ckbetal fused to N-terminus of.HSA.
367 3133 pSac35.y,SP.TA57PP.Ins Variant TA57 propeptide leader followed pSAC35 H 1637 1469 1805 2058 2059 TA57 ulin(GYG).HSA by single chain insulin, followed by variant 1 mature HSA.
368 3134 pSac35.ySP.TA57PP+S. Variant TA57 propeptide leader followed pSAC35 1638 11470 1806 2060 2061 TA57 Insulin(GYG).HSA by single chain insulin, followed by variant 2 mature HSA.
369 3139 pSAC35:INV.CKB1.G2 Invertase signal peptide followed by pSAC35 1639 1471 1807 2062 2063 invertase 8-N93.DA.H.K.HSA amino acids 028-N93 of full length CK.131.
(see, e.g, SEQ IDNO:1788), followed by a 16 amino acid linker derived from the N-I terminus of HSA, followed by mature ________________________________ PISA.

, Table 2 1 Fusion Construct Construct Name f Description Expression SEQ SEQ i SEQ SEQ SEQ I Leader ' No. ID Vector ID ID ID ID ID 1 Sequence =
' NO:Y NO: NO:Z NO: NO:B I .
X A , ., 370 3140 pSAC35:GLP1(mut)DA I GLP-1(7-36(A8G)) (SEQ ID NO:1808) is pSAC35 - 1640 11472 . 1808 ' 2064 2065 1 HSAIkex2 FacHsA linked to mature HSA by a 16 amino acid linker derived from the N-terminus of = IISA. The HSA/kex2 signal sequence is used.
o 371 3141 pSAC35:Wntl Ob/HSA HSA/kex2 leader followed by amino pSAC35 " 1641 - 1473 , 1809 2066 2067 HSA/kex2 0 acids N29 to IC389 of WritlOb followed N' by mature HSA.
0.
1-, _ _ 0 372 3142 i pSAC35:Wntl1THSA IISAIkex2 leader followed by mature -pSAC35 1642 1474 1810 2068 2069 HSA/kex2 ko I Wntl 1 followed by mature HSA.
N., 373 3143 4 pSAC35:berstati 1n/HSA HS.A/Icex2 leader followed by amino pSAC35 1643 -1475 1811 r2070 2071 HSAikex2 1-, w acids T23 to C419 of herstatin followed 1-, by mature HSA.
, 1 374 3144 pSAC35:adrenomedullin HSA/kex2 leader followed by amino pSAC35 1644 1476 1812 2072 2073 HSAIkex2 ko (27-52)/HSA. acids 27-52 of adrenomedullin followed _ by rnature HSA.
375 3149 pSAC35.HSA.C-peptide Full length HSA fused to amino acids E7 pSAC35 1645 1477 1813 2074 2075 IISA.
, tandem to Q37 of SEQ ID NO:1813, tandemly repeated.
376 3152 pSAC35:INV.CY:131.Me Invertase signal peptide followed by a pSAC35 1646 1478 1814 2076 i 2077 invertase t.R27-N93.HSA Met, thllowed by amino acids R27-N93 of full length C1(01, followed by mature HSA.
377 3153 pSAC3511\TV.CI(B1.Me Invertase signal peptide followed by a pSAC35 1647 ' 1479 1815 20781 2079 invertase t.S26-N93.1-1SA Met, followed by amino acids S26-N93 of full length CKf31, followed by mature . HS.A.

Table 2 , _ _______________________________________________________________________________ ___________________________ , _______________________________________________________________________________ _____________________ Fusion Construct Construct Name I Description.
Expression SEQ SEQ I SEQ SEQ 1 SEQ Leader No. II) 1 Vector ID ID , ID ID 1 ID Sequence NO:Y NO: NO:Z NO: NO:B
i X A
t i 378 3154 pSAC35:INV.C101,Me Invertase signal peptide followed by a pSAC35 1648 1480 1816 2080 2081 invertase ' t.S25-N9311SA Met, followed by amino acids S25-N93 of full length CKI31, followed by mature , HSA.
, pSAC35:INV.CKBI.Me Invertase signal peptide followed:by a pSAC35 1649 1481 ' 1817 2082 2083 invertase 0 t.G28-N93.1ISA I Met, followed by amino acids G28-N93 of full length CK 1, followed by mature I
co 0. HSA, - ,' 1-, e:.

pSAC35:L4Y.CKB1.Me Invertase signal peptide followed by a pSAC35 1650 i 1482 1818 2084 2085 invertase ' ko ...3 7!) , t.P29-N93.HSA Met, followed by amino acids 129-N93 of 1..) full length cKpi, followed by mature 1-, HSA.
w , 381 3163 pSAC35:HSA.hGH
,HSA/k.ex2 leader fused upstream of pSAC35 1303 1304 1305 HS.A/kex2 1-, / mature HSA and 191 amino acids of hGli "
, ko 382 ¨ 3165 pSAC35:1-1SA.IFNa HSA fused upstream of IFNct and pSAC35 1300 1301 1302 , HSAikex2 downstream of the HSA/kex2 leader.
also named CID 3165, _ pS.AC35:HSAINFu 383 3166 pC4:MPIF1.A22-Amino acids A49 to N120 of MPIF (SEQ pC4 1651 1483 1819 2086 2087 MPIF
N93.HSA ID NO:1821) is fused downstream of MPIF signal peptide and upstream of mature HSA.
, .
, _ 384 : 3167 pC4:HSA.MP1F1D45- 1-Full length IISA followed by amino acids pC4 1652 1484 NI20 D45 'ft:rough N120 of MPIF.
385 , 3168 PC4:MPIF-1.HSA
Amino acids D4-5 through N120 of WEE pC4 1653 1485 1821 ' 2090 2091 MPIF
fused downstream of the IVIPEF signal , segue= and upstream of mature FISA.

Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ SEQ SEQ Leader No. ID Vector ID ID ID ID = ID Sequence NO:Y NO: ' NO:Z NO: NO:B
X
A
386 3169 pSAC35:KT.CKB1.G28 Killer toxin signal sequence fused pSAC35 1654 1486 1822 Killer toxin -N93.HSA :upstream of amino acids G28 through N93 of CKB1 (fragment shown as amino acids Gl. to N66 of SEQ ID NO:1822) _ and mature HSA.
.
.0 , . .
.
387 3170 pSAC35:1(T.HA.CK.B1. Killer toxin signal sequence followed by pSAC35 ' 1655 1487 1823 * Killer toxin 0 G284493.HSA HA dipeptide and amino acids 028 1..) 0.
through N93 of CKB1 (fragment shown , as amino acids 01 to N66 of SEQ ID
iko .4 ,--, NO:1823) and mature HSA.
1..) D 388 3171 pSAC35:sCNTF(1\41-C-terminal deletion of CNTF (amino acids pSAC35 1656 1488 1824 2092 1 2093 IISA/Icex2 0 1-, 0185):HSA M1 through 0185), fused upstream of . w 1-, mature IBA and codon optimized for 1-, expression in yeast. HSArkex2 signal 1..) ko _ sequence is used.

389 3172 pSAC35:HSA: HSAikex2 signal sequence followed by pSAC35 1657 1489 1825 , 2094 2095 HS.Aikex2 sCINTTF(M1-0185) mature HSA and M1 through 0185 of , CNTF.
, 4 ________________ 390= 3184 ' pC4:HSA.NO0OR.C27- ' Full length HSA followed by amino acids pC4 1658 1490 ' 1826 2096 2097 , HSA
0309 C27 to 0309 of the NOGO receptor.
:
¨
391 3185 pC4.N000R.M1- Amino acids M1-C309 of N000 pC4 1659 { 1491 1827 2098 2099 Native C309.HSA receptor fused upstream of mature HSA.

I
receptor I
, 392 3194 pC4:HSA(A14)- Codon optimized EPO(A28-D192.0140) 'pC4 ' 1-660 E 1492 ' 1828 2100 2101 modified [
EPO(A28- fused downstream of mature HSA with a ;
HSA (A14) , _______________ D192.G140)codon opt modified HSA (A14) sipal sequence.
, , s a =
Table 2 Fusion Construct Construct Name Description Expression SEQ SEQ SEQ ' SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence = NO:Y NO: NO:Z NO: NOB
X
A i 393 3195 pC4:HSA(S14)- ' Codon optimized EPO(A28-D192.0140) pC4 1661 1493 18291 2102 2103 modified EPO(A28- fused downstream of mature HSA and a ' HSA (S14) , , D192.G140)codon opt ' modified HSA (S14) signal sequence. , , 394 3196 pC4:11SA(G14)- , Codon optimized EPO(A28-D192.0140) pC4 1662 1494 1830 2104 2105 modified EPO(A28- fused downstream of mature HSA with a (014) P
D192.0140)codon opt modified (014) USA Opal sequence.

s 395 3197 pC4.MPIF'.Insulin(EAB) A single-chain insulin is downstream of i pC4 1663 1495 1831 MPH' 1..) .HSA the MPIF signal peptide and upstream of 0.
1-.

1 mature human HSA.ko _ . .. ...3 :-.. 396 3198 pSac35.11NV.insulin(EA Single-chain insulin is downstream of the pSAC35 - 1664 1496 1832 invertase ...c) 1..) E).HS A invertase signal peptide and upstream of 1-, mature human IBA
1 w =
1-, 397 3202 pSAC35:AP1.d8CKb1/H HSA/kex2 leader followed by amino acids pSAC35 1665 1497 1833 2106 2107 FISAlkex2 SA "API" followed by d8CKb1 and mature 1..) ko ' USA. The sequence of delta 8 for C.KB1 , is shown in SEQ ID NO:1833.
, 398 3203 pSAC35:ASL.d8CK.b1/ HSA/kex2 leader followed by ammo acids pSAC35 1666 ' 1498 1834 2108 2109 HSA/kex2 HSA "ASL" followed by d8CKbl and mature , HSA.
_ 399 3204 pSAC35:SPY.d8CKb1/ HSA/kex2 leader followed by amino acids 'pSAC35 1667 1499 1835 2110 2111 1 HSA/kex2 USA "SPY" followed by d8CICh1 and mature =
IISA.
400 =r 3205 pSAC35:MSPY.d8CKb HSA/kex2 leader followed by amino acids ' pSAC35 - 1668 1500 1836 2112 2113 HSA/kex2 1/HSA "MSPY" followed by d8CKb1 and mature RSA.=
, ,-- . t.
__________________________________________ -Table 2 õ
- , Fusion Construct Construct Name Description Expression SEQ SEQ SEQ I SEQ SEQ Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
XI
A
401 3206 pSAC35:CPYSC.d8C1( HSA/kex2 leader followed by a five pSAC35 1669 1501 1837 j 2114 2115 HSA/kex 2 b 1/1-ISA amino acid linker followed by d8C1(b1 , __________________________________ and mature HSA.
.
r -402 3207 pSAC35:GPY,d8CKb1/ HSA/kex2 leader followed by amino acids pSAC35 1670 1502 1838 2116 2117 HSA/kex2 I-ISA "GPY" followed by d8CKbl and mature o _HSA.

1..) 403 3208 pSAC35:defensin alpha Amino acids A65-C94 of defensin alpha 1 pSAC35 1285 1286 1287 1288 1289 HS Aikex2 0.
ITHSA fused downstream of the HSA/kex2 leader and upstream of mature HSA.

...3 r; 404 3209 pSAC35:defensin alpha Amino acids C66-C94 of defensin alpha 2 pSAC35 1290 ' 1391 1292 1293 1294 1 HSA/kex2 1..) Q

2/HSA fused downstream of the HSA/kex2 leader w and upstream of mature HSA.

1-, 405 3210 pSAC35:defensin alpha Amino acids 65-94 of SEQ ID N01297, pSAC35 1295 1296 1297 1298 1299 HSAIkex2 L

1-, 3/HSA , with A65D and F921 mutations, fused 1..) =
i downstream of the HSAikex2 leader and I upstream of mature HSA.
_______ .:
406 3232 pSAC35:CART/HSA HSA/kex2 leader followed by processed pSAC35 1671 1503 1839 2118 2119 IISA/1cex2 active cocaine-amphetamine regulated transcript (CART) (amino acids V69 ' tbroush L116) followed by mature HSA.
,,, i.=
407 3238 pSAC35:phosphatonin, Phosphatonin fused upstream of HSA.
pSAC35 1306 1307 1308 Native }ISA
phosphaton , .
in 408 3270 pSAC35:adipokine/HSA HSA/kex2 leader followed by adipokine pSAC35 I 1672 1504 1840 2120 2121 HSAikex2 _followed by mature HSA.
t = , Table 2 Fusion Construct Construct Name Description Expression 1 SEQ SEQ SEQ SEQ SEQ 1 Leader No. ID Vector ID ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
409 3272 pSAC35.1NV:{D}8CKt CKbeta-1 tandem repeat (x2) fusion to the pSAC35 1673 1505 1841 2122 2123 invertase b}1(x2)/HSA N-termal USA. Under the invertase signal peptide.
410 3274 pSAC35:Plpal-12.11SA Plpal-12 pepducin peptide fused upstream pSAC35 1334 1335 1336 HSAAex2 of mature IBA, and downstream of the HSAikex2 leader sequence, 411 3275 pSAC35:P4pal-10.11SA P4pal-10 pepducin peptide fused upstream pSAC35 1337 1338 1339 FTSAIkex2 of mature USA, and downstream of the IISA/kex2 leader sequence, t's.1 412 3281 pSAC35.PY3- PYY3-36 tandem repeat (x2) fused pSAC35 1674 1506 1842 2124 2125 FISAIkex2 36(x2)/liSA upstream of USA and downstream of the HSAikex2 sils al I totide.
413 3282 pSAC35:1ISA/PYY3- PYY3-36 tandem repeat (x2) fused pSAC35 1675 1507 1843 2126 2127 EISA/kex2 36(x2) downstream of mature IISA and HSA/kex2 leader.
414 3298 pSAC35:1121/IISA Amino acids Q30-S162 of IL-21 fused pSAC35 2167 2157 2177 2188 2189 IISAJICex2 upstream of mature IBA and downstream of FISA/kex2 leader 415 3307 pSAC35:1L4/11SA Amino acids 1125-8153 of IL-4 fused pSAC35 2168 2158 2178 2190 2191 HSA/Kex2 upstream of mature }ISA and downstream of IISA/kex2 leader 416 3309 pSAC:K.T.GLP-1(7- Killer toxin leader sequence followed ¨by pSAC35 2170 2160 2180 2194 2195 Killer toxin 36(A80))x2.MSA.E25- GLP-1(7-36(A8G) followed by mature A608 mouse serum albumin.
417 1 3312 - pSAC35:h0CILAISA I 1-ISAike7a leader followed by amino pSAC35 2171 2161 2181 2196 2197 IIS_AiKex2 acids N20 to V149 of hOCIL followed by mature HSA

Table 2 Fusion Construct Construct Name DescriptionExpressi . -on SEQ SEQ SEQ SEQ SEQ Leader No, ID Vector ED ID ID ID ID Sequence NO:Y NO: NO:Z NO: NO:B
X
A
418 7777 T20:HSA T20 fused downstream of full length HSA pC4 419 8888 pe4:BNP.HSA Human B-type-nattiuretic peptide fused pC4 1275 1276 1277 1278 1279 Native upstream of mature HSA.
BNP
420 9999 T1249:HSA T1249 fused downstream of full length pC4 1173 1174 1 1175 HSA
HSA

[0066] Table 2 provides a non-exhaustive list of polynucleotides of the invention comprising, or alternatively consisting of, nucleic acid molecules encoding an albumin fusion protein. The first column, "Fusion No." gives a fusion number to each polynucleotide.
Column 2, "Construct ID" provides a unique numerical identifier for each polynucleotide of the invention. The Construct IDs may be used to identify polynucleotides which encode albumin fusion proteins comprising, or alternatively consisting of, a Therapeutic protein portion corresponding to a given Therapeutic Protein:X listed in the corresponding row of Table I wherein that Construct ID is listed in column 5. The "Construct Name"
column (column 3) provides the name of a given albumin fusion construct or polynucleotide.
[0067] The fourth column in Table 2, "Description" provides a general description of a given albumin fusion construct, and the fifth column, "Expression Vector"
lists the vector into which a polynucleotide comprising, or alternatively consisting of, a nucleic acid molecule encoding a given albumin fusion protein was cloned. Vectors are known in the art, and are available commercially or described elsewhere. For example, as described in the Examples, an "expression cassette" comprising, or alternatively consisting of, one or more of (1) a polynucleotide encoding a given albumin fusion protein, (2) a leader sequence, (3) a -promoter region, and (4) a transcriptional terminator, may be assembled in a convenient cloning vector and subsequently he moved into an alternative vector, such as, for example, an expression vector including, for example, a yeast expression vector or a mammalian expression vector. In one embodiment, for expression in S. cervisiae, an expression cassette comprising, or alternatively consisting of, a nucleic acid molecule encoding an albumin fusion protein is cloned into pSAC35. In another embodiment, for expression in CHO cells, an expression cassette comprising, or alternatively consisting of, a nucleic acid molecule encoding an albumin fusion protein is cloned into pC4. In a further embodiment, a polynucleotide comprising or alternatively consisting of a nucleic acid molecule encoding the Therapeutic protein portion of an albumin fusion protein is cloned into pC4:HSA. In a still further embodiment, for expression in NSO cells, an expression cassette comprising, or alternatively consisting of, a nucleic acid molecule encoding an albumin fusion protein is cloned into pEE12. Other useful cloning and/or expression vectors will be known to the skilled artisan and are within the scope of the invention.
[0068] Column 6, "SEQ ID NO:Y," provides the full length amino acid sequence of the albumin fusion protein of the invention. In most instances, SEQ ID NO:Y shows the unprocessed form of the albumin fusion protein encoded ¨ in other words, SEQ
ID NO:Y

shows the signal sequence, a HSA portion, and a therapeutic portion all encoded by the particular construct.
Specifically contemplated by the present invention are all polynucleotides that encode SEQ 1D NO:Y. When these polynucleotides are used to express the encoded protein from a cell, the cell's natural secretion and processing steps produces a protein that lacks the signal sequence listed in columns 4 and/or 11 of Table 2. The specific amino acid sequence of the listed signal sequence is shown later in the specification or is well known in the art. Thus, most preferred embodiments of the present invention include the albumin fusion protein produced by a cell (which would lack the leader sequence shown in columns 4 and/or 11 of Table 2). Also most preferred are polypeptides comprising SEQ
NO:Y without the specific leader sequence listed in columns 4 and/or 11 of Table 2.
Compositions comprising these two preferred embodiments, including pharmaceutical compositions, are also preferred. Moreover, it is well within the ability of the skilled artisan to replace the signal sequence listed in columns 4 and/or 11 of Table 2 with a different signal sequence, such as those described later in the specification to facilitate secretion of the processed albumin fusion protein.
[0069] The seventh column, "SEQ ID NO:X," provides the parent nucleic acid sequence from which a polynucleotide encoding a Therapeutic protein portion of a given albumin .
fusion protein may be derived. In one embodiment, the parent nucleic acid sequence from which a polynucleotide encoding a Therapeutic protein portion of an albumin fusion protein may be derived comprises the wild type gene sequence encoding a Therapeutic protein shown in Table I. In an alternative embodiment, the parent nucleic acid sequence from which a polynucleotide encoding a Therapeutic protein portion of an albumin fusion protein may be derived comprises a variant or derivative of a wild type gene sequence encoding a Therapeutic protein shown in Table 1, such as, for example, a synthetic codon optimized variant of a wild t-ype gene sequence encoding a Therapeutic protein.
[0070] The eighth column, "SEQ ID NO:Z," provides a predicted translation of the parent nucleic acid sequence (SEQ ID NO:X). This parent sequence can be a full length parent protein used to derive the particular construct, the mature portion of a parent protein, a variant or fragment of a wildtype protein, or an artificial sequence that can be used to create the described construct. One of skill in the art can use this amino acid sequence shown in SEQ
ID NO:Z to determine which amino acid residues of an albumin fusion protein encoded by a given construct are provided by the therapeutic protein. Moreover, it is well within the ability of the skilled artisan to use the sequence shown as SEQ ID NO:Z to derive the construct described in the same row. For example, if SEQ ID NO:2 corresponds to a full length protein, but only a portion of that protein is used to generate the specific CID, it is within the skill of the art to rely on molecular biology techniques, such as PCR, to amplify the specific fragment and clone it into the appropriate vector.
100711 Amplification primers provided in columns 9 and 10, "SEQ ID NO:A"
and "SEQ
ID NO:B" respectively, are exemplary primers used to generate a polynucleotide comprising or alternatively consisting of a nucleic acid molecule encoding the Therapeutic protein portion of a given albumin fusion protein. In one embodiment of the invention, oligonucleotide primers having the sequences shown in columns 9 and/or 10 (SEQ
ID NOS :A
and/or B) are used to PCR amplify a polynucleotide encoding the Therapeutic protein portion of an albumin fusion protein using a nucleic acid molecule comprising or alternatively consisting of the nucleotide sequence provided in column 7 (SEQ ID NO:X)of the corresponding row as the template DNA. PCR methods are well-established in the art.
Additional useful primer sequences could readily be envisioned and utilized by those of ordinary skill in the art.
(00721 in an alternative embodiment, oligonucleotide primers may be used in overlapping PCR reactions to generate mutations within a template DNA sequence. PCR
methods are known in the art.
100731 As shown in Table 3, certain albumin fusion constructs disclosed in this application have been deposited with the ATCCV.

Table 3 Construct ID Construct Name ATCC Deposit No./ Date 1642 pSAC35:GCSF.T31-P204.HSA PTA-3767 .Oct. 5, 2001 1643 pSAC35:HSA.GCSF.T31-P204 PTA-3766 Oct. 5, 2001 1812 pSAC35:11.2.A21-T153.11SA PTA-3759 Oct. 4,2001 1941 pC4:EISAIPTH84(junctioned) PTA-3761 ____________________________________________________ Oct. 4, 2001 1949 pC4:PTH.S1-Q84/11SA Ounctioned) PTA-3762 Oct 4, 2001 1966 pC4:EPO.M1-0192.11SA: PTA-3771 Oct. 5, 2001 also named pC4:EPOM1-D192.HSA
1981 pC4.11SA-E2O.A28-D192 PTA-3770 Oct. 5,2081 1997 pEE12.1:EPOM1-D192.HSA PTA-3768 Oct. 5,2001 2030 pSAC35.ycoIL-2.A21-T153.HSA PTA-3757 Oct. 4, 2001 2031 pSAC35.HSA.yco11,-2.A21-T153 PTA 3758 ____________________________________________________ Oct. 4, 2001 __ 2053 pEE12:1FNb-HSA PTA-3764 Oct. 4, 2001 also named pEE12.1:IFN; -HSA
2054 pEE12:HSA-IFNb PTA-3941 Dec. 19,2001..
2249 pSAC35:117Na2-HSA PTA-3763 Oct. 4, 2001 also named pS AC23 :IFIsTa2.--HSA
2250 pSAC3511SAINSULIN(GYG) PTA-3916 Dec. 07, 2001 also named _____________ p S AC35. HSA.INSULING(GYG).F1-N62 2255 pSAC35:INSIJLIN(GYG).HSA PTA-3917 Dec. 07, 2001 also named pSAC35.1NSULING(GYG).F1-_____________ N62.HSA
2276 pSAC35:HSA.1NSULNGGG) PTA-3918 Dec. 07, 2001 also named pSAC35.HSA.INSULING(GGG) .F1-N58 2298 pEE12.1 EPO.R1400.HSA PTA-3760 Oct. 4, 2001 Construct ID Construct Name ATCC Deposit No./ Date 2294 pC4:EP--6.R140G.HSA PTA-3742 Sept. 28, 2001 also named pC4.EPO.R1406.HSA
2-3-25 pC4.EPO:M1-6192.11SA.Codon opt. PTA-3773 _____________________________________________________ Oct. 5,2001 2343 pSAC35.INV-IFNA2.ELSA PTA-3940 _____________________________________________________ Dec. 19 2001 2363 pC4.GCSF.HS.A.EPO.A.28-D192 - PTA-3740 Sept. .28,2001 2373 pC4.GCSF.HSA.EPO.A28-D192.R140G PTA-3741 Sept. 28, 2001 2381 pC4:HSA-1FNa2(C17-E181) PTA-3942 Dec. 19,2001 ________________________________________________________ 2382 pC4:1FNa2-HSA PTA-3939 2387 pC4:EPO(G140)-HSA-GCSF.T31-P204 RA-3919 _____________________________________________________ Dec. 11, 2001 ====,,k 2414 pC4.EPO:M.1-D192copt.HSA.GCSF.T31-P204 PTA-3924 Dec. 12, 2001 also named _____________ pC4.EPO:M1 -D192copt.HAS.GCSF.T31-P204 2441 pEE12.EPO:M1-D192copt.HSA.GCST.T31- PTA-3923 P204 Dec. 12, 2001 also named:
pEE12.EPO:Ml-D192copt.HAS.GCSF.T31-2492 pC4.IFNb(deltaM22).HSA PTA-3943 Dec. 19,2001 3070 pSAC35:KT.GLP-1(7-36(A8G))x2.121SA PTA-4671 , Sept. 16, 2002 3165 pSAC35:HSA.IFNa PTA-4670 Sept. 16, 2002 _____________ also named CID 3165, pSAC35:11SA.INFa 3163 pSAC35:HSA.bGH PTA-47'70 October 22, 2002 [00741 It is possible to retrieve a given albumin fusion construct from the deposit by techniques known in the art and described elsewhere herein (see, Example 40).
The ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC
deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

[0075] In a further embodiment of the invention, an "expression cassette"
comprising, or alternatively consisting of one or more of (1) a polynucleotide encoding a given albumin fusion protein, (2) a leader sequence, (3) a promoter region, and (4) a transcriptional terminator can be moved or "subcloned" from one vector into another. Fragments to be subcloned may be generated by methods well known in the art, such as, for example, PCR
amplification (e.g., using oligonucleotide primers having the sequence shown in SEQ ID
NO:A or B), and/or restriction enzyme digestion.
[0076] in preferred embodiments, the albumin fusion proteins of the invention are capable of a therapeutic activity and/or biologic activity corresponding to the therapeutic activity and/or biologic activity of the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein listed in the corresponding row of Table 1: In further preferred embodiments, the therapeutically active protein portions of the albumin fusion proteins of the invention are fragments or variants of the protein encoded by the sequence shown in SEQ ID NO:X column of Table 2, and are capable of the therapeutic activity and/or biologic activity of the corresponding Therapeutic protein.
Non-human albumin fusion proteins of growth hormone.
[0077] In one embodiment, the albumin fusion proteins of the invention comprise one or more Serum Albumin proteins of a non-human animal species, fused in tandem and in-frame either at the =Nrterminus or the C-terminus to one or more Growth Hormone proteins of the same non-human animal species. Non-human Serum Albumin and Growth Hormone proteins are well known in the art and available in public databases. For example, Table 4 presents accession numbers corresponding to non-human Serum Albumin sequences (column 2) and non-human Growth Hormone sequences (column 3) found in GenBank. In a preferred embodiment, a Serum Albumin protein from a non-human animal species listed in Table 4 is fused to a Growth Hormone protein from the same non-human animal species.
[0078] In a specific embodiment, the albumin fusion protein of the invention comprises one or more Bos tatuns Serum Albumin proteins listed in Table 4, column 2, fused in tandem and in-frame either at the N-terminus or the C-terminus to one or more Bos taurus Growth Hormone proteins listed in Table 4, column 3.
[0079] Fusion proteins comprising fragments or variants of non-human Sertun Albumin, such as, for example, the mature form of Serum Albumin, are also encompassed by the invention. Fusion proteins comprising fragments or variants of non-human Growth Hormone proteins, such as, for example, the mature form of Growth Hormone, are also encompassed by the invention. Preferably the non-human Growth Hormone fragments and variants retain growth hormone activity.
100801 Polynucleotid.es of the invention comprise, or alternatively consist of, one or more nucleic acid molecules encoding a non-human albumin fusion protein described above.
For example, the polynucleotides can comprise, or alternatively consist of;
one or more nucleic acid molecules that encode a Serum Albumin protein from a non-human animal species listed in Table 4, column 1 (such as, for exaMple, the non-human Serum Albumin reference sequences listed in Table 4, column 2) fused in tandem and in-frame either 5' or 3' to a polynucleotide that comprises, or alternatively consists of, one or more nucleic acid molecules encoding the non-human Growth Hormone protein of the corresponding non-human animal species (for example, the Growth Hormone reference sequences listed in Table 4, column 3).
[0081] The above-described non-human albumin fusion proteins are encompassed by the invention, as are host cells and vectors containing these polynucleotides.
In one embodiment, a non-human albumin fusion protein encoded by a polynucleotide as described above has extended shelf life. In an additional embodiment, a non-human albumin fusion protein encoded by a polynucleotide described above has a longer serum half-life and/or more stabilized activity in solution (or in a pharmaceutical composition) in vitro and/or in vivo than the corresponding unfused Growth Hormone molecule.
[0082] The present invention also encompasses methods of preventing, treating, or ameliorating a disease or disorder in a non-human animal species. In certain embodiments, the present invention encompasses a method of treating a. veterinary disease or disorder comprising administering to a non-human animal species in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a Growth Hormone portion corresponding to a Growth Hormone protein (or fragment or variant thereof) in an amount effective to treat, prevent or ameliorate the disease or disorder.
Veterinary diseases and/or disorders which may be treated, prevented, or ameliorated include growth disorders (such as, for example, pituitary dwarfism), shin soreness, obesity, growth hormone-responsive dermatosis, dilated cardiornyopathy, eating disorders, reproductive disorders, and endocrine disorders.
[0083] Non-human albumin fusion proteins of the invention may also be used to promote healing of skin wounds, corneal injuries, bone fractures, and injuries of joints, tendons, or ligaments.
[00841 Non-human albumin fusion proteins of the invention may also be used to increase milk production in lactating animals. In a preferred embodiment, the lactating animal is a dairy cow.
[0085] Non-human albumin fusion proteins of the invention may also be used to improve body condition in aged animals.
[0086] Non-human albumin fusion proteins of the invention may also be used to increase fertility, pregnancy rates, and reproductive success in domesticated animals.
[00871 Non-human albumin fusion proteins of the invention may also be used to improve the lean-to-fat ratio in animals raised for consumption, as well as to improve appetite, and increase body size and growth rate.
Table 4 Non-Human Non-Human Serum Albumin Non-Human Growth Hormone Species Reference Sequence(s): Reference Sequence(s): GenBank GenBank Protein Accession Protein Accession NOs.
Nos.
Bos taurus ABBOS, CAA76847, P02769: STBO, BAA06379, A29864, CAA41735, 229552, AAF28806, AAF28805, .AAF' 28804, AAA51411 P01246, AAF03132, AAC63901, AAB92549, A36506, 145901, JC1316, CAA23445, CAA00787, CAA00598, AAA30547, AAA30546, AAA30545, AAA30544, AAA30543, A_AA30542 Sus scrofa P08835, CAA30970, 'STPG, PC1017, AAB29947, AAA30988 AA1384359, 146585, 146584, PC1063, A01516, AAB17619, 226829, 225740, CAA37411, CAA00592, AA.A73478, AA_A73477, CAA00356, AAA31046, AAA31045, AAA31044, A.,A.30543 Equus caballus ABHOS, AAG40944, P35747, STHO, P01245, .AAD25992, 227704, ____________ CAA52194 AAA21027 Ovis aries ABSHS, P14639, CAA34903 STSH, AAB24467, AAC48679, 228487, 223932, CAA34098, ______________________________________ CAA31063, CAA00828, AAA31527 Salmo salar ABONS2, ABONS2, STONC, P070-64, Q07221, P48096, CAA36643, CAA43187 P10814, P10607, 151186, S03709, 350179, A23154, S06489, CAA42431, AAB29165, AAB24612, Q91221., Q91222, CAA43942, CAA32481, 738042, 224555, CAA00427, ______________________________________ AAA,50757 AAA49558, AAA49555 2-' """--Non-Human 1 Non-Human Serum Albumin Non-Human Growth Hormone Species Reference Sequence(s): Reference Sequence(s): GenBank GenBank Protein Accession Protein Accession Nos.
Nos.
AAA49553, AAA.49401, AAA49406, AAA49403, AAA49402 Gallus gallus ABCHS, P19121, CAA43 098 BAB62262, I3AB69037, AAK95643, A60509, AAG01029, BAA01365, P08998, 226895, CAA31127, CAA35619, AAA48780 Fells eatus P49064, S57632, CAA59279, JC4632, P46404, AAC00073, =JC4660 AAA96142, AAA67294 Canis P49822, S29749, CAB64867, P33711, 146145, AAF89582, familiaris C.A.A76841, AAB30434 AAF21502, AAD43366, $35790, Polypeptide and Polynueleotide Fragments and Variants Fragments [0088] The present invention is further directed to fragments of the Therapeutic proteins described in Table 1, albumin proteins, and/or albumin fusion proteins of the invention.
[00891 The present invention is also directed to polynucleotides encoding fragments of the Therapeutic proteins described in Table 1, albumin proteins, and/or albumin fusion proteins of the invention.
[00901 Even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the Therapeutic protein, albumin protein, and/or albumin fusion protein of the invention, other Therapeutic activities and/or functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. For example, the ability of polypeptides with N-terminal deletions to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise blown in the art. It is not unlikely that a rnutein with a large number of deleted N-terminal amino acid residues may retain sonic biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
10091] Accordingly, fragments of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the reference polypeptide (i.e., a Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). In particular, N-terminal deletions may be described by the general formula m to q, where q is a whole integer representing the total number of amino acid residues in a reference polypeptide (e.g., a Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein of the invention, or a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotid.e or albumin fusion construct described in Table 2), and m is defined as any integer ranging fiom 2 to q minus 6. Polynucleotides encoding these polypeptides are also encompassed by the invention.
[0092] In addition, fragments of serum albumin polypeptides corresponding to an albumin protein portion of an albumin fusion protein of the invention, include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the reference polypeptide (i.e., serum albumin, or a serum albumin portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion . construct described in Table 2). In preferred embodiments, N-terminal deletions may be described by the general formula m to 585, where 585 is a whole integer representing the total number of amino acid residues in mature human serum albumin (SEQ ID NO:1038), and in is defined as any integer ranging from 2 to 579. Polynucleotides encoding these polypeptides are also encompassed by the invention. In additional embodiments, N-teinainal deletions may be described by the general formula m to 609, where 609 is a whole integer representing the total number of amino acid residues in full length human serum albumin (SEQ ID
NO:1094), and m is defined as any integer ranging from 2 to 603. Polynucleotides encoding these polypeptides are also encompassed by the invention.
[0093] Moreover, fragments of albumin fusion proteins of the invention, include the full length albumin fusion protein as well as polypeptides having one or more residues deleted from the amino terminus of the albumin fusion protein (e.g., an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2; or an albumin fusion protein having the amino acid sequence disclosed in column 6 of Table 2). In particular, N-terminal deletions may be described by the general formula m to q, where q is a whole integer representing the total number of amino acid residues in the albumin fusion protein, and m is defined as any integer ranging from 2 to q minus 6.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[0094] Also as mentioned above, even if deletion of one or more amino acids from the N-tenninus or C-tenninus of a reference polypeptide (e.g., a Therapeutic protein; serum albumin protein; or albumin fusion protein of the invention) results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) and/or Therapeutic activities may still be retained. For example the ability of polypeptides with C-terminal deletions to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking the N-terminal and/or C-terminal residues of a reference polypeptide retains Therapeutic activity can readily be determined by routine methods described herein and/or otherwise known in the art.
[0095j The present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention (e.g., a Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). In particular, C-terminal deletions may be described by the general formula 1 to n, where n is any whole integer ranging from 6 to q minus I, and where q is a whole integer representing the total number of amino acid residues in a reference polypeptide (e.g., a Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). Polynucleotides encoding these polypeptides are also encompassed by the invention.
[00961 In addition, the present invention, provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of an albumin protein corresponding to an albumin protein portion of an albumin fusion protein of the invention (e.g., serum albumin or an albumin protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). In particular, C-terminal deletions may be described by the general formula 1 to n, where n is any whole integer ranging from 6 to 584, where 584 is the whole integer representing the total number of amino acid residues in mature human serum albumin (SEQ ID NO:1038) minus 1.
Polynucleotides encoding these polypeptides are also encompassed by the invention. In particular, C-terminal deletions may be described by the general formula 1 to n, where n is any whole integer ranging from 6 to 608, where 608 is the whole integer representing the total number of amino acid residues in serum albumin (SEQ ID NO:1094) minus 1. Polynucleotides encoding these polypeptides are also encompassed by the invention.
[0097] Moreover, the present invention provides polypeptides having one or more residues deleted from the carboxy terminus of an albumin fusion protein of the invention. In particular, C-terminal deletions may be described by the general formula 1 to n, where n is any whole integer ranging from 6 to q minus 1, and where q is a whole integer representing the total number of amino acid residues in an albumin fusion protein of the invention.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[098] In addition, any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted reference polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m to n of a reference polypeptide (e.g., a Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein of the invention, or a Therapeutic protein portion õ
encoded by a polynucleotide or albumin fusion construct described in Table 2, or serum albumin (e.g., SEQ ID NO:1038), or an albumin protein portion of an albumin fusion protein of the invention, or an albumin protein portion encoded by a polynucleotide or albumin fusion construct described in Table 2, or an albumin fusion protein, or an albumin fusion protein encoded by a polynucleotide or albumin fusion construct of the invention) where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
[0099] The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference polypeptide sequence (e.g., a Therapeutic protein referred to in Table 1, or a Therapeutic protein portion of an albumin fusion protein of the invention, or a Therapeutic protein portion encoded by a polynucleotide or albumin fusion construct described in Table 2, or serum albumin (e.g., SEQ
ID NO: 1038), or an albumin protein portion of an albumin fusion protein of the invention, or an albumin protein portion encoded by a polynucleotide or albumin fusion construct described in Table 2, or an albumin fusion protein, or an albumin fusion protein encoded by a polynucleotide or albumin fusion construct of the invention) set forth herein, or fragments thereof. in preferred embodiments, the application is directed to proteins comprising polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to reference polypeptides having the amino acid sequence of N- and C-terminal deletions as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
101001 Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a Therapeutic activity and/or functional activity (e.g. biological activity) of the polypeptide sequence of the Therapeutic protein or serum albumin protein of which the amino acid sequence is a fragment.
[01011 Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
Variants [01021 "Variant" refers to a polynucleotide or nucleic acid differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof.
Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide.
[01031 As used herein, "variant", refers to a Therapeutic protein portion of an albumin fusion protein of the invention, albumin portion of an albumin fusion protein of the invention, or albumin fusion protein of the invention differing in sequence from a Therapeutic protein (e.g. see "therapeutic" column of Table 1), albumin protein, and/or albumin fusion protein, respectively, but retaining at least one functional and/or therapeutic property thereof as described elsewhere herein or otherwise known in the art. Generally, variants are overall very similar, and, in many regions, identical to the amino acid sequence of the Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein, albumin protein corresponding to an albumin protein portion of an albumin fusion protein, and/or albumin fusion protein. Nucleic acids encoding these variants are also encompassed by the invention.
[01041 The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, the amino acid sequence of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention (e.g., the amino acid sequence of a Therapeutic protein.:X disclosed in Table 1; or the amino acid sequence of a Therapeutic protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 1 and 2, or fragments or variants thereof), albumin proteins corresponding to an albumin protein portion of an albumin fusion protein of the invention (e.g., the amino acid sequence of an albumin protein portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 'I and 2; the amino acid sequence shown in SEQ ID
NO: 1038;
or fragments or variants thereof), and/or albumin fusion proteins. Fragments of these polypeptides are also provided (e.g., those fragments described herein).
Further polypeptides encompassed by the invention are polypeptides encoded by polynucleotides which hybridize to the complement of a nucleic acid molecule encoding an albumin fusion protein of the invention under stringent hybridization conditions (e.g., hybridiriation to filter bound DNA in 6X Sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2X SSC, 0.1% SDS at about 50 - 65 degrees Celsius), under highly stringent conditions (e.g., hybridintion to filter bound DNA in 6X sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.1X SSC, 0.2% SDS at about 68 degrees Celsius), or under other stringent hybridization conditions which are 'mown to those of skill in the art (see, for example, Ausubel, F.M.
et al., eds., 1989 Current protocol in Molecular Biology, Green publishing associates, inc., and John Wiley &
Sons Inc., New York, at pages 6.3.1 - 6.3.6 and 2.10.3). Polynucleotides encoding these polypeptides are also encompassed by the invention.
[01051 By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
[01061 As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of an albumin fusion protein of the invention or a fragment thereof (such as a Therapeutic protein portion of the albumin fusion protein or an albumin portion of the albumin fusion protein), can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al.
(Comp. App. Biosci.6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix¨PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size¨sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size-500 or the length of the subject amino acid sequence, whichever is shorter.
[0107) If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity.
For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N-and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity, calculated by the above FASTDB
program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is What is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
101081 For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends= of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
[0109) The variant will usually have at least 75 % (preferably at least about 80%, 90%, 95% or 99%) sequence identity with a length of normal HA or Therapeutic protein which is the same length as the variant. Homology or identity at the nucleotide or amino acid sequence level is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et aL, Proc. Natl. Acad. Sci. USA .87: 2264-2268 (1990) and Altschul, J. Mol.
Evol. 36:
290-300 (1993)) which are tailored for sequence similarity searching.
[01101 The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al., (Nature Genetics 6: 119-129 (1994)), The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblasin, and tblastx is the BLOSUM62 matrix (Henikoff et at, Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992).
For blastaa, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively. Four blastn parameters may be adjusted as follows: Q=10 (gap creation penalty); R-10 (gap extension penalty); wink=1 (generates word hits at every winkth position along the query); and gapw--16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings were Q=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN-3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.
PIM The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
Moreover, polypeptid.e variants in which less than 50, less than 40, less than 30, less than 20, less than

10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino .acids are substiffited, deleted, or added in any combination are also preferred. Polynueleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human . inRNA to those preferred by a bacterial host, such as, yeast or E. coli).
[011.2] In a preferred embodiment, a polynucleotide of the invention which encodes the albumin portion of an albumin fusion protein is optimized for expression in yeast or mammalian cells, hi a further preferred embodiment, a polynucleotide of the invention which encodes the Therapeutic protein portion of an albumin fusion protein is optimized for expression in yeast or mammalian cells. In a still further preferred embodiment, a polynucleotide encoding an albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells.
101131 In an alternative embodiment, a codon optimized polynucleotide which encodes a Therapeutic protein portion of an albumin fusion protein does not hybridize to the wild type polynucleotide encoding the Therapeutic protein under stringent hybridization conditions as described herein. In a further embodiment, a codon optimized polynucleotide which encodes an albumin portion of an albumin fusion protein does not hybridize to the wild type polynucleotide encoding the albumin protein under stringent hybridization conditions as described herein. In another embodiment, a codon optimized polynucleotide which encodes an albumin fusion protein does not hybridize to the wild type polynucleotide encoding the TheraPeutic protein portion or the albumin protein portion under stringent hybridization conditions as described herein.
101141 In an additional embodiment, a polynucleotide which encodes a Therapeutic protein portion of an albumin fusion protein does not comprise, or alternatively consist of, the naturally occurring sequence of that Therapeutic protein. In a further embodiment, a polynucleotide which encodes an albumin protein portion of an albumin fusion protein does not comprise, or alternatively consist of, the naturally occurring sequence of albumin protein.
In an alternative embodiment, a polynucleotide which encodes an albumin fusion protein does not comprise, or alternatively consist of, the naturally occurring sequence of a Therapeutic protein portion. or the albumin protein portion.
[0115] Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.
(Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants .may be produced by rnutagenesis techniques or by direct synthesis.
[0116] Using known methods of protein engineering and recombinant DNA
technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol.
Chem. 268: 2984-2988 (1993)) reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobel.i et al., J. Biotechnology 7:199-216 (1988).) [01171 Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual LL-la mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that "{rniost of the molecule could be altered with little effect on either [binding or biological activity]." In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
[0118] Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-tenninus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
[0119] Thus, the invention further includes polypeptide variants which have a functional activity (e.g., biological activity and/or therapeutic activity).
In one embodiment, the invention provides variants of albumin fusion proteins that have a functional activity (e.g., biological activity and/or therapeutic activity) that corresponds to one or more biological and/or therapeutic activities of the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein. In another embodiment, the invention provides variants of albumin fusion proteins that have a functional activity (e.g., biological activity and/or therapeutic activity) that corresponds to one or more biological and/or therapeutic activities of the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. Polynucleotides encoding such variants are also encompassed by the invention.
[0120] In preferred embodiments, the variants of the invention have conservative substitutions. By "conservative substitutions" is intended swaps within groups such as replacement of the aliphatic or hydrophobic amino acids Ala, Val, Len and Lie;
replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Gin;
replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Tip, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
[0121] Guidance concerning how to make phenotypically silent amino acid substitutions is provided, for example, in Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
[01221 The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. in contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
[01231 The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
101241 As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpoiar side chains, whereas few features of surface side chains are generally conserved.
Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Gin;
replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His;
replacement of the aromatic residues Phe, Tyr, and Tzp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acid substitution, variants of the present invention include (i) polypeptides containing substitutions of one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) polypeptides containing substitutions of one or more of the amino acid residues having a substituent group, or (iii) polypeptides which have been fused with or chemically conjugated to another compound, such as a compound to increase the stability and/or solubility of the polyp eptide (for example, polyethylene glycol), (iv) polypeptide containing additional amino acids, such as, for example, an IgG Fe fusion region peptide . Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
[01251 For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation.
Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pirickard et al., Clin. Exp. Inununol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
[0126] In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of the amino acid sequence of an albumin fusion protein, the amino acid sequence of a Therapeutic protein and/or human serum albumin, wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Nucleic acids encoding these polypeptides are also encompassed by the invention.
[0127] The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivatiVe, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bend formation, denaethylation, formation of covalent cross-links, formation of cysteine, formation of pyro glutamate, fomiylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation., transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol.
182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
Functional activity [0128] "A polypeptide having functional activity" refers to a polypeptide capable of displaying one or more blown functional activities associated with the full-length, pro-protein, and/or mature form of a Therapeutic protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti-polypeptide antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to fomi multimers with polypeptides of the invention, and ability to hind to a receptor or ligand for a polypeptide.
[0129) "A polypeptide having biological activity" refers to a polypeptide exhibiting activity similar to, but not necessarily 'identical to, an activity of a Therapeutic protein of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than. about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
[0130] In preferred embodiments, an albumin fusion protein of the invention has at least one biological and/or therapeutic activity associated with the Therapeutic protein portion (or fragment or variant thereof) when it is not fused to albumin.

[0131] The albumin fusion proteins of the invention can be assayed for functional activity (e.g., biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Additionally, one of skill in the art may routinely assay fragments of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein, for activity using assays referenced in its corresponding row of Table 1 (e.g., in column 3 of Table 1). Further, one of skill in the art may routinely assay fragments of an albumin protein corresponding to an albumin protein portion of an albumin fusion protein, for activity using assays known in the art and/or as described in the Examples sectfon below.
[01321 For example, in one embodiment where one is assaying for the ability of an albumin fusion protein to bind or compete with a Therapeutic protein for binding to an anti-Therapeutic polypeptide antibody and/or anti-albumin antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, inummofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
[0133] In a preferred embodiment, where a binding partner (e.g., a receptor or a ligand) of a Therapeutic protein is identified, binding to that binding partner by an albumin fusion protein which comprises that Therapeutic protein as the Therapeutic protein portion of the fusion can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et at, Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of an albumin fusion protein to bind to a substrate(s) of the Therapeutic polypeptide corresponding to the Therapeutic protein portion.
of the fusion can be routinely assayed using techniques known in the art.
[0134] In an alternative embodiment, where the ability of an albumin fusion protein to multinaerize is being evaluated, association with other components of the multimer can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affmity blotting. See generally, Phizicky et al., supra.
[0135] In preferred embodiments, an albumin fusion protein comprising -all or a portion of an antibody that binds a Therapeutic protein, has at least one biological and/or therapeutic activity (e.g., to specifically bind a polypeptide or epitope) associated with the antibody that binds a Therapeutic protein (or fragment or variant thereof) when it is not fused to albumin. In other preferred embodiments, the biological activity and/or therapeutic activity of an albumin fusion protein comprising all or a portion of an antibody that binds a Therapeutic protein is the inhibition (i.e., antagonism) or activation (i.e., agonism) of one or more of the biological activities and/or therapeutic activities associated with the polypeptide that is specifically bound by antibody that binds a Therapeutic protein.
[01361 Albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be characterized in a variety of ways. In particular, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be assayed for the ability to specifically bind to the same antigens specifically bound by the antibody that binds a Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein using techniques described herein or routinely modifying techniques known in the art.
[0137] Assays for the ability of the albumin fusion proteins (e.g., comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) to (specifically) bind a specific protein or epitope may be performed in solution (e.g., Houghten, Bio/Techniques 13:412-421(1992)), on beads (e.g., Lam, Nature 354:82.-84 (1991)), on chips (e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (e.g., U.S. Patent No. 5,223,409), on spores (e.g., Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl.
Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g.. Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla et al., Proc..Natl. Acad.
Sci. USA 87:6378-6382 (1990); and Felici, J. Mol. Biol. 222:301-310 (1991)).
Albumin fusion proteins comprising at least a fragment or variant of a Therapeutic antibody may also be assayed for their specificity and affinity for a specific protein or epitope using or routinely modifying techniques described herein or otherwise known in the att.

[01381 The albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be assayed for cross-reactivity with other antigens (e.g., molecules that have sequence/structure conservation with the molecule(s) specifically bound by the antibody that binds a Therapeutic protein (or fragment or variant thereof) corresponding to the Therapeutic protein portion of the albumin fusion protein of the invention) by any method known in the art.
[0139]
Immunoassays which can be used to analyze (imrnunospecific) binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA
(enzyme linked immunosorbent assay), "sandwich" immunoassays, imrnunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, itrummodiffusion assays, agglutination assays, complement-fixation assays, imnaunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).
Exemplary immunoassays are described briefly below (but are not intended by way of [0140]
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Tritori X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium Nanadate), adding the albumin fusion protein of the invention (e.g., comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) to the cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at 40 degrees C, adding sepharog beads coupled to an anti-albumin antibody, for example, to the cell lysate, incubating for about an hour or more at 40 degrees C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the albumin fusion protein to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the albumin fusion protein to an antigen and decrease the background (e.g,, pre-clearing the cell lysate with sepharoS'gbeads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0141] Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polya.crylamide gel (e.g., 8%- 20%
SDS-PAGE
depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tweeir 20), applying the albumin fusion protein of the invention (diluted in blocking buffer) to the membrane, washing the membrane in washing buffer, applying a secondary antibody (which recognizes the albumin fusion protein, e.g, an anti-human serum albumin antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in. Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
[0142) ELISAs comprise preparing antigen, coating the well of a 96-well microtiter plate with the antigen, washing away antigen that did not bind the wells, adding the albumin fusion protein (e.g., comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) of the invention conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells and incubating for a period of time, washing away unbound or non-specifically bound albumin fusion proteins, and detecting the presence of the albumin fusion proteins specifically bound to the antigen coating the well. In ELISAs the albumin fusion protein does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes albumin fusion protein) conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the albumin fusion protein may be coated to the well. In this case, the detectable molecule could be the antigen conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase). One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of FUSAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at

11.2.1.
[0143] The binding affinity of an albumin fusion protein to a protein, antigen, or epitope and the off-rate of an albumin fusion protein-protein/antigen/epitope interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or '251) with the albumin fusion protein of the invention in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the albumin firsion protein for a specific protein, antigen, or epitope and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second protein that binds the same protein, antigen or epitope as the albumin fusion protein, can also be determined using radioimmunoassays. In this case, the protein, antigen or epitope is incubated with an albumin fusion protein conjugated to a labeled compound (e.g., 3H or 1251) in the presence of increasing amounts of an unlabeled second protein that binds the same protein, antigen, or epitope as the albumin fusion protein of the invention.
[01441 In a preferred embodiment, BlAcore kinetic analysis is used to determine the binding on and off rates of albumin fusion proteins of the invention to a protein, antigen or epitope. BlAcore kinetic analysis comprises analyzing the binding and dissociation of albumin fusion proteins, or specific polypeptides, antigens or epitopes from chips with immobilized specific polypeptides, antigens or epitopes or albumin fusion proteins, respectively, on their surface.
[0145j Antibodies that bind a Therapeutic protein corresponding to the Therapeutic protein portion of an albumin fusion protein may also be described or specified in terms of their binding affinity for a given protein or antigen, preferably the antigen which they specifically bind. Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 104 M, 10-2 M, 5 X 104 M, 10-3 M, 5 X 104 M,104 M. More preferred binding affinities include those with a dissociation constant or Kd less than 5 X 104 M, 104 M, 5 X
M, leM, 5 X le NI, 107 M, 5 X 104 M or 104 M. Even more preferred binding affinities include those with a dissociation constant or Kd less than 5 X le M, 10-9 M, 5 X
1040 M, 10-1 NI, 5 X 10-" M, lO M, 5 X 10-12 1 42 rvi, 5 X 10-13 m, io-13 m, 5 X 10-14 M, 10-14 M, 5 X 1045 M, or 1045 M. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, has an affinity for a given protein or epitope similar to that of the corresponding antibody (not fused to albumin) that binds a Therapeutic protein, taking into account the valency of the =
albumin fusion protein (comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) and the valency of the corresponding antibody. In addition, assays described herein (see Examples and Table 1) and otherwise known in the art may routinely be applied to measure the ability of albumin fusion proteins and fragments, variants and derivatives thereof to elicit biological activity and/or Therapeutic activity (either in vitro or in vivo) related to either the Therapeutic protein portion and/or albumin portion of the albumin fusion protein. Other methods will be known to the skilled artisan and are within the scope of the invention.
Albumin 101461 As described above, an albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion.
101471 An additional embodiment comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are linked to one another by chemical conjugation.
[0148] The terms, human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms, "albumin and "serum albumin" are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).
[0149] As used herein, "albumin" refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or moii functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or fragments thereof (see for example, EP 201 239, EP 322 094 WO 97/24445, W095/23857) especially the mature form of human albumin as shown in Figure 1 and SEQ NO:
1038, or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.
101501 In preferred embodiments, the human serum albumin protein used in the albumin fusion proteins of the invention contains one or both of the following sets of point mutations with reference to SEQ ID NO: 1038: Leu-407 to Ala, Leu-408 to Val, Va1-409 to Ala, and Arg-410 to Ala; or Arg-410 to A, Lys-413 to Gin, and Lys-414 to Gin (see, e.g., International publication No. W095/23857).
In even more preferred embodiments, albumin fusion proteins of the invention that contain one or both of above-described sets of point mutations have improved stability/resistance to yeast Yap3p proteolytic cleavage, allowing increased production of recombinant albumin fusion proteins expressed in yeast host cells.
(0151j As used herein, a portion of albumin sufficient to prolong the therapeutic activity or shelf-life of the Therapeutic protein refers to a portion of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity of the protein so that the shelf life of the Therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the shelf-life in the non-fusion state. The albumin portion of the albumin fusion proteins may comprise the full length of the HA sequence as described above, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the HA
sequence or may include part or all of specific domains of HA. For instance, one or more fragments of HA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HA fragment is the mature form of HA.
101521 The albumin portion of the albumin fusion proteins of the invention may be a variant of normal.HA. The Therapeutic protein portion of the albumin fusion proteins of the invention may also be variants of the Therapeutic proteins as described herein. The term "variants" includes insertions, deletions and substitutions, either conservative or non conservative, where such changes do not substantially alter one or more of the oncotic, useful ligand-binding and non-immunogenic properties of albumin, or the active site, or active domain which confers the therapeutic activities of the Therapeutic proteins.
[0153] In particular, the albumin fusion proteins of the invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin, for example those fragments disclosed in EP 322 094 (namely HA (Pn), where n is 369 to 419). The albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be from a different animal than the Therapeutic protein portion.
101541 Generally speaking, an HA fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long. The HA variant may consist of or alternatively comprise at least one whole domain of HA, for example domains 1 (amino acids 1-194 of SEQ ID NO: 1038), domain 2 (amino acids 195-387 of SEQ ID NO: 1038), domain 3 (amino acids 388-585 of SEQ ID NO: 1038), domains 1 and 2 (1-387 of SEQ ID
NO:

1'8), domains 2 and 3 (195-585 of SEQ ID NO: 1038) or domains 1 and 3 (amino acids 1-194 of SEQ 1D NO: 1038 and amino acids 388-585 of SEQ ID NO: 1038). Each domain is itself made up of two homologous subdomains namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with flexible inter-subdomain linker regions comprising residues 148106 to G1u119, G1u292 to Va1315 and G1u492 to Ala511.
[0159 Preferably, the albumin portion of an albumin fusion protein of the invention comprises at least one subdomain or domain of HA or conservative modifications thereof. If the fusion is based on subdomains, some or all of the adjacent linker is preferably used to link to the Therapeutic protein moiety.
Antibodies that Specifically bind Therapeutic proteins are also Therapeutic proteins 101561 The present invention also encompasses albumin fusion proteins that comprise at least a fragment or variant of an antibody that specifically binds a Therapeutic protein disclosed in Table 1, It is specifically contemplated that the term "Therapeutic protein"
encompasses antibodies that bind a Therapeutic protein (e.g., as Described in colutnn I of Table I) and fragments and variants thereof. Thus an albumin fusion protein of the invention may contain at least a fragment or variant of a Therapeutic protein, and/or at least a fragment or variant of an antibody that binds a Therapeutic protein.
Antibody structure and background [01571 The basic antibody structural unit is known to comprise a tetrame,r. Each tetrarner is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa), The amino-terminal portion of each chain includes a variable region f about 100 to 110 or more amino acids primarily responsible for antigen recognition. The earboxy-tenninal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, 1gG, IgA., and TgR, respectively. See generally, Fundamental Immunology Chapters 3-5 (Paul, W., ed., 4th ed. Raven Press, NY.
(1998)). The variable regions of each light/heavy chain pair form the antibody binding site.
[0158] Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.

[01591 The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDR regions, in general, are the portions of the antibody which make contact with the antigen and determine its specificity. The CDRs from the heavy and the light chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains variable regions comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions are connected to the heavy or light chain constant region. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins qf Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).
101601 As used herein, "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen (e.g., a molecule containing one or more CDR regions of an antibody). Antibodies that may correspond to a Therapeutic protein portion . of an albumin fusion protein include, but are not limited to, , monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies (e.g., single chain Fvs), Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies specific to antibodies of the invention), and epitope-binding fragments of any of the above (e.g., VH
domains, VL
domains, or one or more CDR regions).
Antibodies that bind Therapeutic Proteins [01611 The present invention encompasses albumin fusion proteins that comprise at least a fragment or variant of an antibody that binds a Therapeutic Protein (e.g., as disclosed in Table 1) or fragment or variant thereof.
(01621 Antibodies that bind a Therapeutic protein (or fragment or variant thereof) may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken antibodies. Most preferably, the antibodies are human antibodies. As used herein, "htunan" antibodies include antibodies having the amino acid sequence of a human imnaunoglobulin and include antibodies isolated from human immunoglobulin libraries and xenomice or other organisms that have been genetically engineered to produce human antibodies_ [01631 The antibody molecules that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention can be of any type (e.g., IgG, IgE, Ig,M, IgD, IgA and IgY), class (e.g., IgGI, IgG2, Ig03, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. In preferred embodiments, the antibody molecules that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein are IgGI. In other preferred embodiments, the immunoglobulin molecules that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein are IgG2. In other preferred embodiments, the immunoglobulin molecules that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein are IgG4.
101641 Most preferably the antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab and F(ab')2, Fd, single-chain Fvs (seFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CII3 domains.
[01651 The antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a Therapeutic protein or may be specific for both a Therapeutic protein as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360;
WO
92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos.
4,474,893; 4,714,681;
4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Inununol. 148:1547-1553 (1992).
[0166] Antibodies that bind a Therapeutic protein (or fragment or variant thereof) may be bispecific or bifunctional which means that the antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
Hispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann Clin. Exp.
Immunol. 79: 315-321 (1990), Kostelny et al. .1 Irnmunol. 148:1547 1553 (1992). In addition, bispecific antibodies may be formed as "diabodies" (Holliger et al.
"'Diabodies': small bivalent and bispecific antibody fragments" PNAS USA 90:6444-6448 (1993)) or "Janusins"
(Tratmecker et al. "Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on 111V infected cells" EMBO J 10:3655-3659 (1991) and Traunecker et al.
"Janusin: new molecular design for bispecific reagents" Int J Cancer Suppl 7:51-52 (1992)).
101671 The present invention also provides albumin fusion proteins that comprise, fragments or variants (including derivatives) of an antibody described herein or known elsewhere in the art. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, .or less than 2 amino acid substitutions relative to the reference VII domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3. In specific embodiments, the variants encode substitutions of VIICDR3. In a preferred embodiment, the variants have conservative amino acid substitutions at one or more predicted non-essential amino acid residues.
[01681 Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein may be described or specified in terms of the epitope(s) or portion(s) of a Therapeutic protein which they recognize or specifically bind. Antibodies which specifically bind a Therapeutic protein or a specific epitope of a Therapeutic protein may also be excluded. Therefore, the present invention encompasses antibodies that specifically bind Therapeutic proteins, and allows for the exclusion of the same. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, binds the same epitopes as the unfused fragment or variant of that antibody itself.
[0169] Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein may also be described Or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a Therapeutic protein are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% sequence identity (as calculated using methods known in the art and described herein) to a Therapeutic protein are also included in the present invention. In specific embodiments, antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% sequence identity (as calculated using methods known in the art and described herein) to a Therapeutic protein are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3,4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, has similar or substantially identical cross reactivity characteristics compared to the fragment or variant of that particular antibody itself.
(01701 Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide encoding a Therapeutic protein under stringent hybridization conditions (as described herein).
Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5 X le m, 10-2 NI, 5 X
10-3 M, le m, 5 X 104 M, 104M. More preferred binding affinities include those with a dissociation constant or Kd less than 5 x. l0 M, NE, 5 X
10-6 M, 10M, 5 X 10-7 M, 107 M, 5 X leM or 104 M. Even more preferred binding affinities include those with a dissociation constant or Kd less than 5 X le m, 10-9 NI, 5 X le M, 10-1 M, 5 X 1041 M, 10-11 M, 5 X 1042 M, 10-12 Da 5 X 1043 M, 10-13 M, 5 X 10-14 M, 10-14 M, 5 X 10-15 M, or 10-M. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, has an affinity for a given protein or epitope similar to that of the corresponding antibody (not fused to albumin) that binds a Therapeutic protein, taking into account the valency of the albumin fusion protein (comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) and the valency of the corresponding antibody.
[0171) The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of a Therapeutic protein as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, competitively inhibits binding of a second antibody to an epitope of a Therapeutic protein. In other preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, competitively inhibits binding of a second antibody to an epitope of a Therapeutic protein by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
101721 Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention may act as agonists or antagonists of the Therapeutic protein. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art.
For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by Unmunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, has similar or substantially similar characteristics with regard to preventing ligand binding and/or preventing receptor activation compared to an un-fused fragment or variant of the antibody that binds the Therapeutic protein.

91731 The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dirnerization of the receptor. The antibodies may he specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the Therapeutic proteins (e.g. as disclosed in Table 1). The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S.
Patent No.
5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.

58(16):3668-3678 (1998); Harrop et at., J. Immunol. 161(4):1786-1794 (1998);
Zhu et al., Cancer Res. 58(15)3209-3214 (1998); Yoon'et al., J. Inummol. 160(7):3170-3179 (1998);
Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., 3. Immtmol.
Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et at., J. Biol.
Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);
Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996), In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, have similar or substantially identical agonist or antagonist properties as an un-fused fragment or variant of the antibody that binds the Therapeutic protein.
E01 741 Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention may be used, for example, to purify, detect, and target Therapeutic proteins, including both in in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have utility in immunoassays fin- qualitatively and quantitatively measuring levels of the Therapeutic protein in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
Likewise, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, may be used, for example, to purify, detect, and target Therapeutic proteins, including both in vitro and in vivo diagnostic and therapeutic methods.
[0175] Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, fonnylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical amino acids. Albumin fusion proteins of the invention may also be modified as described above.
Methods of Producing Antibodies that bind Therapeutic Proteins [01761 The antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a Therapeutic protein may be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Fretmcl's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysoleeithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dimitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
101771 Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybiidoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Harnmerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 -ilsevier, N.Y., 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
[01781 Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with a Therapeutic protein or fragment or variant thereof, an albumin fusion protein, or a cell expressing such a Therapeutic protein or fragment or variant thereof or albumin fusion protein. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
(0179) Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody. wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
101801 Another well known method for producing both polyclonal and monoclonal human B cell lines is transformation using Epstein Ban. Virus (EBV). Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY.
The source of B cells for transformation is commonly human peripheral blood, but B cells for transformation may also be derived from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues are generally made into single cell suspensions prior to EBV transformation. Additionally, steps may be taken to either physically remove or inactivate T cells (e.g., by treatment with cyclosporin A) in B

cell-containing samples, because T cells from individuals seropositive for anti-EBV
antibodies can suppress B cell immortalization by EBV.
[01811 In general, the sample containing human B cells is innoculated with EBV, and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCC #VR-1492). Physical sips of EBV transformation can generally be seen towards the end of the 3-4 week culture period. By phase-contrast microscopy, transformed cells may appear large, clear, hairy and tend to aggregate in tight clusters of cells. Initially, EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV
lines may become monoclonal or polyclonal as a result of the selective outgrowth of particular B cell clones. Alternatively, polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV
transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human canines (e.g., GM
1500, SKO-007, RPM1 8226, and KR-4). Thus, the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.
[01821 Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(abl2 fragments of the invention may be produced by proteolytic cleavage of irnmunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab)2 fragments).
F(abf)2 fragments contain the 'variable region, the light chain constant region and the CHI domain of the heavy chain.
[01831 For example, antibodies that bind to a Therapeutic protein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which cany the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g,, using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the hage gene III or gene VIII protein. Examples of phage display methods that can be used to make antibodies that bind to a Therapeutic protein include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et at., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. 3. Immunol. 24:952-958 (1994); Persic et at,, Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Nos.
5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;
5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.
[01841 As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(abs)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques

12(6);864-869 (1992); and Sawai et al., ATRI 34:26-34 (1995); and Better et at., Science 240:1041-1043 (1988).
[01851 Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human inununoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229;1202 (1985); Oi et at., BioTechniques 4:214 (1986);
Gullies et at., (1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567;
and 4,816397. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarily determining regions (CDRs) from the non-=

human species and a framework regions from a human immunoglobulin molecule.
Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S.
Patent No.
5,585,089; Riechrnann et al., Nature 332:323 (1988)).
Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO
91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; PadIan, Molecular Immunology 28(4/5):489-498 (1991);
Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO
98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.
[01871 Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain irrununoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the HI region prevents endogenous antibody production.
The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which 7.press human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human irnmunoglobulin transgenes harbored by the transgenic mice rearrange during 13 cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol.

13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT
publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;
5,661,016;
5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598.
In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
101881 Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).
Polynucleotides Encoding Antibodies [0189] The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a Therapeutic protein, and more preferably, an antibody that binds to a polypeptide having the amino acid sequence of a "Therapeutic protein:X" as disclosed in the "SEQ ID NO:Z" column of Table 2.
101901 The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide = =

quence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
101911 Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA
clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art (See Example 107).
[0192] Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
101931 In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA
techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g,, Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
[0194] In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine rnAb and a human itrmlunog 4:Jlin constant region, e.g., humanized antibodies.
[0195] Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778; Bird, Science 242:423- 42 (1988);
Huston et al., Proc.
Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Pv fragments in E. coil may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
Recombinant Expression of Antibodies [0196] Recombinant expression of an antibody, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody or a single chain antibody), requires construction of an expression vector containing a polynucleotide that encodes the antibody.

Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036;
and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
[0197] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody.
Thus, the invention includes host cells containing a polynucleotide encoding an antibody Of the invention, or a heavy or light chain thereof, or a single chain antibody, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immimoglobulin molecule, as detailed below.
[01981 A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coil, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences;
insect cell systems infected with recombinant virus expression vectors (e.g., baculovinis) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CO), in conjunction with a vector such as the major intennediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
101991 In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector p1JR278 (Ruther et al., EMBO J.
2:1791 (1983)), in which the antibody coding sequence may be ligated individually, into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke &
Schuster, J. Biol.
Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
[0200] In an insect system, Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhechin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

[02011 In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be li gated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
Insertion in a non-essential region of the viral genorne (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation rodons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
(02021 In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modificatiOn of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK., 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, lis578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
(0203i For long-term, high-yield production of recombinant proteins, stable =
expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, olyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media_ The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
102041 A number of selection systems may be used, including but not limited to the herpes simplex virus thyrnidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad.
Sci, USA
48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tic-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes:
dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980);
O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, PIM. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside 0-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Phannacol. Toxicol. 32:573-596 (1993);
. Mulligan, Science 260:926-932 (1993); and Morgan. and Anderson, Ann_ Rev.
Biochem.
62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel at al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981).
[02051 The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA
cloning, Vol..3.
(Academic Press, New York, 1987)). When a marker in the vector system expressing =

antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol.
Cell. Biol. 3:257 (1983)).
[0206j Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availability of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative. Glutarnine synthase expression systems can also function in glutamine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications:
W087/04462;
W086/05807; W089/01036; W089/10404; and W091/06657.
Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suppliers, including, for example Lanza Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/teehnology 10:169(1992) and in Biblia and Robinson Biotechnol.
Nog. 11:1 (1995).
[0207) The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986);
Kohler, Proc. Natl. Acad: Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
[02081 Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen rifler Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
Modifications of Antibodies NMI Antibodies that bind a Therapeutic protein or fragments or variants can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc.
Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hernagglutinin tag (also called the "HA
tag"), which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767(1984)) and the "flag" tag.
[02101 The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen.
Detection can he facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tornographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidinibiotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothioeyanate, rhodarnine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes lumina% examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 1251, 1311, 1111n or 99Tc. Other examples of detectable substances have been described elsewhere herein.
102111 Further, an antibody of the invention may be conjugated to a therapeutic moiety such as a cytotaxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A
cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, claunorubicin, dihydroxy anthracin dione, mitoxantrone, mithrarnycin, actinomycin 19, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloreth.amine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (11) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly datmomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinornycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
[0212) The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is n'ot to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, B-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., INF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI
(See, International Publication No. WO 99/23105), a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lympholcines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleulcin-6 ("1.L-6"), gartulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
102131 Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylarnide, nylon, polystyrene, polyvinyl chloride or polypropylene.
(0214) Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Amon et al., "Monoclonal Antibodies For Imrnunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.
(eds.), pp, 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et at., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.
623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchem et at. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immtmol. Rev. 62:119-58(1982).
[02151 Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
102161 An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytoldne(s) can be used as a therapeutic.
Antibody-albumin fusion [02171 Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention include, but are not limited to, antibodies that bind a Therapeutic protein disclosed in the "Therapeutic Protein X"
column of Table 1, or a fragment or variant thereof.
(0218) In specific embodiments, the fragment or variant of an antibody that immunospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VII domain.

In other embodiments, the fragment or variant of an antibody that immunospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, one, two or three VII
CDRs. In other embodiments, the fragment or variant of an antibody that immunospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VH CDRL In other embodiments, the fragment or variant of an antibody that immunospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VH CDR2. In other embodiments, the fragment or variant of an antibody that immunospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VH CDR3.
102191 In specific embodiments, the fragment or variant of an antibody that immunospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VL domain.
In other embodiments, the fragment or variant of an antibody that immunospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, one, two or three VL
CDRs. In other embodiments, the fragment = or variant of an antibody that immunospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic Protein portion of an albumin fusion protein comprises, or alternatively consists of, the VL CDRI. In other embodiments, the fragment or variant of an antibody that inirrturtospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VL CDR2. In other embodiments, the fragment or variant of an antibody that immunospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VL C.DR3.
[02201 In other embodiments, the fragment or variant of an antibody that immunospecifcally binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, one, two, three, four, five, or six VII and/or VL CDRs.
[02211 In preferred embodiments, the fragment or variant of an antibody that immunospecifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, an scFv comprising the VH domain of the Therapeutic antibody, linked to the VL domain of the therapeutic antibody by a peptide linker such as (Gly4Ser)3 (SEQ rlD NO:1092).
Immunophenotyping 102221 The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or fragment or variant thereof) may be utilized for immunophe,notyping of cell lines and biological samples. Therapeutic proteins of the present invention may be useful as cell-specific markers, or more specifically as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of particular cell types.
Monoclonal antibodies (or albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker.
Various techniques can be utilized using monoclonal antibodies (Or albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Patent 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)). , [02231 These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and "non-self' cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
Characterizing Antibodies that bind a Therapeutic Protein and Albumin Fusion Proteins Comprising a Fragment or Variant of an Antibody that binds a Therapeutic Protein [02241 The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or fragment or variant thereof) may be characterized in a variety of ways. In particular, Albumin 176.

'ision proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be assayed for the ability to specifically bind to the same antigens specifically bound by the antibody that binds a Therapeutic protein corresponding to the antibody that binds a Therapeutic protein portion of the albumin fusion protein using techniques described herein or routinely modifying techniques known in the art.
[0225) Assays for the ability of the antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or fragment or variant thereof) to (specifically) bind a specific protein or epitope may be performed in solution (e.g., Houghten, Bio/Technique.s 13:412-421(1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), on chips (e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (e.g., U.S. Patent No. 5,223,409), on spores (e.g., Patent Nos. 5,571,698;
5,403,484; and 5,223,409), on pla.smids (e.g., Cull et al., Proc. Nati. Acad.
Sci. USA
89:1865-1869 (1992)) or on phage (e.g., Scott and Smith, Science 249:386-390 (1990);
Devlin, Science 249:404-406 (1990); Cwirla et al., Proc. NatL Acad. Sci. USA
87:6378-6382 (1990); and Felici, J. Mot. Biol. 222:301-310 (1991)).
The antibodies of the invention or albumin.
fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or fragment or variant thereof) may also be assayed for their specificity and affinity for a specific protein or epitope using or routinely modifying techniques described herein or otherwise known in the art.
[0226) The albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be assayed for cross-reactivity with other antigens (e.g., molecules that have sequence/structure conservation with the molecule(s) specifically bound by the antibody that binds a Therapeutic protein (or fragment or variant thereof) corresponding to the Therapeutic protein portion of the albumin fusion protein of the invention) by any method known in the art.
[02271 Immunoassays which can be used to analyze (inununospecific) binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, BLISA
(enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the at (see, e.g., Ausubel et at, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).
Exemplary immunoassays are described briefly below (but are not intended by way of [0228) Inununoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or TritorX-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaC1, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding an antibody of the invention or albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or fragment or variant thereof) to the cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at 40 degrees C, adding protein A and/or protein G sepharosrbeads (or beads coated with an appropriate anti-idiotypic antibody or anti-albumin antibody in the case when an albumin fusion protein comprising at least a fragment or variant of a Therapeutic antibody) to the cell lysate, incubating for about an hour or more at 40 degrees C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody or albumin fusion protein of the invention to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody or albumin fusion protein to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitafion protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
[0229) Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE
depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Twee7e20), applying the antibody or albumin fusion protein of the invention (diluted in blocking buffer) to the membrane, washing the membrane in washing buffer, applying a secondary antibody (which recognizes the albumin fusion protein, e.g., an anti-human serum albumin antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 3213 or 1250 diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
[0230] ELISAs comprise preparing antigen, coating the well of a 96-well microtiter plate with the antigen, washing away antigen that did not bind the wells, adding the antibody or albumin fusion protein (comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) of the invention conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells and incubating for a period of time, washing away unbound or non-specifically bound albumin fusion proteins, and detecting the presence of the antibody or albumin fusion proteins specifically bound to the antigen coating the well. In ELISAs the antibody or albumin fusion protein does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody or albumin fusion protein, respectively) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, antibody or the albumin fusion protein may be coated to the well. In this case, the detectable molecule could be the antigen conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase).
One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of' ELISAs 'mown in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
[02311 The binding affinity of an albumin fusion protein to a protein, antigen, or epitope and the off-rate of an antibody- or albumin fusion protein-protein/antigen/epitope interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioinununoassay comprising the incubation of labeled antigen (e.g., 3H or 125):) with the antibody or albumin fusion protein of the invention in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
The affinity of the antibody or albumin fusion protein of the invention for a specific protein, antigen, or epitope and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second protein that binds the same protein, antigen or epitope as the antibody or albumin fusion protein, can also be determined using radioimmunoassays. In this case, the protein, antigen or epitope is incubated with an antibody or albumin fusion protein of the invention conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second protein that binds the same protein, antigen, or epitope as the albumin fusion protein of the invention.
[02321 In a preferred embodiment, BlAcore kinetic analysis is used to determine the binding on and off rates of antibody or albumin fusion proteins of the invention to a protein, antigen or epitope. BlAcore kinetic analysis comprises analyzing the binding and dissociation of antibodies, albumin fusion proteins, or specific polypeptides, antigens or epitopes from chips with immobilized specific polypeptides, antigens or epitopes, antibodies or albumin fusion proteins, respectively, on their surface.
Therapeutic Uses =102331 The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein), nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-icliotypic antibodies as described herein), albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, and nucleic acids encoding such albumin fusion proteins. The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a Therapeutic protein, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a Therapeutic protein includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

[02341 In a specific and preferred embodiment, the present invention is directed to antibody-based therapies which involve administering antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein to an animal, preferably a mammal, and most preferably a human, patient for treating one or more diseases, disorders, or conditions, including but not limited to: neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions., and/or as described elsewhere herein. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (e.g., antibodies directed to the frill length protein expressed on the cell surface of a mammalian cell; antibodies directed to an epitope of a Therapeutic protein and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a Therapeutic protein, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a Therapeutic protein includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
[02351 A summary of the ways in which the antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be used therapeutically includes binding Therapeutic proteins locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein for diagnostic, monitoring or therapeutic purposes without undue experimentation.

[02361 The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., 1L-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
102371 The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).

Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred.
Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
102381 It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against Therapeutic proteins, fragments .or regions thereof, (or the albumin fusion protein correlate of such an antibody) for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynueleotides or polypeptides of the invention, including fragments thereof.
Preferred binding affinities include dissociation constants or Rd's less than 5 X 10-2 M, 1(12 M, 5 X 1(12 M, I 0-3 M, 5 X 10-4 M, 10-4 M. More preferred binding affinities include those with a dissociation constant or Rd less than 5 X 10-5 M, 10's M, 5 X 10-6 rs,4, 10-6M, 5 X 10-7 M, 107 M, 5 X 104 M or le M. Even more preferred binding affinities include those with a dissociation constant or Kd less than 5 X 1(19 M, 10-9 M, 5 X 1(110 M, io-io itµ43 5 101" M, 5 X 10-12 M, 1(112 M 5 X 10.13 M, 1O3 M, 5 X 104 M, 10-14 M, S X 1045 M, or 10-15 M.
Gene Therapy [02391 In a specific embodiment, nucleic acids comprising sequences encoding antibodies that bind therapeutic proteins or albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a Therapeutic protein, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.
[02401 Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described in more detail elsewhere in this application.
Demonstration of Therapeutic or Prophylactic Activity [024 1 I The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and 'exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
Therapeutic/Prophylactic Administration and Composition [02421 The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
[02431 Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above;
additional appropriate formulations and routes of administration can be selected from among those described herein below.
[02441 Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradennal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local, hi addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached .to a reservoir, such as an Onunaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
[024511 In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
[02461 In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990);
Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) (0247] In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra;

Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);
Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Srnolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et at., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et at., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
(0248] Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
[0249] In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in viva to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector.
. _ and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see -U.S. Patent No. 4,980,286), or by direct injection, or by use of inicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. ,Natl. Acad. Sci.
USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
102501 The present invention also provides pharmaceutical compositions.
Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. hi a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
[02511 In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a = solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[02521 The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol, histidine, procaine, etc.

[02531 The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a Therapeutic protein can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
[02541 For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably!
mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
Diagnosis and Imaging =
[0255] Labeled antibodies and derivatives and analogs thereof that bind a Therapeutic protein (or fragment or variant thereof) (including albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein), can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of Therapeutic protein. The invention provides for the detection of aberrant expression of a Therapeutic protein, comprising (a) assaying the expression of the Therapeutic protein in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed Therapeutic protein expression level compared to the standard expression level is indicative of aberrant expression.
102561 The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the Therapeutic protein in cells or body fluid of an individual using one or more antibodies specific to the Therapeutic protein or albumin fusion proteins comprising at least a fragment of variant of an antibody specific to a Therapeutic protein, and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed Therapeutic protein gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
[0257] Antibodies of the invention or albumin fusion proteins comprising at least a fragment of variant of an antibody specific to a Therapeutic protein can be used to assay protein levels in a biological sample using classical imrnunohistological methods known to those of skill in the art (e.g., see Jalkanen et al., 1. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include inununoassays, such as the enzyme linked immunosothent assay (ELISA) and the radioirnmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (358), tritium (3H), indium (1121n), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
[0258] One facet of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a Therapeutic protein in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises:
a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the Therapeutic protein is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the therapeutic protein.

Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
[0259] It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mIc. The labeled antibody, antibody fragment, or albumin fusion protein comprising at least a fragment or variant of an antibody that binds a Therapeutic protein will then preferentially accumulate at the location of cells which contain the specific Therapeutic protein. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacolcinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
102601 Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours.
In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
102611 In an embodiment, monitoring of the disease or disorder. is earned out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
102621 Presence of the labeled molecule can be detected in the patient -using methods known in the art for in vivo scanning. These methods depend upon the type of label used.
Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
[0263] In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S.
Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument.
In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magietic resonance imaging (MRI). Antibodies that specifically detect the albumin fusion protein but not albumin or the therapeutic protein alone are a preferred embodiment. These can be used to detect the albumin fusion protein as described throughout the specification.
Kits 102641 The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically inummoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody-may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
102651 In another specific embodiment of the present invention, the kit is a diagnostic kit for use in 'screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit mayµinclude a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically inununoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
102661 In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. in this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
10267] In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody.
The detecting means of the kit may include a second, labeled monoclonal antibody.
Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
102681 In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention.
After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is deermined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
[0269] The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
102701 Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
Albumin Fusion Proteins [02711 The present invention relates generally to albumin fusion proteins and methods of treating, preventing, or ameliorating diseases or disorders. As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a Therapeutic protein (or fragment or variant thereof). An albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion (i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a Therapeutic protein is joined in-frame with a polynucleotide encoding all or a portion of albumin) or to one another. The Therapeutic protein and albumin protein, once part of the albumin fusion protein, may each be referred to as a "portion", "region" or "Moiety" of the albumin fusion protein.
102721 In a preferred embodiment, the invention provides an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 1 or Table 2.
Polynucleotides encoding these albumin fusion proteins are also encompassed by the invention.
[0273j . Preferred albumin fusion proteins of the invention, include, but are not limited to, albumin fusion proteins encoded by a nucleic acid molecule comprising, or alternatively consisting of, a polyriucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of a Therapeutic protein (or fragment or variant thereof); a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of a Therapeutic protein (or fragment or variant thereof) generated as described in Table 1, Table 2 or in the Examples; or a nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) joined in frame to at least one polynucleotide encoding at least one molecule of a Therapeutic protein (or fragment or variant thereof), further comprising, for example, one or more of the following elements: (1) a functional self-replicating vector (including but not limited to, a shuttle vector, an expression vector, an integration vector, and/or a replication system), (2) a region for initiation of transcription (e.g., a promoter region, such as for example, a regulatable or inducible promoter, a constitutive promoter), (3) a region for termination of transcription, (4) a leader sequence, and (5) a selectable marker.
102741 In one embodiment, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein (e.g., as described in Table 1) and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of; a biologically active and/or therapeutically active frag,nient of a Therapeutic protein and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of a Therapeutic protein and a serum albumin protein. In preferred embodiments, the serum albumin protein component of the albumin fusion protein is the mature portion of serum albumin.
[0275] In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein, and a biologically active and/or therapeutically active fragment of serum albumin. In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein and a biologically active and/or therapeutically active variant of serum albumin. In preferred embodiments, the Therapeutic protein portion of the albumin fusion protein is the mature portion of the Therapeutic protein.
[0276) . In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment or variant of a Therapeutic protein and a biologically active and/or therapeutically active fragment or variant of serum albumin. In preferred embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, the mature portion of a Therapeutic protein and the mature portion of serum albumin.
10277) Preferably, the albumin fusion protein comprises HA as the N-terminal portion, and a Therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising HA as the C-terminal portion, and a Therapeutic protein as the N-terminal portion may also be used.
[0278] In other embodiments, the albumin fusion protein has a Therapeutic protein fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the Therapeutic proteins fused at the N- and C- termini are the same Therapeutic proteins. In an alternative preferred embodiment, the Therapeutic proteins fused at the N- and C- termini are different Therapeutic proteins. In another preferred embodiment, the Therapeutic proteins fused at the N- and C- termini are different Therapeutic proteins which may be used to treat or prevent the same or a related disease, disorder, or condition (e.g. as listed in the "Preferred Indication Y" column of Table 1). In another preferred embodiment, the Therapeutic proteins fused at the N- and C- termini are different Therapeutic proteins which may be used to treat, ameliorate, or prevent diseases or disorders (e.g. as listed in the "Preferred indication Y"
column of Table 1) which are known in the art to commonly occur in patients simultaneously, concurrently, or consecutively, or which commonly occur in patients in association with one another.
102791 Exemplary fusion proteins of the invention containing multiple Therapeutic protein portions fused at the N- and C- termini of albumin include, but are not limited to, GCSF-HSA-EPO, EPO-HSA-GCSF, 1FNalpha-HSA-112, L2-FISA-IFNalpha, GCSF-HSA-112, 112-HSA-GCSF, 11.2-HSA-EPO, EPO-HSA-IL2, 1L3-HSA-EPO, EPO-HSA-11,3, GCSF-HSA-GMCSF, GMCSF-HSA-GCSF, 112-HSA-GMCSF, GMCSF-HSA-112, PTH-HSA-Calcitonin, Calcitonin-HSA-PTH, PTH-PTH-HSA-Calcitonin, Calcitoniri-HSA-PTH-PTH, PTH-Calcitonin-HSA-PTH, or PTH-HSA-Calcitonin-PTH.
(02801 Albumin fusion proteins of the invention encompass proteins containing one, two, three, four, or more molecules of a given Therapeutic protein X or variant thereof fused to the N- or C- terminus of an albumin fusion protein of the invention, and/or to the N- and/or C- terminus of albumin or variant thereof. Molecules of a given Therapeutic protein X or variants thereof may be in any number of orientations, including, but not limited to, a 'head to head' orientation (e.g., wherein the N-terminus of one molecule of a Therapeutic protein X is fused to the N-terminus of another molecule of the Therapeutic protein X), or a 'head to tail' orientation (e.g., wherein the C-terminus of one molecule of a Therapeutic protein X is fused to the N-terminus of another molecule of Therapeutic protein X).
(0281I In one embodiment, one, two, three, or more tandemly oriented Therapeutic protein X polypeptides (or fragments or variants thereof) are fused to the N-or C- terminus of an albumin fusion protein of the invention, and/or to the N- and/or C-terminus of albumin or variant thereof.
102821 In a specific embodiment, one, two, three, four, five, or more tandemly oriented molecules of PTH are fused to the N- or C-terminus of albumin or variant thereof.
For example, one, two, three, four, five, or more tandemly oriented molecules of PTH
(including, but not limited to, molecules of PTH comprising, or alternatively consisting of, amino acids I to 34) are fused to the N- or C-terminus of albumin or variant thereof.
Exemplary fusion proteins of the invention containing multiple protein portions of PTH, include, but are not limited to, PTH-PTH-HSA, HSA-PTH-PTH, PTH-PTH-PTH-HSA, HSA-PTH-PTH-PTH, PTH-PTH-PTH-PTH-HSA, or HSA-PTH-PTH-PTH-PTH.

102831 In another specific embodiment, one, two, three, four, five, or more tandemly oriented molecules of GLP- I are fused to the N- or C-terminus of albumin or variant thereof.
For example, one, two, three, four, five, or more tandemly oriented molecules of GLP-1 (including, but not limited to, molecules of GLP-1 comprising, or alternatively consisting of, amino acids 7 to 36, with residue 8 being mutated from an /Mani= to a (ilycine) (See for Example, the mutants disclosed in U.S. Patent No. 5,545,618, are fused to the N- or C-terminus of albumin or variant thereof.
Exemplary fusion proteins of the invention containing multiple protein portions of GLP-1, include, but are not limited to, GL1-GLP1-HSA, IISA-GLP1-GLP I, GLP1mutant-GLP1mutant-HSA, HSA-GLP1mutant-GLP1mutant, GLP I mutant-GLP1-HSA, HSA-GLP1mutant-GLP 1, GLP 1-GLP lmutant-HSA, or HSA-GLP1-GLP1mutant. Particularly preferred embodiments are GLP-1 tandem fusions such as construct 11) #3070 and the protein encoded by such construct.
102841 Albumin fusion proteins of the invention further encompass proteins containing one, two, three, four, or more molecules of a given Therapeutic protein X or vaunt thereof fused to the N- or C- terminus of an albumin fusion protein of the invention, and/or to the N- and/or C- terminus of albumin or variant thereof, wherein the molecules are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat.
No. 5,073,627. Albumin fusion proteins comprising multiple Therapeutic protein X polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology. Linkers are particularly important when fusing a small peptide to the large HSA molecule. The peptide itself can be a linker by fusing tandem copies of the peptide (see for example GLP-1) or other known linkers can be used.
Constructs that incorporate linkers are described in Table 2 or are apparent when examining SEQ JD NO:Y.
[0285] Further, albumin fusion proteins of the invention may also be produced by fusing a Therapeutic protein X or variants thereof to the N-terminal and/or C-terminal of albumin or variants thereof in such a way as to allow the formation of intramolecular and/or intermolecular multimeric forms. In one embodiment of the invention, albumin fusion proteins may be in monomeric or multimeric forms (i.e., dimers, trimers, tetra.mers and higher multimers). In a further embodiment of the invention, the Therapeutic protein portion of an albumin fusion protein may be in Monomeric form or multimeric form (i.e., dimers, trimers, tetramers and higher multimerS). In a specific embodiment, the Therapeutic protein portion =
=

of an albumin fusion protein is in multimeric form (i.e., dimers, trimers, tetramers and higher multimers), and the albumin protein portion is in monomeric form.
[02861 In addition to albumin fusion protein in which the albumin portion is fused N-terminal and/or C-terminal of the Therapeutic protein portion, albumin fusion proteins of the invention may also be produced by inserting the Therapeutic protein or peptide of interest (e.g., a Therapeutic protein X as disclosed in Table 1, or an antibody that binds a Therapeutic protein or a fragment or variant thereof) into an internal region of HA. For instance, within the protein sequence of the HA molecule a number of loops or turns exist between the end and beginning of a-helices, which are stabilized by disulphide bonds. The loops, as determined from the crystal structure of HA (PDB identifiers 1A06, 113J5, MICE, 1BMO, 1E7E to 1E71 and ILJOR) for the most part extend away from the body of the molecule.
These, loops are useful for the insertion, or internal fusion, of therapeutically active peptides, particularly those requiring a secondary structure to be functional, or Therapeutic proteins, to essentially generate an albumin molecule with specific biological activity.
[0287] Loops in human albumin structure into which peptides or polypeptides may be inserted to.generate albumin fusion proteins of the invention include: Va154-Asn61, Thr76-Asp89, A1a92-G1u100, G1nI70-A1a176, His 247 - G1u252, Glu 266 - G1u277, Giu His288, A1a362-G1u368, Lys439-Pro447, Va1462-Lys475, Thr478-Pro486, and Lys560-Thr566. In more preferred embodiments, peptides or polypeptides are inserted into the Va154-Asn61, G1n170-A1a176, and/or Lys560-Thr566 loops of mature human albumin (SEQ
ID NO:1038).
[02881 Peptides to be inserted may be derived from either phage display or synthetic peptide libraries screened for specific biological activity or from the active portions of a molecule with the desired function. Additionally, random peptide libraries may be generated within particular loops or by insertions of randomized peptides into particular loops of the HA molecule and in which all possible combinations of amino acids are represented.
[02891 Such library(s) could be generated on HA or domain fragments of HA
by one of the following methods:
102901 randomized mutation of amino acids within one or more peptide loops of HA
or HA domain fragments. Either one, more or all the residues within a loop could be mutated in this manner;
[02911 replacement of, or insertion into one or more loops of HA or HA
domain fragments (i.e., internal fusion) of a randomized peptide(s) of length X, (where X is an amino acid and n is the number of residues;
102921 N-, C- or N- and C- terminal peptide/protein fusions in addition to (a) and/or (b).
[02931 The HA or HA domain fragment may also be made multifunctional by grafting the peptides derived from different screens of different loops against different targets into the same HA or HA domain fragment.
(0294) In preferred embodiments, peptides inserted into a loop of human serum albumin are peptide fragments or peptide variants of the Therapeutic proteins disclosed in Table 1. More particularly, the invention encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids in length inserted into a loop of human serum albumin.
The invention also encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids fused to the N-terminus of human serum albumin. The invention also encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least% at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids fused to the C-terminus of human serum albumin. For example, short peptides described in Table 1 and 2 (e.g., Therapeutic Y) can be inserted into the albumin loops.
(0295) Generally, the albumin fusion proteins of the invention may have one HA-derived region and one Therapeutic protein-derived region. Multiple regions of each protein, however, may be used to make an albumin fusion protein of the invention. Similarly, more than one Therapeutic protein may be used to make an albumin fusion protein of the invention. For instance, a Therapeutic protein may be fused to both the N- and C-terminal ends of the HA. In such a configuration, the Therapeutic protein portions may be the same or different Therapeutic protein molecules. The structure of bifunctional albumin fusion proteins may be represented as: X-HA-Y or Y-HA-X.
(0296) For example, an anti-BLySTm scFv-HA-1FNa-2b fusion may be prepared to modulate the immune response to IFNa-2b by antiBLySTM scFv. An alternative is making a hi (or even multi) functional dose of HA-fusions e.g. HA-IFNa-213 fusion mixed with HA-anti-BLySTM scFv fusion or other HA-fusions in various ratio's depending on function, half-life etc.
[0297] Bi- or multi-functional albumin fusion proteins may also be prepared to target the Therapeutic protein portion of a fusion to a target organ or cell type via protein or peptide at the opposite terminus of HA.
[0298i As an alternative to the fusion of known therapeutic molecules, the peptides could be obtained by screening libraries constructed as fusions to the N-, C-or N- and C-termini of HA, or domain fragment of HA, of typically 6, 8, 12, 20 or 25 or X
(where X is an amino acid (as) and n equals the number of residues) randomized amino acids, and in which all possible combinations of amino acids were represented. A particular advantage of this approach is that the peptides may be selected in situ on the HA molecule and the properties of the peptide would therefore be as selected for rather than, potentially, modified as might be the case for a peptide derived by any other method then being attached to HA.
[02991 Additionally, the albumin fusion proteins of the invention may include a linker peptide between the fused portions to provide greater physical separation between the moieties and thus maximize the accessibility of the Therapeutic protein portion, for instance, . for binding to its cognate receptor. The linker. peptide may consist of amino acids such that it is flexible or more rigid.
[03001 The linker sequence may be cleavable by a protease or chemically to yield the growth hormone related moiety. Preferably, the protease is one which is produced naturally by the host, for example the S. cerevisiae protease kex2 or equivalent proteases.
[03011 Therefore, as described above, the albumin ftision proteins of the invention may have the following formula R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein RI
is at least one Therapeutic protein, peptide or polypeptide sequence, and not necessarily the same Therapeutic protein, L is a linker and R2 is a serum albumin sequence.
[03021 In preferred embodiments, Albumin fusion proteins of the invention comprising a Therapeutic protein have extended shelf life compared to the shelf life the same Therapeutic protein when not fused to albumin. Shelf-life typically refers to the time period over which the therapeutic activity of a Therapeutic protein in solution or in some other storage formulation, is stable without undue loss of therapeutic activity.
Many of the Therapeutic proteins are highly labile in their unfused state. As described below, the typical shelf-life of these Therapeutic proteins is markedly prolonged upon incorporation into the albumin fusion protein of the invention.
[03031 Albumin fusion proteins of the invention with "prolonged" or "extended"

shelf-life exhibit greater therapeutic activity relative to a standard that has been subjected to the same storage and handling conditions. The standard may be the unfused MI-length Therapeutic protein. When the Therapeutic protein portion of the albumin fusion protein is an analog, a variant, or is otherwise altered or does not include the complete sequence for that protein, the prolongation of therapeutic activity may alternatively be compared to the tmfused equivalent of that analog, variant, altered peptide or incomplete sequence. As an example, an albumin fusion protein of the invention may retain greater than about 100% of the therapeutic activity, or greater than about 105%, 110%, 120%, 130%, 150% or 200% of the therapeutic activity of a standard when subjected to the same storage and handling conditions as the standard when compared at a given time point.
103041 Shelf-life may also be assessed in terms of therapeutic activity remaining after storage, normalized to therapeutic activity when storage began. Albumin fusion proteins of the invention with prolonged or extended shelf-life as exhibited by prolonged or extended therapeutic activity may retain greater than about 50% of the therapeutic activity, about 60%, 70%, 80%, or 90% or more of the therapeutic activity of the equivalent unfused Therapeutic protein when subjected to the same conditions. For example, as discussed in Example 38, an albumin fusion protein of the invention comprising hGH fused to the full length HA sequence may retain about 80% or more of its original activity in solution for periods of up to 5 weeks or more under various temperature conditions.
Expression of Fusion Proteins [03051 The albumin fusion proteins of the invention may be produced as recombinant molecules by secretion from yeast, a microorganism such as a bacterium, or a human or animal cell line. Preferably, the polypeptide is secreted from the host cells.
[0306] A particular embodiment of the invention comprises a DNA construct encoding a signal sequence effective for directing secretion in yeast, particularly a yeast-derived signal sequence (especially one which is homologous to the yeast host), and the fused molecule of the first aspect of the invention, there being no yeast-derived pro sequence between the signal and the mature polypeptide.
03071 The Saecharotnyces cerevisiae invertase signal is a preferred example of a yeast-derived signal sequence.
[0308] Conjugates of the kind prepared by Poznansky et al., (EBBS Lett.
239:18 (1988)), in which separately-prepared polypeptides are joined by chemical cross-linking, are -nt contemplated.
103091 The present invention also includes a cell, preferably a yeast cell transformed to express an albumin fusion protein of the invention. In addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. If the polypeptide is secreted, the medium will contain the polypeptide, with the cells, or without the cells if they have been filtered or centrifuged away. Many expression systems are known and may be used, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevLsiae, Kluyveromyces lactis and Pichia pastoris, filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
[03101 Preferred yeast strains to be used in the production of albumin fusion proteins are D88, DXYI and EXP10. 1)88 Cleu2-3, leu2-122, can), pral, ubc4} is a derivative of parent strain A1122his+ (also known as DBI; see, e.g.., Sleep et aL
Biotechnology 8:42-46 (1990)). The strain contains a leu2 mutation which allows for auxotropie selection of 2 micron-based plasmids that contain the LEU2 gene. D88 also exhibits a derepression of PRB1 in glucose excess. The PRBI promoter is normally controlled by two checkpoints that monitor glucose levels and growth stage. The promoter is activated in wild type yeast upon glucose depletion and entry into stationary phase. Strain 1)88 exhibits the repression by glucose but maintains the induction upon entry into stationary phase. The PRA1 gene encodes a yeast vacuolar protease, YscA endoprotea.se A, that is localized in the ER. The UBC4 gene is in the ubiquitination pathway and is involved in targeting short lived and abnormal proteins for ubiquitin dependant degradation. Isolation of this ubc4 mutation was found to increase the copy number of an expression plasmid in the cell and cause an increased level of expression of a desired protein expressed from the plasmid (see, e.g., International Publication No. W099/00504).
(03111 DXY1, a derivative of 1388, has the following genotype: [leu2-3, leu2-122, can1 , pral, ubc4, ura3::yap3]. In addition to the mutations isolated in D88, this strain also has a knockout of the YAP3 protease. This protease causes cleavage of mostly di-basic residues (RR, RK, KR, KK) but can also promote cleavage at single basic residues in proteins. Isolation of this yap3 mutation resulted in higher levels of full length HSA
production (see, e.g., U.S. Patent No. 5,965,386 and Kerry-Williams et al., Yeast 14:161-169 (1998)).

. )312] BXPIO has the following genotype: ku2-3, ku2-122, caul, pm], ubc4, ura3, yap* URA3, lys2, hsp150::LYS2, pmt1::URA3. In addition to the mutations isolated in DXY1, this strain also has a knockout of the PMT1 gene and the HSP150 gene.
The PMT1 gene is a member of the evolutionarily conserved family of dolichyl-phosphate-D-mannose protein 0-mannosyltransferases (Pmts). The transmembrane topology of Pmtlp suggests that it is an integral membrane protein of the endoplasmic reticulum with a role in 0-linked glycosylation. This mutation serves to reduce/eliminate 0-linked glycosylation of HSA
fusions (see, e.g., International Publication No. W000/44772).
Studies revealed that the Hsp150 protein is inefficiently separated from rHA. by ion exchange chromatography. The mutation in the IISP150 gene removes a potential contaminant that has proven difficult to remove by standard purification techniques. See, e.g., U.S. Patent No. 5,783,423.
[03131 The desired protein is produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid. The yeasts are transformed with a coding sequence for the desired protein in any of the usual ways, for example electroporation. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.
[03141 Successfully transformed cells, Le., cells that contain a DNA
construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce the desired polypeptide. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975).1. MoL Biol.
98, 503 or Berent et al. (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.
[0315] Useful yeast plasmid vectors include pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
Plaamids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Ylps) and incorporate the yeast selectable markers 11IS3, 7RP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).
[0316] Preferred vectors for making albumin fusion proteins for expression in yeast include pPPC0005, pSeCHSA, pScisIHSA, and pC4:HSA which are described in detail in Example I. Figure 2 shows a map of the pPPC0005 plasmid that can be used as the base vector into which polynucleotides encoding Therapeutic proteins may be cloned to form HA-fusions. It contains a PRI31 S. cerevisiae promoter (PRB1p), a Fusion leader sequence (FL), DNA encoding HA (rHA) and an ADH.1 S. cerevisiae terminator sequence. The sequence of the fusion leader sequence consists of the first 19 amino acids of the signal peptide of human serum albumin (SEQ ID NO:1094) and the last five amino acids of the mating factor alpha 1 promoter (SLDKR, see EP-A-387 319).
[0317] The plasmids, pPPC0005, pScCHSA, pSeNHSA, and pC4:HSA were deposited on April 11, 2001 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209 and given accession numbers ATCC PTA-3278, PTA-3276, PTA-3279, and PTA-3277, respectively. Another vector useful for expressing an albumin fusion protein in yeast the pSAC35 vector which is described in Sleep et al., BioTechnology 8:42 (1990) 103181 Another yeast promoter that can be used to express the albumin fusion protein is the MET25 promoter. See, for example, Dominik Mumburg, Rolf Muller and Martin Funk. Nucleic Acids Research, 1994, Vol. 22, No. 25, pp. 5767-5768. The Met25 promoter is 383 bases long (bases ¨382 to ¨1) and the genes expressed by this promoter are also known as Met15, Met17, and YLR303W. A preferred embodiment uses the sequence below, where, at the 5' end of the sequence below, the Not 1 site used in the cloning is underlined and at the 3' end, the ATG start codon is underlined:
GCGGCCGCCGGATGCAAGGGTTCGAATCCUrt _____________________________________ AGCTCTCATTATTTTTTGCTTITT
CTCTTGAGGTCACATGATCGCAAAATGGCAAATGGCACGTGAAGC'TGTCGATATT
GGGGAACTGTGGTGGTTGGCAAATGACTAATTAAGTTAGTCAAGGCGCCATCCTC
ATGAAAACTGTGTAACATAATAACCGAAGTGTCGAAAAGGTGGCACCTTGTCCA
ATTGAACACGCTCGATGAAAAAAATAAGATATATATAAGGTTAAGTAAAGCGTC
TGTTAGAAAGGAAGYITTTCCTTTTTCTTGCTCTCTTGTCTITI _________________________ CATCTACTATTTC
CTTCGTGTAATACAGGGTCGTCAGATACATAGATACAATTCTATTACCCCCATCC
ATACAATO (SEQ ID NO: 2138) [0319] A
variety of methods have been developed to operably link DNA to vectors via complementary cohesive tennimi. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA
segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

103201 Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA
polymerase or E.
coli DNA polymerase I, enzymes that remove protruding, gamma-single-Stranded termini with their 3 5'-exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities.
[03211 The combination of these activities therefore generates blunt-ended DNA
segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA
segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA
segment.
103221 Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CT, USA.
[0323] A desirable way to modify the DNA in accordance with the invention, if, for example, HA variants are to be prepared, is to use the polymerase chain reaction as disclosed by Saiki et a (1988) Science 239, 487-491. In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
103241 Exemplary genera of yeast contemplated to be useful in the practice of the present invention as hosts for expressing the albumin fusion proteins are Pichia (Harisenula), Saccharornyces, Kluyveroinyees, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Deharomyces, Metschunikowia, Rhodosporidium, Leucosporidiutn, Botiyoascus, Sporidiobolus, Endontycopsis, and the like. Preferred genera are those selected from the gaup consisting of Saccharonzyces, Schizosaccharontycesõ
Kluyverotnyces, Pichia and Torulaspora. Examples of Saecharomyces spp. are S. cerePisiae, S. italieus and S. rouxii.
[03251 Examples of lazdyverotnyces spp. are K. fi-agilis, K lactis and K.
marxianus. A
suitable Torulaspora species is T delbrueckii. Examples of Pichia (Hansenula) spp. are?.

angusta (formerly H. polymorpha), P. anomala (formerly H. anomala) and P.
pastoris.
Methods for the transformation of S. cerevisiae are taught generally in EP 251 744, EP 258 067 and WO 90/01063.
[0326] Preferred exemplary species of Saccharotnyces include S.
cerevisiae, S.
italicus, S. diastaticus, and Zygosaccharomyces rouxii. Preferred exemplary species of Kluyveromyces include K. fragilis and K. locus. Preferred exemplary species of Hansenula include H. polytnorpha (now Pichia angusta), H. anomala (now Pichia anomala), and Pichia capsulata. Additional preferred exemplary species of Pichia include P.
pastorts. Preferred exemplary species of Aspergillus include A. niger and A. nidulans. Preferred exemplary species of Yarrowia include 1'. lipolytica. Many preferred yeast species are available from the ATCC. For example, the following preferred yeast species are available from the ATCC
and are useful in the expression of albumin fusion proteins: Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 yap3 mutant (ATCC Accession No. 4022731);
Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 hsp150 mutant (ATCC
Accession No. 4021266); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743pnztl mutant (ATCC .Accession No. 4023792); Saccharomyces cerevisiae Hansen, teleomorph (ATCC Accession Nos. 20626; 44773; 44774; and 62995); Saccharomyces diastaticus Andrews et Gilliland ex van der Walt, teleomorph (ATCC Accession No. 62987);
Kluyveromyces lactis (Dombrowski) van der Walt, teleomorph (ATCC Accession No.

76492); Pichia angusta (Teunisson et al.) Kurtzman, teleomorph deposited as Hansenula polymorpha de Marais et Maia, teleomorph (ATCC Accession No. 26012);
Aspergillus niger van Tieghem, anamorph (ATCC Accession No. 9029); Aspergillus niger van Tieghern, anamorph (ATCC Accession No. 16404); Aspergillus nidulans (Eidam) Winter, anamotph (ATCC Accession No. 48756); and Yarrowia lipolytica (Wickerhatn et al.) van der Walt et von Aix, teleomorph (ATCC Accession No. 201847).
[0327] Suitable promoters for S. cerevisiae include those associated with the PGKI
gene, GALL or GAL10 genes, CYCI, PH05, TRPI, ADHI, ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructoldnase, triose phosphate isomerase, phosphoglucose isomerase, glucokinase, alpha-mating factor pheromone, [a mating factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDI promoter, and hybrid promoters involving hybrids of parts of 5' regulatory regions with parts of 5' regulatory regions of other promoters or with upstream activation sites (e.g. the promoter of EP-A-258 067).

13281 Convenient regulatable promoters for use in Schizosaccharomyces pombe are the thiamine-repressible promoter from the unit gene as described by Maundrell (1990) J.
Biol. Chem. 265, 10857-10864 and the glucose repressible jbp1 gene promoter as described by Hoffman & Winston (1990) Genetics 124, 807-816.
(0329) Methods of transforming Pichia for expression of foreign genes are taught in, for example, Cregg et al. (1993), and various Phillips patents (e.g. US 4 857 467), and Pichia expression kits are commercially available from Invitrogen By, Leek, Netherlands, and Invitrogen Corp., San Diego, California.
Suitable promoters include AOX1 and AOX2. Gleeson et al. (1986) S. Gen. Micmbiol. 132, 3459-3465 include information on Hansenula vectors and transformation, suitable promoters being MOX1 and FMD1; whilst EP 361 991, Fleer et at. (1991) and other-publications from Rhone-Poulenc Rorer teach how to express foreign proteins in Kluyveromyces spp., a suitable promoter being PGKI.
[0330) The transcription termination signal is preferably the 3' flanking sequence of a eulcaryotic gene which contains proper signals for transcription termination and polyadenylation. Suitable 3' flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter.
Alternatively, they may be different in which case the termination signal of the S. cerevisiae ADHI gene is preferred.
(0331) The desired albumin fusion protein may be initially expressed with a secretion leader sequence, which may be any leader effective in the yeast chosen.
Leaders useful in yeast include any of the following:
a) the MPIF-1 signal sequence (e.g., amino acids 1-21 of GenBank Accession number A.AB511.34) MICVSVAALSCLMLVTALGSQA (SEQ ID NO:2132) b) the starmiocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:1054) c) the pre-pro region of the HSA signal sequence (e.g., MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:1176) d) the pre region of the HSA signal sequence (e.g., MICWVTFISLLFLFSSAYS, SEQ
ID NO:1177) or variants thereof, such as, for example, MKWVSF1SLLFLFSSAYS, (SEQ ID NO:1168) e) the invertase signal sequence (e.g., MLLQAFLFLLAGFAAKISA, SEQ ID
NO:1108) I) the yeast mating factor alpha signal sequence (e.g., MRFPSIFTAVLAFAASSALAAPVN _____ [Ti EDETAWAEAVIGYSDLEGDFDV
AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR, SEQ ID NO:1109 or MRFPSEFTAVLAEAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV
AVLPESNSTNNGLLFINTTLASIAAKEEGVSLDKR, SEQ ID NO:1109) g) K. lactis killer toxin leader sequence 11) a hybrid signal sequence (e.g., MKWVSFISLLFLFSSAYSRSLEKII, SEQ ID
NO:1110) i) an HSA/MFac-1 hybrid signal sequence (also known as HSA/kex2) (e.g., MICWVSFISELFLESSAYSRSLDKR, SEQ ID NO:1111) j) a K lactis killer/ MFa-1 fusion leader sequence (e.g., MNIFYIFLELLSEVQGSLDKR, SEQ ID NO:1169) k) the ImmunogIobulin Ig signal sequence (e.g., MGWSCIILFLVATATGVHS, SEQ
ID NO:1095) =
1) the Fibttlin B precursor signal sequence (e.g., MERAAPSRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO:1096) =
= in) the clusterin precursor signal sequence (e.g., MMKTILLFVGLLLTWESGQVIB, SEQ ID NO:1097) n) the insulin-like growth factor-binding protein 4 signal sequence (e.g., MLPLCLVAALLLAAGPGPSLG, SEQ ID NO:1098) o) vaiiants of the pre-pro-region of the USA signal sequence such as, for example, MICWVSFISLLFLESSAYSRG'VERR (SEQ ID No:1167), MICATTFISLITLFAGVLG (SEQ ID NO:1099), MKWVTFISLLELFSGVLG (SEQ ID NO:1100), MICWVTFISLLFLEGGVLG (SEQ ID NO:1101), Modified HSA leader ITSA 464 MICWVTFISLLFLFAGVSG (SEQ NO:2133);
Modified HSA leader HSA #66 MKWVTFISLLFLFGGVSG (SEQ ID NO:2134);
Modified USA (A14) leader ¨
MICWVTFISLLFLFAGVSG (SEQ 11.3 NO: 1102);
Modified USA (S14) leader (also known as modified USA #65) ¨
MKWVTFISLLFLFSGVSG (SEQ ID NO:1103), Modified HSA (014) leader ¨

MKWVTFISLLFLFGGVSG (SEQ ID NO:1104), or MKWVTFISLIILFGGVLGDLHKS (SEQ ID NO:1105) p) a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID
NO:1055) q) acid phosphatase (PH05) leader (e.g., MFKSVVYSILAASLANA SEQ ID
NO:2135) r) the pre-sequence of MFoz-1 s) the pre-sequence of 0 glucartase (BGL2) t) killer toxin leader u) the presequence of killer toxin v) k. lactis killer toxin prepro (29 amino acids; 16 amino acids of pre and 13 amino acids of pro) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO:2136) w) S. diastaticus glucoarnylase II secretion leader sequence x) S. carlsbergensis a-galactosidase (MEL1) secretion leader sequence y) Candida glucoarnylase leader sequence z) The hybrid leaders disclosed in EP-A-387 319 aa) the gp67 signal sequence (in conjunction with baculoviral expression systems) (e.g., amino acids 1-19 of GenBank Accession Number AAA72759) or bb)the natural leader of the therapeutic protein X;
Cc) S. cerevisiae invertase (SUC2) leader, as disclosed in JP 62-096086 (granted as 911036516); or dd) lnulinase MICLAYSLLLPLAGVSASVINYKR (SEQ ID NO:2137).
ea) A modified TA57 propeptide leader variant #1 ¨
MKLICTVRSAVLSSLFASQYLGQPIDDTESQTTSVNLMADDTESAFATQTN
SGGLDVVGL1SMAKR (SEQ ID NO:2128) ft) A modified TA57 propeptide leader variant #2 ¨
MKTICTVRSAVLSSLFASQVLGQPFDDTESQTTSVNLMADDTESAFATQTN
SGGLDVVGLISMAEEGEPKR (SEQ ID NO:2129) Additional Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins [03321 The present invention also relates to vectors containing a polyrmcleotide encoding an albumin fusion protein of the present invention, host cells, and the production of albumin fusion proteins by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
[0333] The polynucleotides encoding albumin fusion proteins of the invention may be joined to a vector containing a selectable marker for propagation in a host.
Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
[0334] The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, tip, phoA and sac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
[03351 As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
[03361 Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pICK223-3, pICK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, p0G44, pXT1 and pSG available from 'fratagene; and pSVI(3, pBPV, pMSG and pSVI, available from Pharmacia.
Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlbad, CA).
Other suitable vectors will be readily apparent to the skilled artisan.
[03371 In one embodiment, polynucleotides encoding an albumin fusion protein of the invention may be fused to signal sequences which will direct the localization of a protein of the invention to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of a protein of the invention from a prokaryotic or eukaryofic cell.
For example, in E. coli, one may wish to direct the expression of the protein to the periplasrnic space.
Examples of signal sequences or proteins (or fragments thereof) to which the albumin fusion proteins of the invention may be fused in order to direct the expression of the polypeptide to the petiplasmic space of bacteria include, but are not limited to, the pelB
signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit, and the signal sequence of alkaline phosphatase. Several vectors are commercially available for the construction of fusion proteins which will direct the localization of a protein, such as the pIVIAL series of vectors (particularly the pMAL-p series) available from New England Biolabs.
In a specific embodiment, polynucleotides albumin fusion proteins of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria. See, U.S. Patent Nos. 5,576,195 and 5,846,818.
[03381 Examples of signal peptides that may be fused to an albumin fusion protein of the invention in order to direct its secretion in mammalian cells include, but are not limited to:
a) the MN:F-1 signal sequence (e.g., amino acids 1-21 of GenBank Accession number AAB51134) MICVSVAALSCLMINTALGSQA (SEQ )1) NO:2132) b) the stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:1054) c) the pre-pro region of the HSA signal sequence (e.g., MKWVTFLSLLFLFSSAYSRGVFRR, SEQ ID NO:1176) d) the pre region of the HSA signal sequence (e.g., MKWVTFISLLFLFSSAYS, SEQ
13) NO:1177) or variants thereof, such as, for example, MKWVSFISLLFLFSSAYS, (SEQ ID NO:1168) e) the invertase signal sequence (e.g., MLLQAFLFLLAGFAAKISA, SEQ ID
Na1108) 0 the yeast mating factor alpha signal sequence (e.g., MRFPSIFTAVLAFAASSALAAPVNTT1tDETAQIPAEAVIGYSDLEGDFDVAVL
PFSNSTNNGLLF1NTTIASIAAKEEGVSLEKR, SEQ ID NO:1109 or MRFPSIFTAVLAFAA.SSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVL
PFSNSTNNGLLFINTTIASIAA10EEGVSLDKR, SEQ ID NO:1109) g) K. lactis killer toxin leader sequence h) a hybrid signal sequence (e.g., MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID
NO:1110) i) an HSA/MFa-1 hybrid signal sequence (also known as HSA/kex2) (e.g., MKWVSFISLLFLFSSAYSRSLD1CR, SEQ ID NO:! Ill) j) a K laths killer/ MFa-1 fusion leader sequence (e.g., MNIFYIFIILLSFVQGSLDICR, SEQ ID NO:1169) k) the Immunoglobulin 1g signal sequence (e.g., MGWSCI1LFLVATATGVHS, SEQ
ID NO:1095) I) the Fibulin B precursor signal sequence (e.g., MERAAPSRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO:1096) m) the clusterin precursor signal sequence (e.g., MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO:1097) -n) the insulin-like growth factor-binding protein 4 signal sequence (e.g., MLPLCLVAALLLAAGPGPSLG, SEQ ID NO:1098) o) variants of the pre-pro-region of the HSA signal sequence such as, for example, MICWVSFISLLFLFSSAYSRGVFRR (SEQ ID NO:1167), MKWVTFISLLFLFAGVLG (SEQ ID NO:1099), MKWVTFISLLFLFSGVLG (SEQ ID NO:1100), MKWVTF1SLLFLFGGVLG (SEQ ID NO:1101), Modified HSA leader HSA #64 MKWVTFISLLFLFAGVSG (SEQ ID NO:2133);
Modified HSA leader HSA #66 MKWVTFISLLFLFGGVSG (SEQ ID NO:2134);
Modified HSA (A14) leader ¨
MKWVTFISLLFLFAGVSG (SEQ ID NO: 1102);

Modified HSA (S14) leader (also known as modified HSA 065) ¨
MKWVTFISLLFLFSGVSG (SEQ ID NO:1103), Modified HSA (014) leader ¨
MKWVTFISLLFLEGGVSG (SEQ ID NO:1104), or MKWVTFISLLFLEGGVLGDLHKS (SEQ ID NO:1105) p) a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ED
NO:1055) q) acid phosphatase (PH05) leader (e.g., MFKSVVYSILAASLANA SEQ ID
NO:2135) r) the pre-sequence of iviEoz-1 s) the pre-sequence of 0 glucanase (EGL2) t) killer toxin leader u) the presequence of killer toxin = k. lactis killer toxin prepro (29 amino acids; 16 amino acids of pre and 13 amino acids of pro) MNIFYIELFLLSEVQGLEHTERRGSLDKR (SEQ ID NO:2136) w) S. diastaticus glucoarnylase fl secretion leader sequence x) S. carlsbergensis a-galactosidase (M.EL1) secretion leader sequence y) Candida glucaantylase leader sequence z) The hybrid leaders disclosed in EP-A-387 319 aa) the gp67 signal sequence (in conjunction with baculoviral expression systems) (e.g., amino acids 1-19 of GenBank Accession Number AAA72759) or bb) the natural leader of the therapeutic protein X;
cc) S. cerevisiae invertase (SUC2) leader, as disclosed in JP 62-096086 (granted as 911036516); or dd) Inulinase IVIKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:2137).
ee) A modified TA57 propeptide leader variant 01 ¨
MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVIsa,MATID _____________________ FESAFATQINSGG
LDVVGLISMAKR (SEQ ID NO:2128) ft) A modified TA57 propeptide leader variant 02 ¨
MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSG
GLDVVGLISMAEEGEPKR (SEQ ID NO:2129) -µ3391 Vectors which use glutamine synthase (GS) or DIIFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availability of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: W087/04462;
W086/05807; W089/01036; W089/10404; and W091/06657.
Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, NH).
Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnot Prog. 11:1(1995).
(03401 The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eulcaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
[03411 Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et at., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptidcs of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
[03421 In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence corresponding to a Therapeutic protein may be replaced with an albumin fusion protein corresponding to the Therapeutic protein), and/or to include genetic material (e.g., heterologous polynucleotide sequences such as for example, an albumin fusion protein of the invention corresponding to the Therapeutic protein may be included). The genetic material operably associated with the endogenous polynucleotide may activate, alter, and/or amplify endogenous polynucleotides.
103431 In addition, techniques known in the art may be used to operably associate heterologous polynucleotides (e.g., polynucleotides encoding an albumin protein, or a fragment or variant thereof) and/or heterologous control regions (e.g., promoter and/or enhancer) with endogenous polynucleotide sequences encoding a Therapeutic protein via homologous recombination. (see, e.g., US Patent Number 5,641,670, issued June 24, 1997;
International Publication Number WO 96/29411; International Publication Number WO
94/12650; Koller et at., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijistm et al., Nature 342:435-438 (1989)).
(0344) Albumin fusion proteins of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, hydrophobic charge interaction chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("1-FPLC") is employed for purification.
[03451 In preferred embodiments the albumin fusion proteins of the invention are purified using Anion Exchange Chromatography including, but not limited to, chromatography on Q-sepharost DEAE sepharosg: poros HQ, poros DEAR, Toyopearl Q, Toyopearl QA.E, Toyopearl DEAR, Resource/Source Q and DRAB, Fractogel Q and DEAR
columns.
[03461 In specific embodiments the albumin fusion proteins of the invention are purified using Cation Exchange Chromatography including, but not limited to, SP-sepharosem, CM sepharosTeA, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource/Source S and CM, Fractogel S and CM columns and their equivalents and comparables.
[03471 In specific embodiments the albumin fusion proteins of the invention are purified using Hydrophobic Interaction Chromatography including, but not limited to, Phenyl, Butyl, Methyl, Octyl, Hexyl-sepharosre poros Phenyl, Butyl, Methyl, Octyl, Hexyl, Toyopearl Phenyl, Butyl, Methyl, Octyl, Hexyl Resource/Source Phenyl, Butyl, Methyl, Octyl, Hexyl, Fractogel Phenyl, Butyl, Methyl, Octyl, Hexyl columns and their equivalents and comparables.
[03481 In specific embodiments the albumin fusion proteins of the invention are purified using Size Exclusion Chromatography including, but not limited to, sepharogSI00, 5200, 5300, superdex resin columns and their equivalents and comparables.
[03491 In specific embodiments the albumin fusion proteins of the invention are purified using Affinity Chromatography including, but not limited to, Mimetic Dye affinity, peptide affinity and antibody affinity columns that are_selective for either the HSA or the "fusion target" molecules.
[03501 In preferred embodiments albumin fusion proteins of the invention are purified using one or more Chromatography methods listed above. In other preferred embodiments, albumin fusion proteins of the invention are purified using one or more of the following Chromatography columns, Q sepharog FF column, SP Sepharoat FF column, Q
Sepharosr High Performance Column, Blue SepharoieFF column , Blue Column, Phenyl SepharoslIFF
column, DEAE SepharosTFF, or Methyl Column.
[03511 Additionally, albumin fusion proteins of the invention may be purified using the process described in PCT International Publication WO 00/44772.
One of skill in the art could easily modify the process described therein for use in the purification of albumin fusion proteins of the invention.
103521 Albumin fusion proteins of the present invention may be recovered from:
products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycoaylated. In addition, albumin fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
[0353] In one embodiment, the yeast Pichia pastoris is used to express albumin fusion proteins of the invention in a eulcaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using 02.
This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for 02. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (A0X1) is highly active. In the presence of methanol, alcohol oxidase produced from the A0X1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See Ellis, S.B., et al., MoL Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al., Yeast 5:167-77 (1989); Tschopp, J.F., et al., Nucl. Acids Res.
15:3859-76 (1987).
Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the A0X1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast gown in the presence of methanol.
[0354] In one example, the plasmid vector pPIC9K is used to express DNA
encoding an albumin fusion protein of the invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology,"
D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong A0X1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
[0355] Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, ' translation, secretion (if desired), and the like, including an in-frame AUG
as required.
[03561 In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide encoding an albumin fusion protein of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
[03571 In addition, albumin fusion proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et at., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosin.e, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglyeine, = cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L
(levorotary).
103581 The invention encompasses albumin fusion proteins of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, arnidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, Nal3H4; acetylation, fonnylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
(03591 Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or 0-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or 0-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The albumin fusion proteins may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
[0360) Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotiri and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodarnine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine (1211, 123/, 1251, 1311), carbon eaL.)¨, sulfur (35S), tritium (311), indium (1111n, 1121n, tumin, "5mIn), technetium (99Tc,99mTc), thallium (2 1Ti), gallium (68Ga, 67Ga), palladium (1 3Fd), molybdenum (98Mo), xenon (133Xe), fluorine (18F), 153Sm, InLu, 159Gd, loPm, 'La, t75yb, 166m, 90y, 47se, 1it B4--v 188Re,142Pr, I05Rh, and 97Ru.
[03611 In specific embodiments, albumin fusion proteins of the present invention or fragments or variants thereof are attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 1771x, 80Y, 166Ho, and 153Sm, to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is 111In.
In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is 8GY. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyc1ododecane-N,M,N1',Nm-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via linker molecule.
Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art - see, for example, DeNardo et al., Clin Cancer Res, 4(10):2483-90 (1998);
Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med.
Biol. 26(8):943-50 (1999).
[03621 As mentioned, the albumin fusion proteins of the invention may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, axnidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, dernethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation., and ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR
PROPERTIES, 2nd Ed.., T. E. Creighton, W. H. Freeman and Company, New York (1993);
POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et at., Meth.
Enzymol. 182:626-646 (1990); Rattan et at., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
103631 Albumin fusion proteins of the invention and ,antibodies that bind a Therapeutic protein or fragments or variants thereof can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gen= et al., Proc. Natl. Acad. Sci.
USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the .fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA"
tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
[03641 Further, an albumin fusion protein of the invention may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A
cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicirt, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, I -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromyein and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, camaustine (BSNU) and lomustine (CCNU), cyclothospharnide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), ble,omycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., viricristine and vinblastine).
103651 The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, 13-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM If (See, International Publication No. WO 97/34911), Pas Ligand (Takahashi et aL, mt. ImmurioL, 6:1567-1574(1994)), VEGI
(See, International Publication No. WO 99/23105), a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lympholcines, interleukin-1 ("IL-I"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Teclufitiues for conjugating such therapeutic moiety to proteins (e.g., albumin fusion proteins) are well known in the art.
103661 Albumin fusion proteins may also be attached to solid supports, which are particularly useful for immunoassays or purification of polypeptides that are bound by, that bind to, or associate with albumin fusion proteins of the invention. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
[03671 Albumin fusion proteins, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic. =

[03681 In embodiments where the albumin fusion protein of the invention comprises only the Vii domain of an antibody that binds a Therapeutic protein, it may be necessary and/or desirable to coexpress the fusion protein with the VL domain of the same antibody that binds a Therapeutic protein, such that the VH-albumin fusion protein and VL
protein will associate (either covalently or non-covalently) post-translationally.
[03691 In embodiments where the albumin fusion protein of the invention comprises only the VL domain of an antibody that hinds a Therapeutic protein, it may be necessary and/or desirable to coexpress the fusion protein with the VII domain of the same antibody that binds a Therapeutic protein, such that the VL-albumin fusion protein and VH protein will associate (either covalcntly or non-covalently) post-translationally.
[03701 Some Therapeutic antibodies are bisPecific antibodies, meaning the antibody that binds a Therapeutic protein is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. In order to create an albumin fusion protein corresponding to that Therapeutic protein, it is possible to create an albumin fusion protein which has an scFv fragment fused to both the N- and C- terminus of the albumin protein moiety. More particularly, the scFv fused to the N-terminus of albumin would correspond to one of the heavy/light (VHNL) pairs of the original antibody that binds a Therapeutic protein and the scFv fused to the C-terminus of albumin would correspond to the other heavy/light (VHNL) pair of the original antibody that binds a Therapeutic protein.
103711 Also provided by the invention are chemically modified derivatives of the albumin fusion proteins of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased irnmunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The albumin fusion proteins may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
[0372] The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about I kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic nrofile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a Therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
[03731 As noted above, the polyethylene glycol may have a branched structure.
Branched polyethylene glycols are described, for example, in U.S. Patent No.
5,643,575;
Morpurgo et al., App!. Biochern. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. /0:638-646 (1999).
[03141 The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on fimctional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), see also Malik et at,, Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
[0375] As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or -ysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
[0376) One may specifically desire proteins chemically modified at the N-terminus.
Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus .with a carbonyl group containing polymer is achieved.
[03771 As indicated above, pegylation of the albumin fusion proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the albumin fusion protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit.
Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. I. of Hematol. 68:1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95106058; and WO 98132466.
[03781 One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monxnethoxy polyethylene glycol (MPEG) using tresylchloride (CISO2CH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
[03791 Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Patent No. 5,612,460, discloses urethane linkers for connecting oolyetbylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1 ,1'-carbonyldi imidazo le, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466.
Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
[03801 The number of polyethylene glycol moieties attached to each albumin fusion protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 1345, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et at., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
[03811 The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
(0382) The presence and quantity of albumin fusion proteins of the invention may be determined using ELISA, a well known immunoassay known in the art. In one EIJSA
protocol that would be useful for detecting/quantifying albumin fusion proteins of the invention, comprises the steps of coating an ELISA plate with an anti-human serum albumin antibody, blocking the plate to prevent non-specific binding, washing the ELISA plate, adding a solution containing the albumin fusion protein of the invention (at one or more different concentrations), adding a secondary anti-Therapeutic protein specific antibody coupled to a detectable label (as described herein or otherwise known in the art), and detecting the presence of the secondary antibody. In an alternate version of this protocol, the ELBA plate might be coated with the anti-Therapeutic protein specific antibody and the labeled secondary reagent might be the anti-human albumin specific antibody.
Uses of the Polynudeotides 103831 Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
[03841 The polynucleotides of the present invention are useful to produce the albumin fusion proteins of the invention.. As described in more detail below, polynucleotides of the invention (encoding albumin fusion proteins) may be used in recombinant DNA
methods useful in genetic engineering to make cells, cell lines, or tissues that express the albumin fusion protein encoded by the polynucleotides encoding albumin fusion proteins of the invention.
(03851 Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner.
Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled "Gene Therapy", and Examples 63 and 64).
Uses of the Polypeptides 103861 Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
[03871 Albumin fusion proteins of the invention are useful to provide immunological probes for differential identification of the tissue(s) (e.g., immunohistochemistry assays such as, for example, ABC immunoperoxidase (Hsu et al., I. Histochem. Cytochem.
29:577-580 (1981)) or cell type(s) (e.g., immunocytochetnisty assays).
103881 Albumin fusion proteins can be used to assay levels of polypeptides in a biological sample using classical inununohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol.
105:3087-3096 (1987)). Other methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (MA). Suitable assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1311, 1251, 1231, 121v,tj , carbon (14C), sulfur (35S), tritium (3H), indium (115mln, 113mln, u21ñ 111In), and technetium (99TC, 99mTc), thallium2(1 010=-=1)= = , gallium (68Ga, 67Ga), palladium (1 3Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, i59Gd, 149pm, risyb, 1661./0, 90y, 4 7sc, I R 86- e, "Re, 142pr, 105,.. , 97 KnRu; luminescent labels, such as lurninol; and fluorescent labels, such as fluorescein and rhodatnine, and biotin.
10389) Albumin fusion proteins of the invention can also be detected in viva by imaging. Labels or markers for in viva imaging of protein include those detectable by X-radiography, nuclear magnetic resonance (11MR) or electron spin relaxtion (ESR). For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the albumin fusion protein by labeling of nutrients given to a cell line expressing the albumin fusion protein of the invention.
103901 An albumin fusion protein which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131 1 12- , 99mTc, (131L 1251, 1231, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (1159n, 113mln, 1121n, 1111n), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (680a, 67Ga), palladium (103pd), molybdenum ("Mo), xenon (133Xe), fluorine (18F, 153Sm, 177Lu, 59Gd, 149pm, 140La, 175 yb, 166E10, 90Y, 7SC, i86Re, MR;

r 1-- ni Rh, 97Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will nonnally range from about 5 to 20 millicuries of 99mTc. The labeled albumin fusion protein will then preferentially accumulate at locations in the body (e.g., organs, cells, extracellular spaces or matrices) where one or more receptors, ligands or substrates (corresponding to that of the Therapeutic protein used to make the albumin fusion protein of the invention) are located. Alternatively, in the case where the albumin fusion protein comprises at least a fragment or variant of a Therapeutic antibody, the labeled albumin fusion protein will then preferentially accumulate at the locations in the body (e.g_, organs, cells, extracellular spaces or matrices) where the polypeptidesiepitopes corresponding to those bound by the Therapeutic antibody (used to make the albumin fusion protein of the invention) arc located. In vivo tumor imaging is described in S.W. Burchiel et al., "Irtununopharmacokinetics of Radiolabeled Antibodies and Their Fragments"
(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A.
Rhodes, eds., Masson Publishing Inc. (1982)). The protocols described therein could easily be modified by one of skill in the art for use with the albumin fusion proteins of the invention.
103911 In one embodiment, the invention provides a method for the specific delivery of albumin fusion proteins of the invention to cells by administering albumin fusion proteins of the invention (e.g., polypeptides encoded by polynucleotides encoding albumin fusion proteins of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a Therapeutic protein into the targeted cell. , In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
[1:13921 In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering albumin fusion proteins of the invention in association with toxins or cytotoxic prodrags.
103931 By "toxin" is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, rich% abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. "Toxin" also includes a cytostatic or cytocidal agent, a therapeutic agent or a rad; -)active metal ion, e.g., alpha-emitters such as, for example, 2I3Bi, or other radioisotopes Itch as, for example, 103pd, I33xe, 131/7 , 68¨e ti "Co, 65Zn, "Sr, "P, "S, 90Y, 153Sm, 153Gd, 169¨

Yb 51Cr, "Mn, "Se, 113Sn, "Yttrium, "Tin, I"Rhenium, 166Holmium, and '"Rhenium;
luminescent labels, such as luminoi; and fluorescent labels, such as fluorescein and rhodamine, and biotin. In a specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 90Y. In another specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope "in, In a further specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 131I.
103941 Techniques known in the art may be applied to label polypeptides of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Patent Nos. 5,756,065; 5,714,631;
5,696,239; 5,652,361;
5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560;
and 5,808,003).
103951 The albumin fusion proteins of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans.
Such disorders include, but are not limited to, those described herein under the section heading "Biological Activities," below.
[03961 Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression level of a certain polypeptide in cells or body fluid of an individual using an albumin fusion protein of the invention; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

0397] Moreover, albumin fusion proteins of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin 13, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
103981 In particular, albumin fusion proteins comprising of at least a fragment or variant of a Therapeutic antibody can also be used to treat disease (as described supra, and elsewhere herein). For example, administration of an albumin fusion protein comprising of at least a fragment or variant of a Therapeutic antibody can bind, and/or neutralize the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein specifically binds, and/or reduce overproduction of the polypeptide to which the Therapeutic antibody used. to make the albumin fusion protein specifically binds.
Similarly, administration of an albumin fusion protein comprising of at least a fragment or variant of a Therapeutic antibody can activate the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein specifically binds, by binding to the polypeptide bound to a membrane (receptor).
10399] At the very least, the albumin fusion proteins of the invention of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
Albumin fusion proteins of the invention can also be used to raise antibodies, which in turn may be used to measure protein expression of the Therapeutic protein, albumin protein, and/or the albumin fusion protein of the invention from a recombinant cell, as a way of assessing transformation of the host cell, or in a biological sample. Moreover, the albumin fusion proteins of the present invention can be used to test the biological activities described herein.

Diagnostic Assays 104001 The compounds of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those described for each Therapeutic protein in the corresponding row of Table 1 and herein under the section headings "Immune Activity,"
"Blood Related Disorders," "Hyperproliferative Disorders," "Renal Disorders,"
"Cardiovascular Disorders," "Respiratory Disorders," "Anti-Angiogenesis Activity,"
"Diseases at the Cellular Level," "Wound Healing and Epithelial Cell Proliferation," "Neural Activity and Neurological Diseases," "Endocrine Disorders," "Reproductive System Disorders," "Infectious Disease," "Regeneration," and/or "Gastrointestinal Disorders," infra.
104011 For a number of disorders, substantially altered (increased or decreased) levels of gene expression can be detected in tissues, cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" gene expression level, that is, the expression level in tissues or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression level of the gene encoding a polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression level(s) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
[0402] The present invention is also useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome 10403] By "assaying the expression level of the gene encoding a polypeptide" is intended qualitatively or quantitatively measuring or estimating the level of a particular polypeptide (e.g. a polypeptide corresponding to a Therapeutic protein disclosed in Table 1) or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA
level) or relatively (e.g., by comparing to the polypeptide level or mRNA
level in a second biological sample). Preferably, the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
104041 By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention (including portions thereof) or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the full length or fragments thereof of a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
[0405i Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidiniurn-thioeyanate-phenol-chlorofonn method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, S1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
[0406] The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides that bind to, are bound by, or associate with albumin fusion proteins of the invention, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polnieptides. Thus, for instance, a diagnostic assay in accordance with the invention for detecting abnormal expression of polypeptides that bind to, are bound by, or associate with albumin fusion proteins compared to normal control tissue samples may be used to detect the presence of tumors.
Assay techniques that can be used to determine levels of a polypeptide that bind to, are bound by, or associate with albumin fusion proteins of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA
assays.
Assaying polypeptide levels in a biological sample can occur using any art-known method.
[0407) Assaying polypeptide levels in a biological sample can occur using a variety of techniques. For example, polypeptide expression in tissues can be studied with classical immtmohistological methods (Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jallcanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987)). Other methods useful for detecting -olypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (EL1SA) and the radioimmunoassay (R1A). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (1121n), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
[04081 The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of interest (such as, for example, cancer). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.
104091 For example, albumin fusion proteins may be used to quantitatively or qualitatively detect the presence of polypeptides that bind to, are bound by, or associate with albumin fusion proteins of the present invention. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled albumin fusion protein coupled with light microscopic, flow cytometric, or fluorimetric detection.
104101 In a preferred embodiment, albumin fusion proteins comprising at least a fragment or variant of an antibody that specifically binds at least a Therapeutic protein disclosed herein (e.g, the Therapeutic proteins disclosed in Table 1) or otherwise !mown in the art may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluoreseence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
104111 The albumin fusion proteins of the present invention may, additionally, be employed histologically, as in immunofluorescence, irrnnunoelectron microscopy or non-immunological assays, for in situ detection of polypeptides that bind to, are bound by, or associate with an albumin fusion protein of the present invention. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention. The albumin fusion proteins are preferably applied by overlaying the labeled albumin fusion proteins onto a biological sample, Through the use of such a procedure, it is possible to determine not only the presence of the polypeptides that bind to, are bound by, or associate with albumin fusion proteins, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
104121 Immunoassays and non-immunoassays that detect polypeptides that bind to, are bound by, or associate with albumin fusion proteins will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
104131 The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled albumin fusion protein of the invention. The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled.
The amount of bound label on solid support may then be detected by conventional means.
[0414] By "solid phase support or carrier" is intended any support capable of binding a polypeptide (e.g., an albumin fusion protein, or polypeptide that binds, is bound by, or associates with an albumin fusion protein of the invention.) Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The , support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polypeptide. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc.
Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
104151 The binding activity of a given lot of albumin fusion protein may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
[04161 In addition to assaying polypeptide levels in a biological sample obtained from an individual, polypeptide can also be detected in vivo by imaging. For example, in one embodiment of the invention, albumin fusion proteins of the invention are used to image diseased or neoplastic cells.
104171 Labels or markers for in vivo imaging of albumin fusion proteins of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly hannfiil to the subject. Suitable markers for NivIR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the albumin fusion protein by labeling of nutrients of a cell line (or bacterial or yeast strain) engineered.
[04181 Additionally, albumin fusion proteins of the invention whose presence can be detected, can be administered. For example, albumin fusion proteins of the invention labeled with a radio-opaque or other appropriate compound can be administered and visualized in viva, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.
[04191 A polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 1311, 1121n, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 991nTc.
The labeled albumin fusion protein will then preferentially accumulate at the locations in the body which contain a polypeptide or other substance that binds to, is bound by or associates with an albumin fusion protein of the present invention. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W.
Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
104201 One of the ways in which an albumin fusion protein of the present invention an be detectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (ETA) (VoIler, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J.E., Meth. Enzymot 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC
Press, Boca Raton, FL,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound to the antibody will react with an appropriate substrate, preferably a clvomogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometrie, fluorimetric or by visual means. Reporter enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidaSe, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
[04211 Albumin fusion proteins may also be radiolabelled and used in any of a variety of other immunoassays. For example, by radioactively labeling the albumin fusion proteins, it is possible to the use the albumin fusion proteins in a radioirmnunoassay (R1A) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986).
The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
[04221 Additionally, chelator molecules, are known in the art and can be used to label the Albumin fusion proteins. Chelator molecules may be attached Albumin fusion proteins of the invention to facilitate labeling said protein with metal ions including radionuclides or fluorescent labels. For example, see Subramanian, R. and Meares, C.F., "Bifunctional Chelating Agents for Radiometal-labeled monoclonal Antibodies," in Cancer Imaging with Radiolabeled Antibodies (13. M. (3oldenberg, Ed.) Kluwer Academic Publications, Boston;
SO., H., "Targeted delivery of radiolabeled imaging and therapeutic agents;
bifunctional radiopharmaceuticals." Grit. Rev. Ther, Drug Carrier Syn. /6:209-244 (1999);
Srivastava S.C. and Mease R.C., "Progress in research on ligands, nuclides and techniques for labeling monoclonal antibodies." Int. Rad. Appl. Instrum. B 18:589-603 (1991); and Liu, S. and Edwards, DS., "Bifunctional chelators for therapeutic lanthanide radiophannaceuticals."
Bioconjug. Chem. 12:7-34 (2001). Any chelator which can be covalently bound to said Albumin fusion proteins may be used according to the present invention. The chelator may further comprise a linker moiety that connects the chelating moiety to the Albumin fusion protein.
[04231 In one embodiment, the Albumin fusion protein of the invention are attached to an acyclic chelator such as diethylene triamine-N,N,N,N",N"-pentaacetic acid (DPTA), analogues of DPTA, and derivatives of DPTA. As non-limiting examples, the chelator may be 2-(p-isothiocyanatobenzy1)-6- methyldiethylenetriaminepentaacetic acid (1B4M-DPTA, also known as MX-DTPA), 2-methyl-6-(rho-nitrobenzy1)-1,4,7- triazaheptane-N,NX,N",N"-pentaacetic acid (nitro-1B4M-DTPA or nitro-MX-DTPA); 2-(p-isothiocyanatobenzy1)-cyclohexyldiethylenetriarnirtepentaacetic acid (CHX-DTPA), or N42-amino-3-(rho-nitrophenyl)propy1}-trans-cyclohexane-1,2-diamine-NX,N"-pentaacetic acid (nitro-CHX-A-DTPA).
[0424] In another embodiment, the Albumin fusion protein of the invention are attached to an acyclic terpyridine chelator such as 6,6"-bisaN,N,N",N"-tetra(carboxymethypaminojrnethyl]-4'-(3-amino-4-methoxyphenyl)-2,2%6',2 "-terpyridine (TMT-amine).
[0425] In specific embodiments, the macrocyclic chelator which is attached to the the Albumin fusion protein of the invention is 1,4,7,10-tetraazacyclododecane-N,N',N",N"-tetraacetic acid (DOTA). In other specific embodiments, the DOTA is attached to the the Albumin fusion protein of the invention via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art -see, for example, DeNardo et al., Clin. Cancer Res. 4(10)2483-90, 1998; Peterson et al., Bioconjug.
Chem. 10(4):553-7, 1999; and Zimmerman et al., Nucl. Med. Biol, 26(4943-50, 1999.
In addition, U.S. Patents 5,652,361 and 5,756,065, which disclose chelating agents that may be conjugated to antibodies, and methods for making and using them.
Though U.S. Patents 5,652,361 and 5,756,065 focus on conjugating chelating agents to antibodies, one skilled in the art could readily adapt the method disclosed therein in order to conjugate chelating agents to other polypeptides.

104261 Bifunctional chelators based on macrocyclic ligands in which conjugation is via an activated arm, or functional group, attached to the carbon backbone of the ligand can be employed as described by M. Moi et al., J. Amer. Chem. Soc. 49:2639 (1989) (2-p-ni trobenzyl-1,4,7,10-tetraazacycl odod ecane-N,N,N" ,1\r-tetraacetic acid);
S. V. Deshpande et al., J. NucL Med. 3/:473 (1990); G. Ruser et al., Bioconj. Chem. 1:345 (1990);
C. J. Broan et a1., J. C. S. Chem. Comm. 23:1739 (1990); and C. J. Anderson et al., J. NucL
Med. 36:850 (1995).
104271 In one embodiment, a macrocyclic chelator, such as polyazamacrocyclic chelators, optionally containing one or more carboxy, amino, hydroxamate, phosphonate, or phosphate groups, are attached to the Albumin fusion protein of the invention.
In another embodiment, the chelator is a chelator selected from the group consisting of DOTA, analogues of DOTA, and derivatives of DOTA.
104281 In one embodiment, suitable chelator molecules that may be attached to the the Albumin fusion protein of the invention include DOXA (1-oxa-4,7,10-triazacyclododecanetriacetic acid), NOTA (1,4,7-trinacyclononanetriacetic acid), TETA
(1,4,8,11-tetraazacyclotetradecanetetraacetic acid), and THT (4'-(3-amino-4-methoxy-pheny1)-6,6"-bis(N',N-dicarboxymethyl-N-methylhydra zino)-2,2%6',2"-terpyridine), and analogs and derivatives thereof. See, e.g., Ohmono et al., J. Med. Chem. 35:

(1992); Kung et al., J. Arucl. Med. 25: 326-332 (1984); Jurisson et al., Chem.
Rev. 93:1137-1156 (1993); and U.S. Patent No. 5,367,080. Other suitable chelators include chelating agents disclosed in U.S. Patent Nos. 4,647,447; 4,687,659; 4,885,363; EP-A-71564;
W089/00557; and EP-A-232751.
[04291 In another embodiment, suitable inacrocyclic carboxylic acid chelators which can be used in the present invention include 1,4,7,10-tetraancyclododecane-N,AP,N",/r-tetraacetic acid (DOTA); 1,4,8,12-tetraazacyclopentadecane-N,N,N",W"-tetraacetic acid (15N4); 1,4,7-triazacyclononane-N,M,N"-triacetic acid (9N3); 1,5,9-tiazacyclododecane-N,N,N"-triacetic acid (12N3); and 6-bromoacetamido-benzy1-1,4,8,11-tetraazacyclotetradecane- N,1%11,N",/%1"1-tetraacetic acid (BAT).
104301 A preferred chelator that can be attached to the Albumin Fusion protein of the invention is a-(5-isothiocyanato- 2-methoxypheny1)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, which is also known as Me0-DOTA-NCS. A salt or ester of a-(5-isothiocyanato- 2-methoxypheny1)- 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid may also be used.

104311 Albumin fusion proteins of the invention to which chelators such as those decribed are covalently attached may be labeled (via the coordination site of the chelator) with radionuclides that are suitable for therapeutic, diagnostic, or both therapeutic and diagnostic purposes. Examples of appropriate metals include Ag, At, Au, Bi, Cu, Ga, Ho, In, Lu, Pb, Pd, Pm, Pr, Rb, Re, Rh, Sc, Sr, Tc, TI, Y, and Yb. Examples of the radionuclide used for diagnostic purposes are Fe, Gd, 111In, 67Ga, or "Ga. In another embodiment, the radionuclide used for diagnostic purposes is 111In, or 67Ga. Examples of the radionuclide used for therapeutic purposes are 1661.1o, 165Dy, 90y, 115m 52r,-=e, or 72Ga. In one embodiment, the radionuclide used for diagnostic purposes is I66Ho or 90Y. Examples of the radionuclides used for both therapeutic and diagnostic purposes include 153Sm, 177Lu, I59Gd, I75Yb, or 47Sc.
In one embodiment, the radionuclide is I 53Sm, mLu, 175Yb, or I59Gd.
[04321 Preferred metal radionuclides include 90Y, 99mTc, 47Sc, 67Ga, "Cr, 177mSn, CU, 167TM, 97Ru, I88Re, 177Lu, I99Au, 47Sc, 67Ga, 5ICr, 177MSn, 67C11, 167TM, 95R11, 188Re, 1771.11, 199A11, 203Pb and "ICC.
04331 In a particular embodiment, Albumin fusion proteins of the invention to which chelators are covalently attached may be labeled with a metal ion selected from the group consisting of 90Y, "In, 177Lu, 'Ho, 215- =, and 225Ac.
[04341 Moreover, y-emitting radionuclides, such as 99mTc, 111In, 67, and 169Th have been approved or under investigation for diagnostic imaging, while 0-emitters, such as 6701, "IA& I86Re, and 90Y are useful for the applications in tumor therapy Also other useful radionuclides include y-emitters, such as 9 mTc, 1111n, 67Ga, and 169Yb, and 13-emitters, such as 67Cu, inAg,'86Re,lag --Re and 90Y. as well as other radionuclides ofinterest such as 211At, 212Bi, 'Lu, 86Rb , foRh, 153sm, 198Au, 149pm, 85sr, 142pr, 214pb, 109r.,a 166--Ito, 208T1, and Sc.44 Albumin fusion proteins of the invention to which chelators are covalently attached may be labeled with the radionuclides described above.
[0435J In another embodiment, Albumin fusion proteins of the invention to which chelators are covalently attached may be labeled with paramagnetic metal ions including ions of transition and lanthanide metal, such as metals having atomic numbers of 21-29, 42, 43, 44, or 57-71, in particular ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. The paramagnetic metals used in compositions for magnetic resonance imaging include the elements having atomic numbers of 22 to 29, 42, 44 and 58-70.

[04361 In another embodiment, Albumin fusion proteins of the invention to which chelators are covalently attached may be labeled with fluorescent metal ions including lanthanides, in particular La, Ce, Pr, Nd, Pm, Sm, Eu (e.g., 152Eu), Gd, Tb, Dy, Ho, Er, Tna, Yb, and Lu.
104371 In another embodiment, Albumin fusion proteins of the invention to which chelators are covalently attached may be labeled with heavy metal-containing reporters may include atoms of Mo, Bi, Si, and W.
[04381 It is also possible to label the albumin fusion proteins with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phyc,oerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
104391 The albumin fusion protein can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[04401 The albumin fusion proteins can also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiltuninescent-tagged albumin fusion protein is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium estgr, imidazole, acridinium salt and oxalate ester.
[04411 Likewise, a bioluminescent compound may be used to label albumin fusion proteins of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
Transgenic Organisms [04421 Transgenic organisms that express the albumin fusion proteins of the invention are also included in the invention. Transgenic organisms are genetically modified organisms into which recombinant, exogenous or cloned genetic material has been transferred. Such genetic material is often referred to as a transgene. The nucleic acid sequence of the transgene may include one or more transcriptional regulatory sequences and other nucleic acid sequences such as introns, that may be necessary for optimal expression and secretion of the encoded protein. The transgene may be designed to direct the expression of the encoded protein in a manner that facilitates its recovery from the organism or from a product produced by the organism, e.g. from the milk, blood, urine, eggs, hair or seeds of the organism. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal. The transgene may be integrated either at a locus of a gcnome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.
[04431 The term "germ cell line transgenic organism" refers to a transgenic organism in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic organism to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic organisms. The alteration or genetic information may be foreign to the species of organism to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
[0444) A transgenic organism may be a transgenic animal or a transgenic plant.
Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307;
Mullins et al. (1993) Hypertension 22(4):630-633; Brenin et a/. (1997) Surg.
Oncol. 6(2)99-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1997)). The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307.
[0445i A number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Patent No.
4,736,866); express simian SV40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon gulatory factor 1 (IRF-1) (U.S. Patent No. 5,731,490); exhibit dopaminergic dysfunction (U.S, Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent No.
5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Patent No. 5,602,307);
possess a bovine growth hormone gene (Clutter et al. (1996) Genetics 143(4):1753-1760); or, are capable of generating a fully human antibody response (McCarthy (1997) The Lancet 349(9049):405).
104461 While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al. (1997) Mol. Reprod. Dev. 46(4):515-526;
Houdebine (1995) Reprod. Nuir. Dev. 35(6):609-617; Petters (1994) Reprod.
Fertil. Dev.
6(5):643-645; Schnieke et al. (1997) Science 278(5346):2130-2133; and Amoah (1997) Y.
Animal Science 75(2):578-585).
[04471 To direct the secretion of the transgene-encoded protein of the invention into the milk of transgenic mammals, it may be put under the control of a promoter that is preferentially activated in mammary epithelial cells. Promoters that control the genes encoding milk proteins are preferred, for example the promoter for casein, beta lactoglobulin, whey acid protein, or lactalbumin (see, e.g., DiTullio (1.992) BioTechnology 10:74-77; Clark et al. (1989) BioTechnology 7:487-492; Gorton et al. (1987) BioTechnology 5:1183-1187;
and Soulier et al. (1992) FEBS Letts. 297:13). The transgenic mammals of choice would produce large volumes of milk and have long lactating periods, for example goats, cows, camels or sheep.
104481 An albumin fusion protein of the invention can also be expressed in a transgenic plant, e.g. a plant in which the DNA transgene is inserted into the nuclear or plastidic genome. Plant transformation procedures used to introduce foreign nucleic acids into plant cells or protoplasts are known in the art. See, in general, Methods in Enzymology Vol. 153 ("Recombinant DNA Part D") 1987, Wu and Grossman Eds., Academic Press and European Patent Application EP 693554. Methods for generation of genetically engineered plants are further described in US Patent No. 5,283,184, US Patent No. 5, 482,852, and European Patent Application EP 693 554.

Pharmaceutical or Therapeutic compositions [04491 The albumin fusion proteins of the invention or formulations thereof may be administered by any conventional method including parenteral (e.g.
subcutaneous or intramuscular) injection or intravenous infusion. The treatment may consist of a single dose or a plurality of doses over a period of time.
[04501 While it is possible for an albumin fusion protein of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the albumin fusion protein and not deleterious to the recipients thereof.
Typically, the carriers will be water or saline which will be sterile and pyrogen free.
Albumin fusion proteins of the invention are particularly well suited to formulation in aqueous carriers such as sterile pyrogen free water, saline or other isotonic solutions because of their extended shelf-life in solution. For instance, pharmaceutical compositions of the invention may be formulated well in advance in aqueous form, for instance, weeks or months or longer time periods before being dispensed.
104511 For example, formulations containing the albumin fusion protein may be prepared taking into account the extended shelf-life of the albumin fusion protein in aqueous formulations. As discussed above, the shelf-life of many of these Therapeutic proteins are markedly increased or prolonged after fusion to HA.
104521 In instances where aerosol administration is appropriate, the albumin fusion proteins of the invention can be formulated as aerosols using standard procedures. The term "aerosol" includes any gas-borne suspended phase of an albumin fusion protein of the instant invention which is capable of being inhaled into the bronchioles or nasal passages.
Specifically, aerosol includes a gas-borne suspension of droplets of an albumin fusion protein of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition of a compound of the instant invention suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Harwood (19 87); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburri eta!,. (1992) Pharmacol. Taxied. Methods 27:143-159.
104531 The formulations of the invention are also typically non-immunogenic, in part, because of the use of the components of the albumin fusion protein being derived from the proper species. For instance, for human use, both the Therapeutic protein and albumin portions of the albumin fusion protein will typically be human. In some cases, wherein either component is non human-derived, that component may be humanized by substitution of key amino acids so that specific epitopes appear to the human immune system to be human in nature rather than foreign.
[04541 The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the albumin fusion protein with the carrier that constitutes one or more accessory ingredients. hi general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
104551 Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation appropriate for the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampules, vials or syringes, and may be stored in a freeze-dried (I yophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders. Dosage formulations may contain the Therapeutic protein portion at a lower molar concentration or lower dosage compared to the non-fused standard formulation for the Therapeutic protein given the extended serum half-life exhibited by many of the albumin fusion proteins of the invention.
[04561 As an example, when an albumin fusion protein of the invention comprises one of the proteins listed in the "Therapeutic Protein:X" column of Table 1 as one or more of the Therapeutic protein regions, the dosage form can be calculated on the basis of the potency of the albumin fusion protein relative to the potency of hGH, while taking into account the prolonged serum half-life and shelf-life of the albumin fusion proteins compared to that of native hGH. Growth hormone is typically administered at 0.3 to 30.0 RI/kg/week, for example 0.9 to 12.0 IU/kg/week, given in three or seven divided doses for a year or more. In an albumin fusion protein consisting of full length HA fused to full length GH, an equivalent dose in terms of units would represent a greater weight of agent but the dosage frequency can be reduced, for example to twice a week, once a week or less.
[04571 Formulations or compositions of the invention may be packaged together with, or included in a kit with, instructions or a package insert referring to the extended shelf-life of the albumin fusion protein component. For instance, such instructions or package inserts may address recommended storage conditions, such as time, temperature and light, taking into account the extended or prolonged shelf-life of the albumin fusion proteins of the invention.
Such instructions or package inserts may also address the particular advantages of the albumin fusion proteins of the inventions, such as the ease of storage for formulations that may require use in the field, outside of controlled hospital, clinic or office conditions. As described above, formulations of the invention may be in aqueous form and may be stored under less than ideal circumstances without significant loss of therapeutic activity.
[0458] Albumin fusion pmteins of the invention can also be included in nutraceuticals. For instance, certain albumin fusion proteins of the invention may be administered in natural products, including milk or milk product obtained from a transgenic mammal which expresses albumin fusion protein. Such compositions can also include plant or plant products obtained from a transgenic plant which expresses the albumin fusion protein. The albumin fusion protein can also be provided in powder or tablet form, with or without other known additives, carriers, fillers and diluents. Nutraceuticals are described in Scott Hegenhart, Food Product Design, Dec. 1993.
(0459J The invention also provides methods of treatment and/or prevention of diseases or disorders (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of an albumin fusion protein of the invention or a polynucleotide encoding an albumin fusion protein of the invention ("albumin fusion polynucleotide") in a pharmaceutically acceptable carrier.
[0460] The albumin fusion protein and/or polynucleotide will be formulated and dosed in a fashion consistent with good Medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the albumin fusion protein and/or polynucleotide alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners.
The "effective amount" for purposes herein is thus determined by such considerations.
E04611 As a general proposition, the total pharmaceutically effective amount of the albumin fusion protein administered parenterally per dose will be in the range of about lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
If given continuously, the albumin fusion protein is typically administered at a dose rate of about 1 ug/lcg/hour to about 50 ug/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
[0462] Albumin fusion proteins and/or polynucleotides can be are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray.
"Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intraarticular injection and infusion.
[0463] Albumin fusion proteins and/or polynucleotides of the invention are also suitably administered by sustained-release systems. Examples of sustained-release albumin fusion proteins and/or polynucleotides are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray.
"Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intraarticular injection and infusion.
Additional examples of sustained-release albumin fusion proteins and/or polynucleotides include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
[0464] Sustained-release matrices include polylactides (U.S. Pat. No.
3,773,919, EP
58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl methacrylate) (Langer et al., J.
Bioined. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D- (+3-hydroxybutyric acid (EP 133,988).
[0465] Sustained-release albumin fusion proteins and/or polynucleotides also include liposomally entrapped albumin fusion proteins and/or polynucleotides of the invention (see generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposonzes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317 -327 and 353-365 (1989)). Liposomes containing the albumin fusion protein and/or polynucleotide are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc.
Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad.
Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;
Japanese Pat. App!. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.
Ordinarily, the liposornes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.
[0466] In yet an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).
[0467] Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
[0468] For parenteral administration, in one embodiment, the albumin fusion protein and/or polynucleotide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.
[0469] Generally, the formulations are prepared by contacting the albumin fusion protein and/or polynucleotide uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation.
Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
[0470] The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypcptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaceharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, maltose, or dextrins; chelating agents such as EDTA;
sugar alcohols such as rnannitol or sorbitol; counterions such as sodium;
and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
[04711 The albumin fusion protein is typically formulated in such vehicles at a concentration of about 0.1 mg/m1 to 100 mg/ml, preferably 1-10 mg/ml, at a pH
of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.
[04721 Any pharmaceutical used for therapeutic administration can be sterile.
Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Albumin fusion proteins and/or polynucleotides generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
[94731 Albumin fusion proteins and/or polynucleotides ordinarily will be stored in unit, or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered( 1% (w/v) aqueous albumin fusion protein a' polynucleotide solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized albumin fusion protein and/or polynucleotide using bacteriostatic Water-for-Injection.
[04741 In a specific and preferred embodiment, the Albumin fusion protein formulations comprises 0.01 M sodium phosphate, 0.15 rriM sodium chloride, 0.16 micromole sodium octanoate/milligram of fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2. In another specific and preferred embodiment, the Albumin fusion protein formulations consists 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromole sodium octanoate/milligam of fusion protein; 15 micrograms/milliliter polysorbate 80, pH 7.2. The pH and buffer are chosen to match physiological conditions and the salt is added as a tonicifier. Sodium octanoate has been chosen due to its reported ability to increase the thermal stability of the protein in solution. Finally, polysorbate has been added as a generic surfactant, which lowers the surface tension of the solution and lowers non-specific adsorption of the albumin fusion protein to the container closure system.
[04751 The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the albumin fusion proteins and/or polynucleotides of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the albumin fusion proteins and/or polynucleotides may be employed in conjunction with other therapeutic compounds.
104761 The albumin fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, alum, alum plus deoxycholate (IrnimmoAg), MTP-P13 (Biocine Corp.), (Genentech, Inc.), BCG (e.g., THERACYS IVIPL
and nonviable preparations of Corynebacterium parvum. In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with alum. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRLI005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the albumin fusion proteins and/or ,polynucleotides of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanusidiptheria, hepatitis A, hepatitis B, Haemaphilus influenzae 13, whooping cough, pneumonia, influenza, Lyme's Disease, rotavinis, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis.
Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed by the second.

104771 The albumin fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with other therapeutic agents. Albumin fusion protein and/or polynucleotide agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include but not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional imrnunotherapeutic agents, and/or therapeutic treatments described below.
Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed by the second.
(04781 In one embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an anticoagulant.
Anticoagulants that may be administered with the compositions of the invention include, but are not limited to, heparin, low molecular weight heparin, warfarin sodium (e.g., COUMADINO), dicumarol, 4-hydroxycoumarin, anisindione (e.g., M1RADONrm), acenocoumarol (e.g.,, nicounialone, SINTIIRDIVIETK), indan-1,3-dione, phenprocoumon (e.g., MARCUMARTm), ethyl biscoumacetate (e.g., TR,OMEXANn), and aspirin. In a specific embodiment, compositions of the invention are administered in combination with heparin and/or warfarin.
In another specific embodiment, compositions of the invention are administered in combination with warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin and aspirin. In another specific embodiment, compositions of the invention are administered in combination with heparin. In another specific embodiment, compositions of the invention are administered in combination with heparin and aspirin.
[04791 In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with thrombolytic drugs.
Thrombolytic drugs that may be administered with the compositions of the invention include, but are not limited to, plasminogen, lys-plasminogen, alpha2-antiplasmin, streptokinae (e.g., KAB1K1NASETN9, antirespla.ce (e.g., EMINASE7m), tissue plasminogen activator (t-PA, altevase, ACTIVASErm), urokinase (e.g., ABBOIONASErm), saumplase, (Prourokinase, single chain urokinase), and aminocaproic acid (e.g., AMICARTm). In a specific embodiment, compositions of the invention are administered in combination with tissue plasminogen activator and aspirin.
[04801 In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with antiplatelet drugs.
Antiplatelet drugs that may be administered with the compositions of the invention include, but are not limited to, aspirin, dipyridamole (e.g., PERSANTINEnt), and ticlopidine (e.g., TICLIDTm).
[04811 In specific embodiments, the use of anti-coagulants, thrombolytic and/or antiplatelet drugs in combination with albumin fusion proteins and/or polynucleotides of the invention is contemplated for the prevention, diagnosis, and/or treatment of thrombosis, arterial thrombosis, venous thrombosis, thrombo embolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischcmic attack, unstable angina. In specific embodiments, the use of anticoagulants, thrombolytic drugs and/or antiplatelet drags in combination with albumin fusion proteins and/or polynucleotides of the invention is contemplated for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke. in patients with atrial fibrillation including nomteumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the therapeutics of the invention, alone or in .combination with antiplatelet, anticoagulant, and/or thrombolytic drugs, include, but are not limited to, the prevention of occlusions in extracorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
[04821 In certain embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTis), and/or protease inhibitors (Pis). NRITs that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, RETROVIaTu (zidovudine/AZT), VIDEXT4 (didanosineiddl), HLVIIYTM
(zalcitabine/ddC), ZERITrm (stavudine/d4T), EPIVIRTM (larnivudine/3TC), and COMBIVIRT'A
(zidovudine/lamivudine). NNRTIs that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, VIRAMUNETm (nev-irapine), RESCRIPTORTm (delavirdine), and SUSTIVATm (efavirenz).
Protease inhibitors that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, (indinavir), NORVIRTM (ritonavir), INVIRASErm (saquinavir), and VIRACEPTT"
(nelfinavir).
In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with albumin fusion proteins and/or polynucleotides of the invention to treat AIDS and/or to prevent or treat HIV infection.
(0483) Additional NRTIs include LODENOSENErm (F-ddA; an acid-stable adenosine NRTI; Triangle/Abbott; COVIRACILTM (emtricitabine/FTC; structurally related to lamivudine (3TC) but with 3- to 10-fold greater activity in vitro;
Triangle/Abbott); dOTC
(3CH-10652, also structurally related to lamivudine but retains activity against a substantial proportion of lamivudine-resistant isolates; Biochem Pharma); Adefovir (refined approval for anti-HIV therapy by FDA; Gilead Sciences); PREVEONO (Adefovir Dipivoxil, the active' prodrug of adefovir, its active form is PMEA-pp); TENOFOVIRT" (bis-POC PMPA, a PIVIPA prodrug; Gilead); DAPD/DXG (active metabolite of DAPD;
Triangle/Abbott); D-D4FC (related to 3TC, with activity against AZT/3TC-resistant virus);
GW420867X (Glaxo Wellcome); ZIAGENTm (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3'azido-2',3'-dideoxyuridine; WO 99/66936); and S-acy1-2-thioethyl (SAlh)-bearing prodrug forms of 13-L-FD4C and 13-L-FddC (WO 98/17281).
104841 Additional NNRTIs include COACTINONT" (Emivirine/MKC-442, potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINET" (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation;
Agouron);
PNU-142721 (has 20- to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N
mutation;
DuPont); GW-420867X (has 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOL1DE A (naturally occurring agent from the latex tree; active against viruses containing either or both the Y181C and K1 03N mutations);
and Propolis (WO 99/49830).
[0485] Additional protease inhibitors include LOPINAVIRTM (ABT378/r;
Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIRTN
(PNU-140690,, a non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myers Squibb);
L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea compound; Avid &
DuPont);
AG-1776 (a peptidomimetic with in vitro activity against protease inhibitor-resistant viruses;
Agouron); VX-175/GW-433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); and AGENPRASETM (amprenavir; Glaxo Wellcome Inc.).
[0486) Additional antiretroviral agents include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain which binds to gp41 in its resting state and prevents -transformation to the fusogenic state; Trimeris) and 1-1249 (a second-generation fusion inhibitor; Trimeris).
(04871 Additional antiretroviral agents include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140;

antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCRS/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Also included are _ CCR2B, CCR3, and CCR6 antagonists. Chemoldne recpetor agonists such as RANTES, SDF-1, MW-la, Mr?-113, etc., may also inhibit fusion.
[04881 Additional antiretroviral agents include integrase inhibitors.
Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIRT (AR
177, an oligonucleotide that probably acts at cell surface rather than being a true integrase inhibitor;
Arondex); and naphthols such as those disclosed in WO 98/50347.
(04891 Additional antiretroviral agents include hydrOxyurea-like compunds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOXTM (Molecules for Health); inosine rnonophosphate dehydrogenase (IMPDH) inhibitors sucha as VX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolate mofetil; Roche).
[04901 Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral genome nuclear translocation such as arylene bis(methylketone) compounds;
inhibitors of HIV entry such as AOP-RANTES, NNY-RANTES, RANTES-1gG fusion protein, soluble complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100;

nucleocapsid zinc finger inhibitors such as dithiane compounds; targets of HIV
Tat and Rev;
and pharmacoenhancers such as ABT-378.
[0491] Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP- la, M1P-113, SDF-la, EL-2, PROLEUKINTM
(aldesleukin/L2-7001;
Chiron), 1L-4, IL-10, IL-12, and 11-13; interferons such as IFN-alpha2a, EFN-alpha2b, or IFN-beta; antagonists of TNFs, NFKB, GM-CSF, M-CSF, and 11-10; agents that modulate immune activation such as cyclosporin and prcdnisone; vaccines such as RemuneTu ([ITV
Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments, bivalent (I3/E) =
recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-competent irnmunogens, and Gag, Pol, Nef, and Tat vaccines;
gene-based therapies such as genetic suppressor elements (GSEs; WO 98/54366), and intrakines (genetically modified CC chemokines targetted to the ER to block surface expression of newly synthesized CCR5 (Yang et aL, PNAS 94:11567-72 (1997);
Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PAS, PA9, PAIO, PAH, PA12, and PA14, the -anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-521), 257-1), 268-13 and 50.1, anti-Tat antibodies, anti-TNF-a antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor agonists and antagonists such as TCDD, 3,3',4,4',5-pentachlorobiphenyl; 3,3',4,4'-tetrachlorobiphenyl, and a-naphthoflavone (WO 98/30213); and antioxidants such as y-L-glutamyl-L-cysteine ethyl ester (y-GCE; WO
99/56764).
[0492] In a further embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an antiviral agent.
Antiviral agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, remantidine, maxamine, or thymalfasin. Specifically, interferon albumin fusion protein can be administered in combination with any of these agents. Moreover, interferon alpha albumin fusion protein can also be admistered with any of these agents, and preferably, interferon alpha 2a or 2b albumin fusion protein can be administered with any of these agents. Furthermore, interferon beta albumin fusion protein can also be adiriistered with any of these agents.
Additionally, any of the EFN hybrids albumin fusion proteins can be administered in combination with any of these agents.
[0493] In a most preferred embodiment, interferon albumin fusion protein is administered in combination with ribavirin. In a further preferred embodiment, interferon alpha albumin fusion protein is administered in combination with ribavirin. In a further preferred embodiment, interferon alpha 2a albumin fusion protein is administered in combination with ribavirin. In a further preferred embodiment, interferon alpha 2b albumin fusion protein is administered in combination with ribavirin. In a further preferred embodiment, interferon beta albumin fusion protein is administered in combination with ribavirin. In a further preferred embodiment, hybrid interferon albumin fusion protein is administered in combination with ribavirin.
[0494) In other embodiments, albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLET", DAPSONE1", PENTAMIDINEn', ATOVAQUONET'A, ISONIAZIDT", RIFAMPINT'A, PYRAZINAMIDET", ETHAMI3UTOLT", RIFABUTINT", CLARIT'HROMYCINT", AZITHROMYCINT", GANCICLOVIRT", FOSCARNETT", CIDOFOVIRTM, FLUCONAZOLET", ITRACONAZOLET", KETOCONAZOLET", ACYCLOVIRTM, FAMCICOLVIRT", PYRIMETHAMINET", ' LEUCOVORINT", NEUPOGENTM (filgrastim/G-CSF), and LEUKINETM
(sargramostim/GM-CSF). In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLET", DAPSONETM, PENTAMIDINET"4, and/or ATOVAQUONETm' to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with IS ONLAZIDT", RIFAMPINT", PYRAZINAMIDET", and/or ETHAMBUTOLTu to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with RIFABLTTINT", CLARITHROMYCINT", and/or AZITHROMYCINT" to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with GANCICLOVIRTm, FOSCARNETTm, and/or CIDOFOVIRTm to prophylactically treat or prevent an opportunistic cytomegalovirus infection, in another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with FLUCONAZOLETM, ITRACONAZOLETm, and/or KETOCONAZOLET" to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with ACYCLOVIRT" and/or FAMCICOLVIRT" to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type 11 infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with PYRIMETHAMINET" and/or LEUCOVORINTM to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with LEUCOVORINT" and/or NEUPOGENTM to prophylactically treat or prevent an opportunistic bacterial infection.
104951 In a further embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindarnycin, chloramphenicol, cephalosporins, ciprofioxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sulfonamide, etracyclines, trimethoprim, trimethoprim-sulfamethoxvole, and vancomycin.
[0496] In other embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with immunestirnulants.
Immunostimulants that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, levarnisole (e.g., ERGAMISOL114), isoprinosine (e.g. NOSIPLEN74), interferons (e.g. interferon alpha), and interleulcins (e.g., IL-2).
[04971 In other embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with inununosuppressive agents.
Irnmunosuppressive agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells. Other immunosuppressive agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, lefiunomide, mizoribine (BREDIN1Nrm), brequinar, deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT 3 (muromonab-CD3), SANDIMMUNETu, NEORALTM, SANGDYATM (cyclosporine), PROGRAR4 (FK506, tacrolimus), CELLCEPTIll (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURANTm (azathioprine), glucocorticosteroids, adrenocortical steroids such as DELTASONErm (prednisone) and HYDELTRASOLTm (prednisolone), FOLEXrm and 1VIEXATETm (methotrxate), OXSORALEN-ULTRATm (methoxsalen) and RAPAMUNE'rm (sirolimus). In a specific embodiment, inununosuppre.ssants may be used to prevent rejection of organ or bone marrow transplantation.
[04981 In an additional embodiment, albumin fusion proteins and/or polynueleotides of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but not limited to, GAMMART", IVEEGAWTM, SANDOGLOBULINT", GAMMAGARD S/DTM, ATGAIVITm (antithyrnocyte glubulin), and GAMIMUNETH. In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
104991 In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered alone or as part of a combination therapy, either in vivo to patients or in vitro to cells, for the treament of cancer. In a specific embodiment, the albumin fusion proteins, particularly IL-2-albumin fusions, are administered repeatedly during passive ininumotherapy for cancer, such as adoptive cell transfer therapy for metastatic melanoma as described in Dudley et al. (Science Express, 19 September 2002).
[0500] In certain embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered alone or in combination with an anti-inflammatory agent.

Anti-inflammatory agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, corticosteroids (e.g.
betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs (e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamio acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicarn, tiapmfenic acid, and tolmetin.), as well as antihistamines, aminomylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosyhnethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazar, benzydamine, bucolome, difenpirarnide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.
[0501] In an additional embodiment, the compositions of the invention are administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that may be administered with the compositions of the invention include, but are not limited to, Artgiostatin (Entremed, Rockville, MD), Troponin-1 (Boston Life Sciences, Boston, MA), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter "d group" transition metals.
[0502] Lighter "d group" transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
[0503] Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
105041 Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
105051 A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include, but are not limited to, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, (1991)); Sulphated Polysaccharide Peptidoglycan Complex (SP- PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staumsporine;
modulators of matrix, metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propy1-5-(4-pyridiny1)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin;
Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloft et al., J. Rio. Chem. 267:17321-17326, (1992));
Chymostatin (Tornkinson et al., Biochem J. 286:475-480, (1992));
Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, (1990)); Gold Sodium Thiomalate (''GST"; Matsubara and Ziff, J. Clin. Invest.
79:1440-1446, (1987)); antic,ollagenase-scrum; alpha2-antiplasmin (Holmes et al., J.
Biol. Chem.
262(4):1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit disodiurn (N-(2)-carboxypheny1-4- chloroanthronilie acid disodiuna or "CCA"; (Takeuchi et al., Agents Actions 36:312-316, (1992)); and metalloproteinase inhibitors such as BB94.
[05061 Additional anti-angiogenic factors that may also be utilized within the context of the present invention include Thalidomide, (Celgene, Warren, NJ);
Angiostatic steroid;
AGM-1470 (H. Brem and J. Follanan J Pediatr. Surg. 28:445-51(1993)); an integrin alpha v beta 3 antagonist (C. Storgard et at., J Clin, Invest. 103:47-54 (1999));
carboxynarninolinidazole; Carboxyamidotriazole (CAT) (National Cancer Institute, Bethesda, MID); Conbretastatin A-4 (CA4P) (OXiGENE, Boston, MA); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, PA); TNP-470, (Tap Pharmaceuticals, Deerfield, IL);
ZD-0101 AstraZenera (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555);
CGP-41251 (PKC 412); CM101; Dexrazox ane (ICR F187); DMXAA; Endo statin;
Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (Somatostatin);
Panretin; Penacillarnine; Photopoint; P1-88; Prinomastat (AG-3340) Purlytin;
Suradista (FCE26644); Tamoxi fen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and 5-Fluorouracil.
[05071 Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the compositons of the invention include, but are not limited to, AG-3340 (Agouron, La Jolla, CA), BAY-12-9566 (Bayer, West Haven, CT), BMS-275291 (Bristol Myers Squibb, Princeton, NJ), CGS-27032A (Novartis, East Hanover, NJ), Marimastat (British Biotech, Oxford, UK), and Metastat (Aetema, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the compositons of the invention include, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, CA/Medimmune, Gaithersburg, MD). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the compositons of the invention include, but are not limited to, Angiozyme (Ribozyrne, Boulder, CO), Anti-VEGF antibody (Genentech, S. San Francisco, CA), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. San Francisco, CA), SU-5416 (Sugen/ Pharmacia Upjohn, Bridgewater, NJ), and SU-6668 (Sugen). Other anti-angiogenie agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the compositons of the invention include, but are not limited to, IM-862 (Cytran, Kirkland, WA), Interferon-alpha, IL-12 (Roche, Nutley, Ni), and Pentosan polysulfate (Georgetown University, Washington, DC).
105081 In particular embodiments, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of an autoimmune disease, such as for example, an autoimmune disease described herein.
[05091 In a particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of arthritis. In a more particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.
105101 In another embodiment, the polynucleotides encoding a polypeptide of the present invention are administered in combination with an angiogenic protein, or polynucleotides encoding an angiogenic protein. Examples of angiogenic proteins that may be administered with the compositions ,of the invention include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
[05111 In additional embodiments, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered .with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to allcylating agents such as nitrogen mustards (for example, Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines and methylmelamines (for example, Ifexamethylmelamine and Thiotepa), alkyl sulfonates (for example, Busulfan), nitrosoureas (for example, Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), and treptozocin (streptozotocin)), triazenes (for example, Dacarbazine (DTIC;
dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for example, Methotrexate (amethopterin)), pyrimidine analogs (for example, Fluorouacil (5-fluorouracil;
5-FU), Floxuridine (fluorodeoxyuridine; FudR), and Cytarabine (cytosine arabinoside)), purine analogs and related inhibitors (for example, Mercaptopurine (6-mercaptopurine;
6-MP), Thioguanine (5-thioguanine; TG), and Pentostatin (2'-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB, vinblastine sulfate)) and Vincristine (vincristine sulfate)), epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics (for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin; rubidomycin), Doxontbicin, Bleomycin, Plicamycin (mithramycin), and Mitomycin (mitomycin C), enzymes (for example, L-Asparaginase), biological response modifiers (for example, Interferon-alpha and interferon-alpha-2b), platinum coordination compounds (for example, Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone), substituted ureas (for example, Hydroxyurea), rnethylhydrazine derivatives (for example, Procarbazine (N-methylhydrazine;
MEH), adrenocorticosteroids (for example, Prednisone), progestins (for example, Hydroxyprogesterone caproatc, Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol acetate), estrogens (for example, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate, Estradiol, and Ethinyl estradiol), antiestrogens (for example, Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone), antiandrogens (for example, Flutamide), gonadotropin-releasing horomone analogs (for example, Leuprolide), other hormones and hormone analogs (for example, methyltestosterone, estranaustine, estramustine phosphate sodium, chlorotrianisene, and testolactone), and others (for example, dicarbazine, glutamic acid, and mitotane).
[0512] In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: infliximab (also known as RemicadeTM Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflun.omide (also known as AravaTm from Hoechst Marion Roussel), KineretTm (an IL-1 Receptor antagonist also }mown as Analcinra from Amgen, Inc) [05131 In a specific embodiment, compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. In one embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with tositumomab. In a further embodiment, compositions of the invention are administered with tositurnomab and CHOP, or tositumomab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.
105141 In another specific embodiment, the compositions of the invention are administered in combination ZevalinTm. In a further embodiment, compositions of the invention are administered with ZevalinT" and CHOP, or ZevalinTM and any combination of one or more of the components of CHOP, particularly cyclophosphamide andior prednisone.
ZevalinTm may be associated with one or more radisotopes. Particularly preferred isotopes are 9 Y and "
[05151 In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with cytokines. Cytokines that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, IL2, ru, I1L4, IL5, 1L6, IL7, IL10, 1L12, 1L13, 1L15, anti-CD40, CD4OL, IFN-gamma and TNF-alpha. In another embodiment, albumin fusion proteins and/or polynucleotides of the invention may be administered with any interleukin, including, but not limited to, 11..- 1 alpha, IL- I beta, IL-2, IL-3, 1L-4, IL-5, IL-6, )1-7, IL-8, IL-9, IL-10, 1L-11, 114-12, IL-13, 1L-14, IL-17, IL-18, IL-19, IL-20, and IL-21.
10516i In one embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, soluble forms of TNF-alpha, lyrnphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD3OL, CD4OL, 4-1BBL, DcP3, OX4OL, 'DM-gamma (International Publication No WO 96/14328), AIM-I
(International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO

98/07880), OPG, and neutrokine-alpha (International Publication No. WO
98/18921, 0X40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO
97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), 'FRANK, TR9 (International Publication No. WO
98/56892),TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.

S17) In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with angiogenic proteins.
Angiogenic proteins that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110;
Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317;
Placental Growth Factor (PIGF), as disclosed in International Publication Number WO
92/06194; Placental Growth Factor-2 (P1GF-2), as disclosed in Hauser et at., Growth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515;
Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO
98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601.
[0518] In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
105191 In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostin3, LEUKINEThi, PROKINETm), granulocyte colony stimulating factor (G-CSF) (filuastina, NEUPOGENTm), macrophage colony stimulating factor (M-CSF, CSP-1) erythropoietin (epoetin alfa, =

EPOGENTM, PROCRIrrm), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colony stimulating factor, P1XY321 (a GMCSF/IL-3 fusion protein), interleukins, especially any one or more of 1L-1 through 1L-12, interferon-gamma, or thrombopoietin.
[05201 In certain embodiments, albumin fusion proteins and/or polynucleotides of the present invention are administered in combination with adrenergic blockers, such as, for example, acebutolol, atenolol, betaxolol, bisoprolol, carteAol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol.
[0521] In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an antiarrhythmic drug (e.g., adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine, moricizine, phenytoin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocainide, and verapamil).
105221 In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with diuretic agents, such as carbonic anhydrase-inhibiting agents (e.g., acetazolamide, dichlorphenarnide, and methazolamide), osmotic diuretics (e.g., glycerin, isosorbide, mannitol, and. urea),, diuretics that inhibit Nat symport (e.g., furosernide, bumetanide, azosernide, piretanide, tripamide, ethacrynic acid, muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g., bendroflurnethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide, trichormethiazide, chlorthalidone, indapamide, metolazone, and quinethazone), potassium sparing diuretics (e.g., amiloride and triarnterene), and mineralcorticoid 're¨c-eptor antagonists (e.g., spironolactone, canrenone, and potassium camenoate).
[05231 In one embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with treatments for endocrine and/or hormone imbalance disorders. Treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, 1271, radioactive isotopes of iodine such as 131I and 1231; recombinant growth hormone, such as HLIMATROPETm (recombinant somatropin); growth hormone analogs such as PROTROPIW" (somatrerri); dopamine agonists such as PARLODELTm (bromocriptine); somatostatin analogs such as SANDOSTAT1Nn4 (octreotide);
gonadotropin preparations such as PREGNYLTm, A.P.L.TM and PROFASITm (chorionic gonadotropin (CG)), PERGONALTM (menotropins), and METRODENTrm (urofollitropin (uFSH)); synthetic human gonadotropin releasing hormone preparations such as FACTRELT" and LUTREPULSET"

(gonadorelin hydrochloride); synthetic gonadotropin agonists such as LUPRONT"
(leuprolide acetate), SIJPPRELINTM (histrelin acetate), SYNARELT" (nafarelin acetate), and ZOLA.DEXTM (goserelin acetate); synthetic preparations of thyrotropin-releasing hormone such as RELEFACT TRIPA and THYPlNONETm (protirelin); recombinant human TSH
such as THYROGENTm; synthetic preparations of the sodium salts of the natural isomers of thyroid hormones such as L-T4T", SYNTHROIDTm and LEVOTHROIDTm (levothyroxine sodium), L-T3Tm, CYTOMELim and TRIOSTATTm (liothyroine sodium), and THYROLARTm (liotrix);

antitbyroid compounds such as 6-n-propylthiouracil (propylthiouracil), I -methyl-2-mercaptoimidazole and TAPAZOLETm (methimazole), NEO-MERCAZOLETm (carbimazole);

beta-adrenergic receptor antagonists such as propranolol and esmolol; Ca' channel blockers;
dexamethasone and iodinated radiological contrast agents such as TELEPAQUET"
(iopanoic acid) and ORAGRAFINTm (sodium ipodate).
[05241 Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, estrogens or congugated estrogens such as ESTRACETm (estradiol), ESTINYLTm (ethinyl estradiol), PREMARINffi, ESTRATABTm, ORTHO-EST, OGENTm and estropipate (estrone), ESTROVLST (quinestrol), ESTRADERMTm (estradiol), DELESTROGENTm and VALERGENTm (estradiol valerate), DEPO-ESTRADIOL
CYPIONATETm and ESTROJECT LATm (estradiol cypionate); antiestrogens such as NOLVADEXTM (tamoxifen), SEROPHENET" and CLOMH)Tm (clomiphene); progestins such as D'URALUTINTm (hydroxyprogesterone caproate), MPATM and DEPO-PROVERAT'A
(medroxyprogesterone acetate), PROVERATm and CYCRINTm (NEPA), MEGACETm (megestrol acetate), NORLUTINTm (norethindrone), and NORLUTATETm and AYGESTINTm (norethindrone acetate); progesterone implants such as NORPLANT SYSTEMTm (subdermal implants of norgestrel); antiprogestins such as RU 4861" (mifepristone);
hormonal contraceptives such as ENOVJDTM (norethynodrel plus mestranol), PROGPSTASERTTm (intrauterine device that releases progesterone), LOESTRINTm, BREVICONTm, MODICONTm, GENORATM, NBLONATM, NORJNYLTM, OVACON-35TM and OVACON-5OTM (ethinyl estradiollnorethindrone), LEVLENTm, NORDETTETm, TRI-LEVLENTM and TR1PHASIL-21T"
(ethinyl estradioUlevonorgestrel) LO/OVRALTm and OVRALTm (ethinyl estradiol/norgestrel), DEMULENTm (ethinyl estradiollethynodiol diacetate), NORINYLTm, ORTHO-NOVUMTm, NORETHINT", GENORAT", and NELOVATM (norethindrone/mestranol), DESOGENI" and ORTI-10-CEPTT" (ethinyl estradiol/desogestrep, ORTHO-CYCLENT" and ORTHO-TRICYCLENT" (ethinyl estradiol/norgestimate), MICRONORTM and NOR-QDTM
(norethindrone), and OVRETTET" (norgestrel).
[05251 Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, testosterone esters such as methmolone acetate and testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-541"

(testosterone), TESTEXT" (testosterone propionate), DELATESTRYLT"
(testosterone enanthate), DEPO-TESTOSTERONET" (testosterone cypionate), DANOCRNET"
(daxiazol), HALOTESTINT" (fluoxymesterone), ORETON METHYLT", TESTREDT and VIRILONT"
(methyltestosterone), and OXANDR1NT" (oxandrolone); testosterone transdermal systems such as TESTODERIvIT"; androgen receptor antagonist and 5-alpha-reductase inhibitors such as ANDROCURT" (cyproterone acetate), EULEXIW" (flutamide), and PROSCARTM
(finasteride); adrenocorticotropic hormone preparations such as CORTROSYNT"
(cosyntropin); adrenocortical steroids and their synthetic analogs such as ACLOVATET"
(alclornetasone dipropionate), CYCLOCORTT" (arncinonide), BECLOVENV" and VANCERILT" (beclomethasone dipropionate), CELESTONE"' (betamethasone), BENISONET" and uncoRrm (betamethasone benzoate), DIPROSONET" (betarnethasone dipropionate), CELESTONE PHOSPHATE"' (betamethasone sodium phosphate), CELESTONE SOLUSPANT" (betamethasone sodium phosphate and acetate), BETA-VALT"
and VALISONET" (betamethasone valerate), TEMOVATET" (clobetasol propionate), CLODERMT (clocortolone pivalate), CORTEFT" and HYDROCORTONE". (cortisol (hydrocortisone)), HYDROCORTONE ACETATET" (cortisol (hydrocortisone) acetate), LOCOMT" (cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATE"' (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORTT" and SOLU CORTEFT"
(cortisol (hydrocortisone) sodium succinate), WESTCORTT" (cortisol (hydrocortisone) valerate), CORTISONE ACETATET (cortisone acetate), DESOWENT" and TRIDESILONT" (desonide), TOPICORr" (desoximetasone), DECADRONTM
(dexamethasone), DECADRON LA'" (dexamethasone acetate), DECADRON
PHOSPHATE"' and HEXADROL PHOSPHATE"' (dexamethasone sodium phosphate), FLORONET and MAXIFLORTM (diflorasone diacetate), FLORINEF ACETATE"' (fludrocortisone acetate), AEROBIDTM and NASALIDETM (flunisolide), FLUONIDTM
and SYNALARTM (fluocinolone acetonide), LII)EXTM (fluocinonide), FLUOR-OPTM and FMLT"
(fluorometholone), CORDRANT" (flurandrenolide), HALOGTM (halcinonide), HMS
LIZITIFILMT" (medrysone), MEDROLTM (methylprednisolone), DEPO-MEDROLT" and MEDROL ACETATErm (methylprednisone acetate), A-/vfETHAPREDTm and SOLUMEDROLT" (methylprednisolone sodium succinate), ELOCONT" (mometasone furoate), HALDRONETM (paramethasone acetate), DELTA-CORTEFTm (prednisolone), ECONOPREDTM (prednisolone acetate), HYDELTRASOLT" (prednisolone sodium phosphate), HYDELtRA-T.B.ATm (prednisolone tebutate), DELTASONErm (prednisone), ARISTOCORTTm and KENACORTT" (triamcinolone), KENALOGTM (triamcinolone acetonide), ARISTOCORTTm and KENACORT DIACETATET (triamcinolone diacetate), and ARISTOSPANTm (tdamcinolone hexacetonide); inhibitors of biosynthesis and action of adrenocortical steroids such as CYTADRENTm (aminoglutethimide), NIZORALTm (ketoconazole), MODRASTANErm (trilostane), and METOPIRONETm (metyrapone);
bovine, porcine or human insulin or mixtures thereof; insulin analogs; recombinant human insulin such as HUMUUNTm and NOVOLINut oral hypoglycemic agents such as ORAMIDETm and ORINASETM (tolbutamide), DIABJNESETM (chlorpropamide), TOLAMIDElm and TOLINASETm (tolazamide), DYMELORTm (acetohexamide), glibenclamide, MICRONASETM, DIBETAT and GLYNASETm (glyburide), GLUCOTROLTm (glipizide), and DIAMICRONTm (gliclazide), GLUCOPHAGETM (metfonnin), ciglitazone, pioglitazone, and alpha-glucosidase inhibitors; bovine or porcine glucagon; somatostatins such as SANDOSTATINTm (octreotide); and diazoxides such as PROGLYCEMTm (diazoxide).
[05261 In one embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with treatments for uterine motility disorders.
Treatments for uterine motility disorders include, but are not limited to, estrogen drugs such as conjugated estrogens (e.g., PREMARIN and ESTRATAB ), estradiols (e.g., CLIMARA and ALORA ), estropipate, and chlomtrianisene; progestin drags (e.g., AMEN
(medroxyprogesterone), IvHCRONOR (norethidronc acetate), PROMETRIUM
progesterone, and megestrol acetate); and estrogen/progesterone combination therapies such as, for example, conjugated estrogenshnedroxyprogesterone (e.g., PUNT:PROT"
and PREMPHASE ) and norethindrone acetate/ethinyl estsradiol (e.g., FEMIIRTTm).

[0527] in an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with drugs effective in treating iron deficiency and hypochromic anemias, including but not limited to, ferrous sulfate (iron sulfate, FEOSOLTm), ferrous fumarate (e.g., FEOSTATTm), ferrous gluconate (e.g., FERGONTm), polysaccharide-iron complex (e.g., NTEREXTm), iron dextran injection (e.g., INFEDTm), cupric sulfate, pyroxidine, riboflavin, Vitamin B12, cyancobalamin injection (e.g., REDISOLTM, RUBRAMIN PCTm), hydroxocobalarnin, folic acid (e.g., FOLVITETm), leucovorin (folinic acid, 5-CHOH4PteG1u, citrovorum factor) or WELLCOVORIN
(Calcium salt of leucovorin), transferrin or ferritin.
(05281 In certain embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with agents used to treat psychiatric disorders.
Psychiatric drugs that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, phnozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antimanic agents (e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithium citrate), antidepressants (e.g., arnitriptyline, amoxapine, bupropion, citalopram, clomipramine, desiprarnine, doxepin, fluvoxamine, fluoxetine, imipmmine, isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, sertraline, tranylcypromine, trazodone, trirnipramine, and venlafaxine), antianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate, diazepam, halazeparn, lorazepam, oxazcpam, and prazepam), and stimulants (e.g., d-amphetamine, methylphenidate, and pemoline).
105291 In other embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with agents used to treat neurological disorders. Neurological agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, antiepileptic agents (e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline, amantidine, bromocriptine, pergolide, ropinirole, pramipexole, benztropine; biperidcn; ethopropazine;
procyclidine;
trihexyphenidyl, tolcapone), and ALS therapeutics (e.g. riluzole).

105301 In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with vasodilating agents and/or calcium channel blocking agents. Vasodilating agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverinc, isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindoptil, quinapril, ramipril, spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbide dinitrate, isosorbide mononitrate, and nitroglycerin). Examples of calcium channel blocking agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil.
[05311 In certain embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with treatments for gastrointestinal disorders.
Treatments for gastrointestinal disorders that may be administered with the albumin fusion protein and/or polynucleotide of the invention include, but are not limited to, Hz histamine receptor antagonists (e.g., TAGAMETTNI (cimetidine), ZANTACTm (ranitidine), PEPCIDTm (farnotidine), and AYJDIN (nizatidine)); inhibitors of II+, K+ ATPase (e.g., PREVACIDI'm (lansoprazole) and PRILOSECTm (omeprazole)); Bismuth compounds (e.g., PEPTO-BISMOLTm (bismuth subsalicylate) and DE-NOLThl (bismuth subcitrate)); various antacids;
sucralfate; prostaglandin analogs (e.g. CYTOTECTm (misoprostol)); muscarinic cholinergic antagonists; laxatives (e.g., surfactant laxatives, stimulant laxatives, saline and osmotic laxatives); antidiarrheal agents (e.g., LOMO fl.LTm (diphenoxylate), MOTOFENTm (diphenoxin), and INIODIUNITm (loperamide hydrochloride)), Synthetic analogs of sontatostatin such as SANDOSTATINTm (octreotide), antiemetic agents (e.g., ZOFRANTM
(ondansetron), KYTRILTm (granisetron hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, perphenazine, prochlorperazine, promethazine, thiethylperazine, triflupromazine, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol, and nabilone); D2 antagonists (e.g., metoclopramide, trimethobenzamide and chlorpromazine); bile salts;
chenodeoxycholic acid; =
ursodeoxycholic acid; and pancreatic enzyme preparations such as pancreatin and pancrelipase.
[05321 In additional embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
105331 The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions comprising albumin fusion proteins of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or salefor human administration.
Gene Therapy [05341 Constructs encoding albumin fusion proteins of the invention can be used as a part of a gene therapy protocol to deliver therapeutically effective doses of the albumin fusion protein. A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, encoding an albumin fusion protein of the invention.
Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
105351 Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous nucleic acid molecules encoding albumin fusion proteins in vivo. These vectors provide efficient delivery of nucleic acids into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:27 1). A
replication defective retrovinis can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, P.M. et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
[05361 Another viral gene delivery system useful in the present invention uses adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., Bio Techniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et aL, Cell 68:143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992) cited supra).
Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., cited supra; Haj-Ahsnand et al., J. Virol. 57:267 (1986)).
[05371 In another embodiment, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject nucleotide molecule by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes. In a representative embodiment, a nucleic acid molecule encoding an albumin fusion Protein of the invention can be entrapped in liposornes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno at al. (1992) No Shinkei Geka 20:547-5 5 1; PCT publication W091/06309;
Japanese patent application 1047381; and European patent publication EP-A-43075).
l05381 Gene delivery systems for a gene encoding an albumin fusion protein of the invention can be introduced into a patient by any of a number of methods. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g.
by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being uite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by Stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3 054-3 05 7). The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Where the albumin fusion protein can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the albumin fusion protein.
Additional Gene Therapy Methods 105391 Also encompassed by the invention are gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of an albumin fusion protein of the invention.
This method requires a polynucleotide which codes for an albumin fusion protein of the present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the fusion protein by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, W090/11092.
105401 Thus, for =example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide encoding an albumin fusion protein of the present invention ex vivo, with the engineered cells then being provided to a patient to be treated with the fusion protein of the present invention. Such methods arc well-known in the art. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J.
Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990);
Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et at., Cancer Gene Therapy 3: 31-38 (1996)).
In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
105411 As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like).

he polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
[05421 In one embodiment, polynucleotides encoding the albumin fusion proteins of the present invention is delivered as a naked polynucleotide.
The term "naked"
polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, polynucleotides encoding the albumin fusion proteins of the present invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859.
10543] The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, p0G44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL
available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
[0544j Any strong promoter known to those skilled in the art can be used for driving the expression of the polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter, the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters;
the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine Idnase promoters, such as the Herpes Simplex thyrnidine lcinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the gene corresponding to the Therapeutic protein portion of the albumin fusion =
proteins of the invention.
[0545] Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

105461 The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels.
Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells.
They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts.
In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
10547] For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 rrig,/lcg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection.
The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
105481 The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
[05491 The naked polynueleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called "gene guns". These delivery methods are known in the art.

15501 The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofcctin, precipitating agents, etc. Such methods of delivery are known in the art.
105511 in certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et at., Proc. Natl.
Acad. Sci. USA (1987) 84:7413-7416); mRNA
(Malone et al., Proc. Natl. Acad. Sci, USA (1989) 86:6077-6081);
and purified transcription factors (Debs et at., I. Biol. Chem.
(1990)265:10189-10192), in functional form.
[0552] Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propylj-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc. Nati Acad. Sci. USA (1987) 84:7413-7416).
Other commercially available liposomes include trarasfectace (DDAB/DOPE) and DOTAP/DOPE (Boeluinger), [0553] Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication No. WO

for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimetlaylammonio)propane) liposomes. Preparation of DOTMA
liposomes is explained in the literature, see, e.g., P. Feigner et at., Proc. Natl.
Acad. Sci. USA
84:7413-7417.
Similar methods can be used to prepare liposomes from other cationic lipid materials.
[0554] Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ma.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyi choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

(0555] For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size.
Other methods are known and available to those of skill in the art.
[0556] The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527.
For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 naM
Tris/NaC1, sonicated, and then the preformed liposomes are mixed directly with the DNA.
The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca2+-EDTA
chelation (Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483;
Wilson et al., Cell 17:77 (1979)); ether injection (Dcamer, D. and Bangham, A., Biochim.
Biophys. Acta 443:629 (1976); Ostro et at., Biochem. Biophys. Res. Conunun. 76:836 (1977);
Fraley et al., Proc. Natl. Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad.

:i. USA 75:145 (1978); Schaefer-Ridder et al., Science 215:166 (1982)).
105571 Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
105581 U.S. Patent No. 5,676,954 reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice.
U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to mammals.
[0559] In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding an albumin fusion protein of the present invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
[0560] The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCMP, GP+E-86, GP+envAm.12, and DAN cell lines as described in Miller, Human Gene Therapy 1:544 (1990). The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
[0561] The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding an albumin fusion protein of the present invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced ettkgryotic cells will express a fusion protin of the present .vention.
10562] In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotide contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses fusion protein of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovinis expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Mn. Rev. Respir. Dis.109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-l-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434;
Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).
[0563] Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993);
Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); 'Wilson et al., Nature 365:691-692 (1993); and U.S. Patent No. 5,652,224. For example, the adenovirus vector Aci2 is useful and can be grown in human 293 cells. These cells contain the El region of adenovirus and constitutively express Ela and Bib, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.
[0564] Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: El a, Bib, E3, E4, E2a, or LI. through U.
105651 In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr.
Topics in licrobiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb.
Methods for producing and using such AAVs are known in the art. See, for example, U.S.
Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.
105661 For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et at., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc.
Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses.
Once the packaging cells are transfected and infected, they will produce infectious .AAV viral particles which contain the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express a fusion protein of the invention.
[05671 Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. endoding a polypeptide of the present invention) via homologous recombination (see, e.g., U.S. Patent No.
5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994;
Koller et at., Proc. Natl. Mad. Sci. USA 86:8932-8935 (1989); and Zijlstra et at., Nature 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.
105681 Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the =

5' end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.
[05691 The promoter and the targeting sequences can be amplified using PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5' and 3' ends. Preferably, the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction site as the 3' end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.
[0570] The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
[0571) The promoter-targeting sequence construct is taken up by cells.
Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.
105721 The polynucleotide encoding an albumin fusion protein of the present invention may contain a secretory signal sequence that facilitates secretion of the protein.
Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards Zir at the 5' end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
[0573] Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a rotein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).
[05741 A prefenta method of local administration is by direct injection.
Preferably, an albumin fusion protein of the present invention cornplexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
105751 Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.
[05761 Therapeutic compositions useful in systemic administration, include fusion proteins of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site. In specific embodiments, suitable delivery vehicles for use with systemic administration comprise liposomes comprising albumin fusion proteins of the invention for targeting the vehicle to a particular site.
[0577] Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al, Proc. Natl. Acad. Sci.
USA
189:11277-11281, 1992). Oral delivery can be performed by complexing a polynucleotide construct of the present invention, to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DM80) that is capable of passing into the skin.
[05781 Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.
105791 Albumin fusion proteins of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.
Biological Activities [0580] Albumin fusion proteins and/or polynucleotides encoding albumin fusion proteins of the present invention, can be used in assays to test for one or more biological activities. If an albumin fusion protein and/or polynucleotide exhibits an activity in a particular assay, it is likely that the Therapeutic protein corresponding to the fusion portent may be involved in the diseases associated with the biological activity. Thus, the fusion protein could be used to treat the associated disease.
[05811 In preferred embodiments, the present invention encompasses a method of = treating a disease or disorder listed in the "Preferred Indication Y"
column of Table I
comprising administering to a patient in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a Therapeutic protein portion corresponding to a Therapeutic protein disclosed in the 'Therapeutic Protein X"
column of Table 1 (in the same row as the disease or disorder to be treated is listed in the "Preferred Indication Y" column of Table I) in an amount effective to treat, prevent or ameliorate the disease or disorder.
[0582] In a further preferred embodiment, the present invention encompasses a method of treating a disease or disorder listed for a particular Therapeutic protein in the "Preferred Indication:Y" column of Table 1 comprising administering to a patient in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a Therapeutic protein portion corresponding to the Therapeutic protein for which the indications in the Examples are related in an amount effective to treat, prevent or ameliorate the disease or disorder.
105831 Specifically contemplated by the present invention are albumin fusion proteins produced by a cell when encoded by the polynucleotides that encode SEQ rD
NO:Y. When these polynucleotides are used to express the encoded protein from a cell, the cell's natural secretion and processing steps produces a protein that lacks the signal sequence explicitly listed in columns 4 and/or 11 of Table 2. The specific amino acid sequence of the listed signal sequence is shown in the specification or is well known in the art.
Thus, most preferred embodiments of the present invention include the albumin fusion protein produced by a cell (which would lack the leader sequence shown in columns 4 and/or Ii of Table 2).
Also most preferred are polypeptides comprising SEQ ID NO:Y without the specific leader sequence listed in columns 4 andior 11 of Table 2. Compositions comprising these two preferred embodiments, including pharmaceutical compositions, are also preferred. These albumin fusion proteins are specifically contemplated to treat, prevent, or ameliorate a disease or disorder listed for a particular Therapeutic protein in the `Preferred Indication:Y" column of Table 1.
[0584] In preferred embodiments, fusion proteins of the present invention may be used in the diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders relating to diseases and disorders of the endocrine system (see, for example, "Endocrine Disorders" section below), the nervous system (see, for example, "Neurological Disorders"
section below), the immune system (see, for example, "Immune Activity" section below), respiratory system (see, for example, "Respiratory Disorders" section below), cardiovascular system (see, for example, "Cardiovascular Disorders" section below), reproductive system (see, for example, "Reproductive System Disorders" section below) digestive system (see, for example, "Gastrointestinal Disorders" section below), diseases and/or disorders relating to cell proliferation (see, for example, "Hyperproliferative Disorders" section below), and/or diseases or disorders relating to the blood (see, for example, "Blood-Related Disorders"
section below).
[0585] In certain embodiments, an albumin fusion protein of the present invention may be used to diagnose and/or prognose diseases and/or disorders associated with the tissue(s) in which the gene corresponding to the Therapeutic protein portion of the fusion protein of the invention is expressed.
[0586] Thus, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention are useful in the diagnosis, detection and/or treatment of diseases and/or disorders associated with activities that include, but are not limited to, prohormone activation, neurotransmitter activity, cellular signaling, cellular proliferation, cellular differentiation, and cell migration.
[0587] More generally, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention may be useful for the diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated with the following systems.
Immune Activity [0588] Albumin fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention may be usefill in treating, preventing, diagnosing and/or prog,nosing diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoirrimune diseases, acquired (e.g., by chemotherapy or toxins), or infectious.
Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as a marker or detector of a particular immune system disease or disorder.
[0589] In 'another embodiment, a fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention, may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune.
response generated by cells associated with the tissue(s) in which the polypeptide of the invention is expressed.
(05901 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, diagnosing, and/or prognosing irrununodeficiencies, including both congenital and acquired immunodeficiencies. Examples of B cell irnmunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agam.maglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XL?), aganunaglobulinemia including congenital and acquired agarnmaglobulinernia, adult onset agammaglobulinemia, late-onset agarnmaglobulinemia, dysgammaglobulinemia, hypogarnmaglobulinemia, unspecified hypogarrunaglobulinemia, recessive agammaglobulinemia (Swiss type), Selective IgM

deficiency, selective IgA deficiency, selective IgG subclass deficiencies, IgG
subclass deficiency (with or without IgA deficiency), Ig deficiency with increased IgM, IgG and IgA
deficiency with increased IgM, antibody deficiency with normal or elevated Igs, Ig heavy chain deletions, kappa chain deficiency, 13 cell lymphoproliferative disorder (BLPD), common variable immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired), and transient hypogammaglobulinemia of infancy.
(0591) In specific embodiments, ataxia-telangieciasia or conditions associated with ataxia-telangiectasia are treated, prevented, diagnosed, and/or prognosing using the, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
(0592) Examples of congenital immunodeficiencies in which T cell and/or B
cell function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MIIC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich sYndrome, and ataxia telarigie,ctasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4-1- T-Iymphocytopenia, immunodeficiency with predominant T
cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.
(0593) In specific embodiments, DiGeorge anomaly or conditions associated with DiGeorge anomaly are treated, prevented, diagnosed, and/or proposed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
[05941 Other immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, chronic granulomatous disease, Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lyrnphoproliferative syndrome (XL?), leukocyte adhesion deficiency, complement component deficiencies (including Cl, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphopla.sia-aplasia, immunodeficiency with thyrnoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs.
[05951 In a preferred embodiment, the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
10596I In a preferred embodiment fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an agent to boost imrnunoresponsiveness among B cell and/or T cell immunodeficient individuals.
105971 The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, diagnosing and/or prognosing autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue.
Therefore, the administration of fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of 'F-cells, may be an effective therapy in preventing autoimmune disorders.
105981 Autoinunune diseases or disorders that may be treated, prevented, diagnosed and/or .prognosed by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashirnoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoinixnune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g., Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellitus.
105991 Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.
[06001 Additional disorders that are likely to have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsornes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by Immoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispennatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes), bullous pemphigoid (often characterized, e.g., by IgG
and complement in basement membrane), Sjogten's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).
[06011 Additional disorders that may have an autoinanunet. component that may be treated, prevented, diagnosed and/or prognosed with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondria antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-M1 (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG
and IgM
antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM
antibodies to IgE), and many other inflammatory, granulomatous, degenerative, and atrophic disorders.

106021 In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using for example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, and/or diagnosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
106031 In another specific preferred embodiment, systemic lupus erythematosus is treated, prevented, and/or diagnosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. In another specific preferred embodiment. idiopathic thrombocytopenia purpura is treated, prevented, and/or diagnosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
WWI In another specific preferred embodiment IgA nephropathy is treated, prevented, and/or diagnosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
106051 In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumirefusion proteins of the invention.
[06061 In preferred embodiments, fusion proteins of the invention and/or polynucleotides 1Z-fit-coding albumin fusion proteins of the invention are used as a inuriunosuppressive agent(s).
[06071 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, proposing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stern cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.
[06081 Allergic reactions and eianditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Moreover, these molecules can be used to teat, prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
106091 Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose and/or prognose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rbinitis, and eczema. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to modulate IgE concentrations in vitro or in vivo.
106101 Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention have uses in the diagnosis, prognosis, prevention, and/or treatment of inflammatory conditions. For example, since fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., 'septic shock, sepsis, or systemic inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast);
CNS disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's disease); AIDS-related dementia; and prion disease); cardiovascular disorders (e.g., atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); as well as many additional diseases, conditions, and disorders that are characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma, pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosus, diabetes mellitus, and allogenic transplant rejection).
[06111 Because inflammation is a fundamental defense mechanism, inflammatory disorders can effect virtually any tissue of the body. Accordingly, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, have uses in the treatment of tissue-specific inflammatory disorders, including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis, rnyocarditis, myosititis, myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis, scierochoroiditis, scrotitis, sinusitis, spondylitis, steaties, stornatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis.
106121 In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, are useful to diagnose, prognose, prevent, and/or 'treat organ transplant rejections and graft-versus-host disease.
Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD. hi specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of 'f-cells, may be an effective therapy in preventing experimental allergic and hyperacute xeno graft rejection.
[06131 In other embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, are useful to diagnose, prognose, prevent, and/or treat immune complex diseases, including, but not limited to, serum sickness, post streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.
106141 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat, detect, and/or prevent infectious agents. For example, by increasing the immune response, particularly increasing the proliferation activation and/or differentiation of 13 and/or T cells, infectious diseases may be treated, detected, and/or prevented. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.

Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response.
[06151 In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a vaccine adjuvant that enhances immune responsiveness to an antigen. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin -fusion proteins of the invention are used as an adjuvant to enhance tumor-specific immune responses.
[06161 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of:
AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of:
HIV/AIDS, respiratory syneytial virus, Dengue, rotavirus, Japanese B
encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
106171 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
106181 In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B
streptococcus, Shigella spp., Enterotoxigenic Escherichia coil, Enterohemorrhagic E. coil, and Borrelia burgdorferi.
[0619] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a;parasite. in another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an irnmune response to Plasmodium (malaria) or Leishmania.
106201 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis; for example, by preventing the recruitment and activation of mononuclear phagocytes.
[06211 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.
106221 In one embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are administered to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgIVI, and IgE), to induce higher affinity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
[06231 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a stimulator of B cell responsiveness to pathogens.
[0624] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an activator of T cells.
[0625] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.
[0626] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to induce higher affinity antibodies.
106271 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to increase serum inmiunoglobulin concentrations.
[0628] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to accelerate recovery of immunocompromised individuals.
[0629] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to boost inununoresponsiveness among aged populations and/or neonates.
[0630] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of 1-cell populations, In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.
[0631] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to boost immunoresponsiveness among individuals having an acquired loss of B
cell function. Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
[0632] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency.
Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteine of the invention, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.
[0633] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention enhance antigen presentation or antagonize antigen presentation in vitro or in vivo. Moreover, in related embodiments, this enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.
10634j In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to direct an individual's immune system towards development of a humoral response (i.e.
TH2) as opposed to a Till cellular response.

[06351 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.
106361 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable lnununodificiency.
106371 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect. In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used in the pretreatment of bone marrow samples prior to transplant. .
[06381 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a gene-based therapy for genetically inherited disorders resulting in inummo-incompetence/immunodeficiency such as observed among SCLD patients.
106391 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.
106401 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of regulating secreted cytokines that are elicited by polypeptides of the invention.
[0641) In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used in one or more of the applications decribed herein, as they may apply to veterinary medicine.
[06421 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of blocking various aspects of immune responses to foreign agents or self.
Examples of diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimmune disorders such as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury and diseases/disorders associated with pathogens.
[06431 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for preventing the B cell proliferation and Ig secretion associated with autoinunune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.
[06441 In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention invention are used as a inhibitor of B and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.
[06451 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for chronic hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gazrunopathies, and plasmacytomas.
[0646) In another specific embodiment, albumin fusion proteins of the invention and/or polynucielnides encoding albumin fusion proteins of the invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain autoinimune and chronic inflammatory and infective diseases. Examples of autoinunune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes.
[0647) The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing eosinophil production and migration.
[0648] In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to enhance or inhibit complement mediated cell Iysis.

[06491 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.
[06501 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.
(06511 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be employed to treat adult respiratory distress syndrome (ARDS).
106521 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to stimulate the regeneration of mucosal surfaces.
10653j In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to diagnose, prognose, treat, and/or prevent a disorder characterized by primary or acquired immunodeficiency, deficient serum inununoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoirnmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV
disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii.
Other diseases and disorders that may be prevented, diagnosed, prognosed, andfor treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, HIV infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.
[06541 In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat, and/or diagnose an individual having common variable immunodeficiency disease ("CVID"; also known as "acquired agarnmaglobulinemia" and "acquired hypogarnmaglobulinemia") or a subset of this disease.
(0655) In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to diagnose, prognose, prevent, and/or treat cancers or neoplasms including immune cell or immune tissue-related cancers or neoplasms. Examples of cancers or neoplasms that may be prevented, diagnosed, or treated by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and disorders described in the section entitled "Hyperproliferative Disorders"
elsewhere herein, 106561 In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.
[0657) In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.
[0658) In specific embodiments, the compositions of the invention are used as an agent to boost innnunoresponsiveness among B cell irnmunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.
Blood-Related Disorders (0659) The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to modulate hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity. For example, by increasing hemostatic or throMbolytic activity, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia), blood platelet diseases, disorders, and/or conditions (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting.
These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, or scarring.
106601 In specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to prevent, diagnose, prognose, and/or treat thrombosis, arterial thrombosis, venous thrombosis, airomboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina_ In specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for ,reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, the prevention of occlusions in extrcorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
(0661] In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to prevent, diagnose, prognose, and/or treat diseases and disorders of the blood and/or blood forming organs associated with the tissue(s) in which the polypeptide of the invention is expressed.
(0662] The fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to modulate hematopoietie activity. (the formation of blood cells). For example, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to increase the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (13 or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of anemias and leukopenias described below. Alternatively, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may he used to decrease the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets.. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of leukocytoses, such as, for example cosinophilia.
(06631 The fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to prevent, treat, or diagnose blood dyscrasia.
106641 Anemias are conditions in which the number of red blood cells or amount of hemoglobin (the protein that carries oxygen) in them is below normal. Anemia may be caused by excessive bleeding, decreased red blood cell production, or increased red blood cell destruction (hemolysis). The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias. Anemias that may be treated prevented or diagnosed by the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include iron deficiency anemia, hypochromic anemia, microcytic anemia, chlorosis, hereditary siderob;astic anemia, idiopathic acquired sideroblastic 'anemia, red cell aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin B12 deficiency) and folic acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoinuntme helolytic anemia, rnicroangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria). The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias associated with diseases including but not limited to, anemias associated with systemic lupus erythematosus, cancers, lymphomas, chronic renal disease, and enlarged spleens. The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias arising from drug treatments such as anemias associated with methyldopa, dapsone, and/or sulfadrugs. Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias associated with abnormal red blood cell architecture including but not limited to, hereditary spherocytosis, hereditary elliptocytosis, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.
[0665] The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing hemoglobin abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C disease, hemoglobin S-C disease, and hemoglobin E disease).
Additionally, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating thalassemias, including, but not limited to, major and minor forms of alpha-thalassemia and beta-thalassemia.
[0666] In another embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating bleeding disorders including, but not limited to, thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet disorders (e.g., storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bemard-Soulier syndrome), hemolytic-uremic syndrome, heinophelias such as hemoph.elia A or Factor VII deficiency and Christmas disease or Factor IX deficiency, Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation.
[06671 The effect of the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention on the clotting time of blood may be monitored using any of the clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee-White Clotting time.
106681 Several diseases and a variety of drugs can cause platelet dysfunction. Thus, in a specific embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating acquired platelet dysfunction such as platelet dysfunction accompanying kidney failure, leukemia, multiple myeloma, cirrhosis of the liver, and systemic lupus erythematosus as well as platelet dysfunction associated with drug treatments, including treatment with aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs (used for arthritis, pain, and sprains), and penicillin in high doses.
[06691 In another embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may he useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders characterized by or associated with increased or decreased numbers of white blood cells.
Leukopenia occurs when the number of white blood cells decreases below normal. Leukopenias include, but are not limited to, neutropenia and lymphocytopenia. An increase in the number of white blood cells compared to normal is known as leukocytosis. The body generates increased numbers of white blood cells during infection. Thus, leukocytosis may simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis may be an indicator of injury or other disease such as cancer. Leokocytoses, include but are not limited to, eosinophilia, and accumulations of macrophages. In specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating leukopenia. In other specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating leukocytosis.
106701 Leukopenia may be a generalized decreased in all types of white blood cells, or may be a specific depletion of particular types of white blood cells. Thus, in specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating decreases in neutrophil numbers, known as neutropenia.
Neutropenias that may be diagnosed, prognosed, prevented, and/or treated by the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic neutropenia, neutropenias resulting from or associated with dietary deficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency), neutropenias resulting from or associated with drug treatments (e.g., antibiotic regimens such as penicillin treatment, sulfonamide treatment, anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer chemotherapy), and neutropenias resulting from increased neutrophil destruction that may occur in association with some bacterial or viral infections, allergic disorders, autoinunune diseases, conditions in which an individual has an enlarged spleen (e.g., Felty syndrome, malaria and sarcoidosis), and some drug treatment regimens.

106711 The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating lymphocytopenias (decreased numbers of B and/or T
lymphocytes), including, but not limited to, lymphocytopenias resulting from or associated with stress, drug treatments (e.g., drug treatment with corticosteroids, cancer chemotherapies, and/or radiation therapies), AIDS infection and/or other diseases such as, for example, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infections, some viral infections and/or hereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndome, severe combined immunodeficiency, ataxia telangiectsia).
[0672] The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders associated with macrophage numbers and/or macrophage function including, but not limited to, Gaucher's disease, Niemann-Pick disease, Letterer-Siwe disease and Hand-Schuller-Christian disease.
[0673] In another embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or , treating diseases and disorders associated with eosinophil numbers and/or eosinophil function including, but not limited to, idiopathic hypereosinophilic syndrome, eosinophilia-myalgia syndrome, and Hand-Schuller-Christian disease.
106741 In yet another embodiment, the albumin fusion ptoteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating leukemias and lymphomas including, but not limited to, acute lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, myelogenous, myeloblastic, or myelomoriocytic) leukemia, chronic lymphocytic leukemia (e.g., B cell leukemias, T cell leukemias, Sezary syndrome, and Hairy cell leukenia), chronic myelocytic (myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma, non-hodgIcin's lymphoma, Burldtes lymphoma, and mycosis fimgoides.
[0675] In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders of plasma cells including, but not limited to, plasma cell dyscrasias, monoclonal ganunaopathies, monoclonal gammopathies of undetermined significance, multiple myeloma, macroglobulineinia, Waldenstrom's macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon.
106761 In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing myeloproliferative disorders, including but not limited to, polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, apogenic myelod metaplasia, thrombocythernia, (including both primary and seconday thrombocythemia) and chronic myelocytic leukemia.
[0677] In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as a treatment prior to surgery, to increase blood cell production.
106781 In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to enhance the migration, phagocytosis, superoxide production, antibody dependent cellular cytotoxicity of neutrophils, eosionophils and macrophages.
[06791 In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase the number of stem cells in circulation prior to stem cells pheresis. In another specific embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase the number of stem cells in circulation prior to platelet pheresis.
[06801 In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides' elitoding albumin fusion proteins of the invention may be useful as an agent to increase cytoldne production.
106811 In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in preventing, diagnosing, and/or treating primary hematopoietic disorders.
Hyperproliferatire Disorders 106821 hi certain embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat or detect hyperproliferative disorders, including neoplasms. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may proliferate other cells which can inhibit the hyperproliferative disorder.
106831 For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
106841 Examples of hyperproliferative disorders that can be treated or detected by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
106851 Similarly, other hyperproliferative disorders can also be treated or detected by fusion proteins of the invention and/or polynucleotides encoding. albumin fusion proteins of the invention. Examples of such hyperproliferative disorders include, but are not limited to:
Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Gliama, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Med ul lob lastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, bitraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulimemia, Male Breast . Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Hi stiocytom a, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheochrornocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinohlastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulva' Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
[06861 In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to diagnose, prognose, prevent, and/or treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above. Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. 13.
Saunders Co., Philadelphia, pp. 68-79.) 106871 Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
Hyperplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited .to, angiofollicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cernentum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hypeiplasia of the breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular hyperplasia of prostate, nodular regenerative hyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, and verrucous hyperplasia.
[06881 Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell.
Metaplastic disorders which can be diagnosed, proposed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, rnetaplastie polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squarnous metaplasia of amnion, and symptomatic myeloid metaplasia.
106891 Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells.
Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit ple,omorphism.
Dysplasia characteristically occurs where there exists chronic irritation or inflammation. Dysplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopuhnonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniametaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hernimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial white folded dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral dysplasia, odontogenic dysplasia, ophthalmomandibulonielic dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia, pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia, spondyloepiphystal dysplasia, and ventriculoradial dysplasia.
[06901 Additional pre-neoplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
106911 In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to diagnose and/or propose disorders associated with the tissue(s) in which the polypeptide of the invention is expressed.
[06921 In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat cancers and neoplasms, including, but not limited to, those described herein. In a further preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat acute myelogenous leukemia.
[0693] Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may affect apoptosis, and therefore, would be useful in treating 4 number of diseases associated with increased cell survival or the inhibition of apoptosis. For example, diseases associated with increased cell survival or the inhibition of apoptosis that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, tnyoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcomaõ chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Beheet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
[06941 In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.

[06951 Additional diseases or conditions associated with increased cell survival that could be diagnosed, prognosed, prevented, andlor treated by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoina, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcorna, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinonia, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastorna, craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
106961 Diseases associated with increased apoptosis that could be diagnosed, prognosed, prevented, and/or treated by fusion proteins of the invention and/or polynueleotides encoding albumin fusion proteins of the invention, include AIDS;
neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct .,njury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
[0697) Hyperproliferative diseases and/or disorders that could be diagnosed, prognosed, prevented, and/or treated by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, neoplasms located in the liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
,[0698) Similarly, other hyperproliferative disorders can also be diagnosed, prognosed, prevented, and/or treated by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Examples of such hypeiproliferative disorders include, but are not limited to: hyperganunaglobulinemia, lymphoproliferative disorders, paraproteinemias, ptupura, sarcoidosis, Sezary Syndrome, Waldenstron's rnacroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
[0699] Another preferred embodiment utilizes polynucleotides encoding albumin fusion proteins of the invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.
[07001 Thus, the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide encoding an albumin fusion protein of the present invention, wherein said polynucleotide represses said expression.
[07011 Another embodiment of the present invention provides a method of treating cell-proliferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA
construct comprising a recombinant expression vector effective in expressing a DNA
sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the fusion protein of the present invention is inserted into cells to be treated utilizing a retrovirus, or more preferably an adenoviral vector (See G
J. Nabel, et. al., PNAS 1999 96: 324-326). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only =

proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e.
magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.
[07021 Folynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By "repressing expression of the oncogenic genes " is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.
[07031 For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, ,but not limited to, retroviral vectors (Gilboa, I. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl.
Acad. Sci. U.S.A. '85:3014), vaccirtia virus system (Chakrabarty et at,, Mol.
Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art_ In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus Will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle.
Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.
107041 The polynucleotides of the present invention may be delivered directly to cell ,eoliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.
[07051 By "cell proliferative disease" is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.
[07061 Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polymicleotide of the present invention simultaneously to the same site. By "biologically inhibiting" is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and, cell cultures, or any other method known to one of ordinary skill in the art.
[07071 Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as .a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph 1B, et al. J Nat! Cancer Inst, 90(21):1648-53 (1998)).
[07081 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. These fusion protieins and/or polynucleotides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (INF) receptor-1, CD95 (Fas/AP0-1), TNF-receptor-related apoptosis-mediated protein (TRAM?) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et.al., Eur J Biochem 254(3):439-59 (1998)).

e/loreover, in another preferred embodiment of the present invention, these fusion proteins and/or polynucleotides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of these proteins, either alone or in combination with small molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins (See for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem Biol Interact. Apr 24;111-112:23-34 (1998), I Mol Med.76(6):402-12 (1998), hit J Tissue React;20(1):3-15 (1998)).
(07091 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering these albumin fusion proteins and/or polynucleotides, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbic)].
Immunol 1998;231:125-41). Such therapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
[07101 In another embodiment, the invention provides a method of delivering compositions containing the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to targeted cells expressing the a polypeptide bound by, that binds to, or associates with an albumin fuison protein of the invention. Albumin fusion proteins of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
107111 Albumin fusion proteins of the invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the albumin fusion proteins of the invention 'vaccinated' the immune response to respond to proliferative antigens and inamunogens, or indirectly, such as in activating the expression of proteins known to enhance the. immune response (e.g.
chemoldnes), to said antigens and immunogens.
Renal Disorders [0712] Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose disorders of the renal system. Renal disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, but are not limited to, kidney failure, nephritis, blood vessel disorders of kidney, metabolic and congenital kidney disorders, urinary disorders of the kidney, autoimmune disorders, sclerosis and necrosis, electrolyte imbalance, and kidney cancers.
107131 Kidney diseases which can be diagnosed, prognosecl, prevented, and/or treated with compositions of the invention include, but are not limited to, acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases of the kidney (e.g., acute glomerulonephritis, postinfectious glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis I
and II, mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronic nephritis, interstitial nephritis, and post-streptococcal glomerulonephritis), blood vessel disorders of The :kidneys (e.g., kidney infarction, atheroembolic kidney disease, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis, renal underperfusion, renal retinopathy, renal ischemia-reperfusion, renal artery embolism, and renal artery stenosis), and kidney disorders resulting form urinary tract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal lithiasis, nephrolithiasis), reflux nephropathy, urinary tract infections, urinary retention, and acute or chronic unilateral obstructive uropathy.) [0714) In addition, compositions of the invention can be used to diagnose, prognose, prevent, and/or treat metabolic and congenital disorders of the kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glycosuria, nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (=al rickets), Hartnup disease, Bartter's syndrome, Liddle's syndrome, polycystic kidney disease, medullary cystic disease, medullary sponge kidney, Alport's syndrome, nail-patella syndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and membranous nephropathy), and autoimmune disorders of the kidney (e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis).

(0715f Compositions of the invention can also be used to diagnose, prognose, prevent, and/or treat sclerotic or necrotic disorders of the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal segmental glomcrulo sclerosis (FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), cancers of the kidney (e.g., nephroma, hypemephroma, nephroblastoma, renal cell cancer, transitional cell cancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypematremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).
[0716) Compositions of the invention may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. - Such methods are known in the art Compositions of the invention may be administered as part of a Therapeutic, described in more detail below.
Methods of delivering polynucleotides of the invention are described in more detail herein.
Cardiovascular Disorders 107171 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose cardiovascular disorders, including, but not limited to, peripheral artery disease, such as limb ischemia.
[0718) Cardiovascular disorders include, but are not limited to, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
Congenital heart defects include, but are not limited to, aortic coarctation, cor triatriaturn, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.

[07191 Cardiovascular disorders also include, but are not limited to, heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardinm, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
[07201 Arrhythmias include, but are not limited to, sinus arrhytlunia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
[07211 Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, initial valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
[07221 Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperftision injury, and myocarditis.
[07231 Myocardial ischemias include, but are not limited to, coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
[07241 Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Flippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Talcayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythroniclalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.
[07251 Aneurysms include, but are not limited to, dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
[0726) Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
[07271 Cerebrovascular disorders include, but are not limited to, carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, Cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxlmoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, Migraine, and vertebrobasilar insufficiency.
[07281 Embolisms include, but are not limited to, air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include, but are not limited to, coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
[0729) Ischemic disorders include, but are not limited to, cerebral ischemia, ischernic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet'S" Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
137301 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoarn sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Methods of delivering polynucleotides are described in more detail herein.
=
Respiratory Disorders [0731) Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat, prevent, diagnose, and/or prognose diseases and/or disorders of the respiratory system.
[0732) Diseases and disorders of the respiratory system include, but are not limited to, nasal vestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyps, and sinusitis, juvenile angiofibromas, cancer of the nose and juvenile papillomas, vocal cord polyps, nodules (singer's nodules), contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer of the nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g., squarnous cell carcinoma, small cell (oat cell) carcinoma, large cell carcinoma, and adenocarcinoma), allergic disorders (eosinophilic pneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergic alveolitis, allergic interstitial pnettmonitis, organic dust pneumoconiosis, allergic bronchopulrnonary aspergillosis, asthma, Wegenees granulomatosis (granulomatous vasculitis), Goodpasture's syndrome)), pneumonia (e.g., bacterial pneumonia (e.g., Streptococcus pneumoniae (pneurnoncoccal pneumonia), Staphylococcus aureus (staphylococcal pneumonia), Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and Pseudomas spp.), Mycoplasma pneumoniae pneumonia, liemophilus influenzae pneumonia, Legionella pneumophila (Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), and viral pneumonia (e.g., influenza, chickenpox (varicella).

[0733] Additional diseases and disorders of the respiratory system include, but are not limited to bronchiolitis, polio (poliomyelitis), croup, respiratory syncytial viral infection, mumps, erythema infectiosum (fifth disease), roseola infantum, progressive rubella panencephalitis, german measles, and subacute sclerosing panencephalitis), fungal pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal infections in people with severely suppressed immune systems (e.g., cryptococcosis, caused by Clyptococcus neoformans; aspergillosis, caused by Aspergillus spp.;
candidiasis, caused by Candida; and mucormycosis)), Pneumocystis carinii (pneumocystis pneumonia), atypical pneumonias (e.g., hfycoplasma and Chlamydia spp.), opportunistic infection pneumonia, nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonia, pleural disorders (e.g., pleurisy, pleural effusion, and pneumothorax (e.g., simple spontaneous pneumothorax, complicated spontaneous pneumothorax, tension pneumothorax)), obstructive airway diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis, black lung (coal workers' pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis, and benign pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary fibrosis. (e.g., fibrosing alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial pneumonia, lymphoid interstitial pneunaonia, histiocytosis X
(e.g., Letterer-Siwe disease, Hand-Schtiller-Christian disease, eosinophilic granuloma), idiopathic pulmonary hemothderosis,.sarcoidosis and pulmonary alveolar proteinosis), Acute respiratory distress syndrome (also called, e.g., adult respiratory distress syndrome), edema, pulmonary embolism, bn'ancliitis (e.g., viral, bacterial), bronchiectasis, atelectasis, lung abscess (caused by, e.g., Staphylococcus aureus or Legionella pneumophila), and cystic fibrosis.
Anti-Angiogenesis Activity [07341 - The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate.
Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail.
Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al,, Biotech. 9:630-634 (1991);
Foliation et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et aL, J.
Microvasc. Res.
29:401-411 (1985); Foliation, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalrnot 94:715-(1982); and FoHunan et al., Science 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).
[07351 The present invention provides for treatment of diseases or disorders associated with neovascularization by administration of fusion proteins of the invention and/or polynucleotides encoding albumin fusi )ti proteins of the invention.
Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise blown in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. Et. Lippincott Co., Philadelphia (1985)).Thus, the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention. For example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor. Cancers which may be treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endornetrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas;
glioblastoma;
Kaposi's sarcoma; leiomyosarcoma; non- mull cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may he delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.
[0736] Within yet other aspects, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.
[0737] Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating other disorders, besides cancers, which involve angiogenesis. These disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenie granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascula.r glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis;
delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderrna; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis;
Osler-Webber Syndrome; plaque neovascularization; telangjectasia; hemophiliac joints;
angiofibrotna; fibrornuscular dysplasia; wound granulation; Crohn's disease;
and atherosclerosis.
[0738] For example, within one aspect of the present invention methods are provided for treating hypertrophic scars and keloids, comprising the step of administering albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to a hypertrophic scar or keloid.
(0739] Within one embodiment of the present invention fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are 'mown to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
[07401 Moreover, Ocular disorders associated with neovascularization which can be treated with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to:
neovascuks glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal.
85:704-710 (1978) and Gartner etal., Surv. Ophthal. 22:291-312 (1978).
[07411 Thus, within one aspect of the present invention methods are provided for treating neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (e.g., fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels.
In certain pathological conditions however, capillaries may extend into the cornea from the pericomeal vascular plexus of the linabus. When the .cornea becomes vasculaxized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of disorders can result in corneal neovascularization, including for example, corneal infections (e.g, trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali bums, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.
197421 Within particularly preferred embodiments of the invention, may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily.
Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea.
Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical bums). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.
[0743] Within other embodiments, the compounds described above may be injected directly into the comeal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to "protect" the cornea from the advancing blood vessels. This method may also be utilized shortly after a conical insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.
[0744] Within another aspect of the present invention, methods are provided for treating neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma.
Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor.
Within another aspect of the present invention, methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention to the eyes, such that the formation of blood vessels is inhibited.
107451 Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.
107461 Within another aspect of the present invention, methods are provided for treating retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.
[0747] Additionally, disorders which can be treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
[0748] Moreover, disorders and/or states, which can be treated, prevented, diagnosed, and/or prognosed with the the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention of the invention include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crolufs disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.
107491 In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a "morning after" method. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endornetriosis.
107501 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be incorporated into surgical sutures in order to prevent stitch granulomas.
[07511 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or filial) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogeriesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a =
surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.
[0752] Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.
0753] . Within one aspect of the present invention, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.
[07541 The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-ang;iogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter "d group"
transition metals.
[0755] Lighter "d group" transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
[0756] Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
[0757) Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdie acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
10758! A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4;
protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP- PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, arninopropionitrile fiimarate; 4-propy1-5-(4-pyridiny1)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum;
ChIMP-3 (Pavloff et at., J. Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al., Biochem 3. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate;
Eponemycin;
Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987));
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem.
262(4):1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4- chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions 36:312-316, (1992)); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole;
and metalloproteinase inhibitors such as BB94.
Diseases at the Cellular Level [07591 Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated, prevented, diagnosed, and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastorna, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) arid viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
[0760] In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.
[0761] Additional diseases or conditions associated with increased cell survival that could be treated or detected by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myelobla.stic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphoeytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogertic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytorna, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

[0762I Diseases associated with increased apoptosis that could be treated, prevented, diagnosed, and/or prognesed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, AIDS;
neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogrenis syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemiaireperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
Wound Healing and Epithelial Cell Prohferation 107631 In accordance with yet a further aspect of the present invention, there is provided a process for utilizing fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the derrais and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, derma' ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, bums resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to promote dermal reestablishment subsequent to dermal loss [07641 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are types of grafts that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dennic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosa' graft, Oilier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used to promote skin strength and to improve the appearance of aged skin.
107651 It is believed that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could promote proliferation of epithelial . cells. such as sebocytes, hair follicles, hepatocytes, type II
pneumocytes, mucineproducing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. Albumin fusion = proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
107661 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may have a cytoprotective effect on the small intestine mucosa.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteini of the invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.
[0767] Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including bums, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly.
Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to promote the resurfacing of the mucosa' surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to treat diseases associate with the under expression.
107681 Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to prevent and heal damage to the lungs due to various pathological states. Albumin fusion proteins of the invention, and/or polynucleotides encoding albumin fusion proteins of the invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and bums, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using polynucleotides or polypeptides, agonists or antagonists of the present invention. Also fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to stimulate the proliferation of and differentiation of type II pneurnacytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.
[07691 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
07701 In addition, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and 11 diabetes, where some islet cell function remains, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.
Neural Activity and Neurological Diseases (0771j The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used for the diagnosis and/or treatment of diseases, disorders, damage or injury of the brain and/or nervous system.
Nervous system disorders that can be treated with the compositions of the invention (e.g., fusion proteins of .
the invention and/or polynucleotides encoding albumin fusion proteins of the invention), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (I) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to, degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or arnyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including, but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;
(7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;
(8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) dernyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
[0772] In one embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to protect neural cells from the damaging effects of hypoxia. In a further preferred embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia.
According to this embodiment, the compositions of the invention are used to treat or prevent neural cell injury associated with cerebral hypoxia. In one non-exclusive aspect of this embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, are used to treat or prevent neural cell injury associated with cerebral ischemia. In another non-exclusive aspect of this embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury associated with cerebral infarction.
[0773] In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury associated with a stroke. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent cerebral neural cell injury associated with a stroke.
[0774] In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury associated with a heart attack. In a specific embodiment, albumin fusion proteins of the invention and/or polynueleotides encoding albumin fusion proteins of the invention are used to treat or prevent cerebral neural cell injury associated with a heart attack.
[0775] The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture either in the presence or absence of hypoxia or hypoxic conditions; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in viva. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, in Zhang et al., Proc Nat! Acad Sci USA 97:3637-42 (2000) or in Arakawa et. al., J. Neurosci., 10:3507-15 (1990); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the niethods set forth in Pestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann. Rev. Neurosa, 4:17-42 (1981);
increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
[0776] In specific embodiments, motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
[07771 Further, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may play a role in neuronal survival;
synapse formation; conductance; neural differentiation, etc. Thus, compositions of the invention (including fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention) may be used to diagnose and/or treat or prevent diseases or disorders associated with these roles, including, but not limited to, learning and/or cognition disorders. The compositions of the invention may also be useful in the treatment or prevention of neurodegenerative disease states and/or behavioural disorders.
Such neurodegenerative disease states and/or behavioral disorders include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, compositions of the invention may also play a role in the treatment, prevention and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders.
[07781 Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be useful in protecting neural cells from diseases, damage, disorders, or injury, associated with cerebrovascular disorders including, but not limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid stenosis, or Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subdural hernatoma, or subarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome, or vertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct), leukomalacia, periventricular, and vascular headache (e.g., cluster headache or migraines).
107791 In accordance with yet a further aspect of the present invention, there is provided a process for utilizing fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, for therapeutic purposes, for example, to stimulate neurological cell proliferation and/or differentiation. Therefore, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat and/or detect neurologic diseases. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used as a marker or detector of a particular nervous system disease or disorder.
[07801 Examples of neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, brain diseases, such as metabolic brain diseases which includes phenylketormia such as maternal phenylketonuria, pyruvate carboxylase deficiency, pymvate debydrogenase complex deficiency, Wernicke's Encephalopathy, brain edema, brain neoplasms such as cerebellar neoplasms which include infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar diseases such as cerebellar ataxia which include spinocerebellar degeneration such as ataxia telangiectasia, cerebellar dyssynergia, niederich's Ataxia, Machado-Joseph Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as infratentorial neoplasms, diffuse cerebral sclerosis such as encephalitis periaxialis, globoid cell leukodystrophy, metachromatic leukodystrophy and subacute sclerosing panencephalitis.
[07811 Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include cerebrovascular disorders (such as carotid artery diseases which include carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis such as carotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and subaraehnoid hemorrhage, cerebral infarction, cerebral isehemia such as transient cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar insufficiency, vascular dementia such as multi-infarct dementia, periventricular leukomalacia, vascular headache such as cluster headache and migraine.
[07821 Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include dementia such as ADS Dementia Complex, presenile dementia such as Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such as multi-infarct dementia, encephalitis which include encephalitis periaxialis, viral encephalitis such as epidemic encephalitis, Japanese Encephalitis, St. Louis Encephalitis, tick-borne encephalitis and West Nile Fever, acute disseminated encephalomyelitis, meningoencephalitis such as uveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis, encephalomalacia such as periventricular leukomalacia, epilepsy such as generalized epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsy such as complex partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy, status epilepticus such as Epilepsia Partialis Continua, and Hailervorden-Spatz Syndrome.
[07831 Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include hydrocephalus such as Dandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri pseudottunor, Rett Syndrome, Reyes Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranig tuberculoma and Zellweger Syndrome, central nervous system infections such as AIDS
Dementia Complex, Brain Abscess, subdural empyema, encephalomyelitis such as Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, and cerebral malaria.
[0784] Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include meningitis such as arachnoiditis, aseptic meningtitis such as viral meningtitis which includes lymphocytic choriomeningitis, Bacterial meningtitis which includes Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such as Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural effusion, meningoencephantis such as uvemeningoencephalitic syndrome, myelitis such as transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-Jakob Syndrome, Bovine Spongiforrn Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebral toxoplasmosis.
107851 Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include central nervous system neoplasms such as brain neoplasms that include cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral sceloris which includes adrenoleukodystrophy, encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic leukodystrophy, allergic encephalomyelitis, necroti2ing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple sclerosis, central pontine myelinolysis, transverse myelitis, neuromyelitis optics, Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal cord diseases such as arnyotonia congenita, amyotrophic lateral sclerosis, spinal muscular atrophy such as Werdnig-Hoffinann Disease, spinal cord compression, spinal cord neoplasms such as = epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mental retardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(MI), Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria, Laurence-Moon- Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such as. fucosidosis, neuronal ceroid-lipofuscinosis, oculocerehrorenal syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervous system abnormalities such as holoprosencephaly, neural tube defects such as anencephaly which includes hydrangencephaly, Arnold-Chain Deformity, encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina bifida cystica and spina bifida occulta.
10786) Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include hereditary motor and sensory neuropathies which include Charcot-Marie Disease, Hereditary optic atrophy, Refsum's Disease, hereditary spastic paraplegia, Werdnig-Hoffinann Disease, Hereditary Sensory and Autonomic Neuropathies such as Congenital Analgesia and Familial Dysautonornia, Neurologic manifestations (such as agnosia that include Getstmann's Syndrome, Amnesia such as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, communicative disorders such as hearing disorders that includes deafness, partial hearing loss, loudness recruitment and tinnitus, language disorders such as aphasia which include agraphia, anomia, braes aphasia, and Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language development disorders, speech disorders such as aphasia which includes anomia, broca aphasia and Wernicke Aphasia, articulation disorders, communicative disorders such as speech disorders which include dysarthria, echolalia, mutism and stuttering, voice disorders such as aphonia and hoarseness, decerebrate state, delirium, fasciculation, hallucinations, meningism, movement disorders such as angelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle hypotonia, myoclonus, tic, torticollis and tremor, muscle hypertonia such as muscle rigidity such as stiff-man syndrome, muscle spasticity, paralysis such as facial paralysis which includes Herpes Zoster liens, Gastroparesis, Herniplegia, ophthaknoplegia such as diplopia, Duane's Syndrome, Homer's Syndrome, Chronic progressive external ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis,' Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome, , quadriplegia, respiratory paralysis and vocal cord paralysis, paresis, phantom limb, taste disorders such as ageusia and dysgeusia, vision disorders such as amblyopia, blindness, color vision defects, diplopia, hemianopsia, seotoma and subnormal vision, sleep disorders such as hypersonmia which includes Kleine-Levin Syndrome, insomnia, and somnambulism, spasm such as trismus, unconsciousness such as coma, persistent vegetative state and syncope and vertigo, neuromuscular diseases such as amyotonia congenita, arnyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscular atrophy such as spinal muscular atrophy, Charcot-Marie Disease and Werdnig-Hoffmatut Disease, Postpoliornyetitis Syndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous system diseases such as acrodynia, amyloid neuropathies, autonomic nervous system diseases such as Ache's Syndrome, Barre-Lieou Syndrome, Familial Dysautonomia, Homer's Syndrome, Reflex Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as Acoustic Nerve Diseases such as Acoustic Neuroma which includes Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders which includes amblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia such as Duane's Syndrome, Homer's Syndrome, Chronic Progressive External Ophthatmoplegia which includes Kearns Syndrome, Strabismus such as Esotropia and Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such as Optic Atrophy which includes Hereditary Optic Atrophy, Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases such as Neuromyelitis Optica and Swayback, and Diabetic neuropathies such as diabetic foot.
[0787] Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as polyra\diculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and Familial Dysautonon3ia, POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).
Endocrine Disorders 1078µ81 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose disorders and/or diseases related to hormone imbalance, and/or disorders or diseases of the endocrine system.
[0789] Hormones secreted by the glands of the endocrine system control physical growth, sexual function, metabolism, and other functions. Disorders may be classified in two ways: disturbances in the production of hormones, and the inability of tissues to respond to hormones. The etiology of these hormone imbalance or endocrine system diseases, disorders or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins), or infectious. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as a marker or detector of a particular disease or disorder related to the endocrine system and/or hormone imbalance.
[0790] Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including, but not limited to: complications with pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders andlor diseases of the menstrual cycle (e.g., dysmenorrhea and endornetriosis).
[0791] Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases of the pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytomassislet cell tumor syndrome;
disorders and/or diseases of the adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome, hyperaldosteronism, pheochromacytoma; disorders and/or diseases of the pituitary gland, such as, for example, hyperpituitarism, hyppituitarism, pituitary dwarfism, pituitary =
adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases of the thyroid, including but not limited to, hyperthyroidism, hypothyroidism, Plummer's disease, Graves' disease (toxic diffuse goiter), toxic nodular goiter, thyroiditis (HashiMoto's thyroiditis, subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis), Pendred's . syndrome, myxedema, cretinism; thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia, Hurthle cell tumours of the thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma; disorders and/or diseases of the parathyroid, such as, for example, hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the hypothalamus.
[0792]
In addition, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes or ovaries, including cancer.
Other disorders and/or diseases of the testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelier's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydies cells, cr3rptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias of the testis and neo-testis.
[0793] Moreover, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
[0794] In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to diagnose, prognose, prevent, and/or treat endocrine diseases and/or disorders associated with the tissue(s) in which the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin protein of the invention is expressed, Reproductive System Disorders [0795j The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used for the diagnosis, treatment, or prevention of diseases and/or disorders of the reproductive system.
Reproductive system disorders that can be treated by the compositions of the invention, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders which result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.
[07961 Reproductive system disorders and/or diseases include diseases and/or disorders of the testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral and bilateral), anorchia, ectopic testis, epididyrnitis and orchitis (typically resulting from infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal tumors (e.g., Leydig cell tumors), hydrocele, hematocele, varicocele, specmatocele, inguinal hernia, and disorders of sperm production (e.g., immotile cilia syndrome, =
aspermia, asthenozoospermia, azoospermia, oligospermia, and teratozoospennia).
[0797] . Reproductive system disorders also include disorders of the prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous prostatitis, malacoplalcia, benign prostatic hypertrophy or hypetplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squarnous cell carcinomas.
10798) Additionally, the compositions of the invention may be useful in the diagnosis, treatment, and/or prevention of disorders or diseases of the penis and urethra, including inflammatory disorders, such as balanoposthitis, balanitis xerotica obfiterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum, condyloma laturn, and pearly penile papules; urethral abnormalities, such as hypospadias, epispadias, and phimosis; premalipant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile cancers, including squarnous cell carcinomas, carcinoma in situ, vemicous carcinoma, and disseminated penile carcinoma; urethral neoplastic disorders, including penile urethral carcinoma, bulbomembranous urethral carcinoma, and prostatic urethral carcinoma; and erectile disorders, such as priapism, Peyronie's disease, erectile dysfunction, and impotence.
[07991 Moreover, diseases and/or disorders of the vas deferens include vasculititis and CBAVD (congenital bilateral absence of the vas deferens); additionally, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the seminal vesicles, including hydatid disease, congenital chloride diarrhea, and polycystic kidney disease.
[08001 Other disorders and/or diseases of the male reproductive system include, for example, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple sclerosis, and gynecomastia.
(0801) Further, the polynucleotides, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the vagina and vulva, including bacterial vaginosis, candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, =
vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis, condyloma acuminatum, syphilis, molluscum contagiosurn, atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvar vestibulitis, and neoplastic disorders, such as squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas, cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.
(0802) Disorders and/or diseases of the uterus include dysmenonhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidifonn moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas. Additionally, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as a marker or detector of, as well as in the diagnosis, treatment, and/or prevention of congenital uterine abnormalities, such as bicomuate uterus, septate uterus, simple unicornuate uterus, unicomuate uterus with a noncavitary rudimentary ham, unicomuate uterus with a non-communicating cavitary rudimentary horn, unicomuate uterus with a communicating cavitary born, arcuate uterus, uterine didelfus, and T-shaped uterus.
[0803[ Ovarian diseases and/or disorders include anovulation, polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian overproduction of androgens, right ovarian vein syndrome, amenorrhea, hirutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and Ovarian =
Krukenberg tumors).
[0804] Cervical diseases and/or disorders include cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).
[0805] Additionally, diseases and/or disorders of the reproductive system include disorders and/or diseases of pregnancy, including miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, abruptio placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes .gestationis, and urticaria of pregnancy.
Additionally, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used in the diagnosis, treatment, and/or prevention of diseases that can complicate pregnancy, including heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, high blood pressure, anemia, kidney disease, infectious disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, ehlarnydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis of the liver, primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder disorders,and obstruction of the intestine.
[08061 Complications associated with labor and parturition include premature rupture of the membranes, pre-term labor, post-term pregnancy, postmaturity, labor that progresses too slowly, fetal distress (e.g., abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position), shoulder dystocia, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.
[08071 Further, diseases and/or disorders of the postdelivery period, including endometritis, myometritis, parametsitis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.
[0808] Other disorders and/or diseases of the female reproductive system that may be diagnosed, treated, and/or prevented by the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, for example, Turner's syndrome, psendoherrnaphroditisme premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia, ruptured fallopian tube, and Mittelschmerz.
Infectious Disease [0809 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
08101 Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picomaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae ITTLV-Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to:
arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B
encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g.. AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.
Albumin fusion , proteins of the invention and/or polynucleotides encoding albumin fusion prateins. of the invention, can be used to treat or detect any of these symptoms or diseases.
In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis . (e.g., hepatitis B). In an additional specific embodiment fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat AIDS.
[08111 Similarly, bacterial and fungal agents that can cause disease or symptoms and that can be treated or detected by albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but not limited to, the following Gram-Negative and Gram-positive bacteria, bacterial families, and fungi:
Aotinomyees (e.g., Norcardia), Acinetobacter, Coptococcus neoformans, Aspergillus, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdoiferi), Bmcella, Candidia, Carripylobacter, Chlamydia, Clostridium (e.g., Clostridium botulinum, Clostridium d'tficile, Clostridium perfringens, Clostridium tetani), Coccidioides, Corynebacterium (e.g., Coomebacterium diptheriae), Cryptococcus, Dennatocycoses, E. coli (e.g., Enterotoxigenic E.
coli and Enterohemorrhagic E. coli), Enterobactcr (e.g. Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g., Haenzophilus influenza type B), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae), Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus (e.g., Sktphylococcus aureus), Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci), and Ureaplasmas. These bacterial, parasitic, and fungal families can cause diseases or symptoms, including, but not limited to:
antibiotic-resistant infections, bacteremia, endocarditis, septicemia, eye infections (e.g., conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial diarrhea, opportunisdc infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, dental caries, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery, paratyphoid fever, food poisoning, Legionella disease, chronic and acute inflammation, erythema, yeast infections, typhoid;
pneumonia, gonorrhea, meningitis (e.g., mengitis types A and B), chlamydia, syphilis, diphtheria, leprosy, brucellosis, peptic ulcers, anthrax, spontaneous abortions, birth defects, pneumonia, lung infections, ear infections, deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea, Crolin's disease, colitis, vaginosis, sterility, pelvic inflammatory diseases, candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections, noscomial infections.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat: tetanus, diptheria, botulism, and/or meningitis type B.
10812]
Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardias, Helminthiasis, Leishrnaniasis, Schistisoma, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Ttichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium nzalariae and Plasmodium vale), These parasites can cause a variety of diseases or symptoms, including, but not limited to:
Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat, prevent, and/or diagnose malaria.
[08131 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could either be by administering an effective amount of an albumin fusion protein of the invention to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
Regeneration [08141 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997)). The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g.
osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytoldne damage.
[0815] Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculatuxe (including vascular and lymphatics), nervous, hematopoletic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.
108161 Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
[08171 Similarly, nerve and brain tissue could also be regenerated by using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and -stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neurop'athy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
Gastrointestinal Disorders 108181 Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose gastrointestinal disorders, including inflammatory diseases and/or conditions, infections, cancers (e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of the small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.
[0819] Gastrointestinal disorders include dysphagia, odynophagia, inflammation of the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and stricturing, Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric retention disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of the stomach, autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, atrophic, plasma cell, and Menetrierts), and peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric lyrnphadenitis, mesenteric vascular occlusion, parmiculitis, neoplasms, peritonitis, pneurnoperitoneurn, bubphrenic abscess).
[08201 Gastrointestinal disorders also include disorders associated with the small intestine, such as malabsorption syndromes, distension, irritable bowel syndrome, sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of the ileum, Meckers diverticulum, multiple diverticula, failure of complete rotation of the small and large intestine, lymphoma, and bacterial and parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid, cholera, infection by Roundworms (Aseariasts luntbrieokles), Hookworms (Ancylostoma duodenale), Threadworms (Enterobius vermicularis), Tapeworms (Taenia saginata, Echinococcus granulosus, Diphyllobothrium spp., and T.
soliwn).
08211 Liver diseases and/or disorders include intrahepatic cholestasis (alaelle syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome), hepatic vein thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopuhnonaly syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis 13, hepatitis C, hepatitis D, drug induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varices, hepatic cncephalopafhy, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangiornas, bile stones, liver failure (hepatic encephalopathy, acute liver failure), and liver neoplasms (angiomyolipoma, calcified liver metastases, cystic liver metastases, epithelial tumors, fibrolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liver hemangioendothelioma, mesenchyrnal harnartoma, mesenchymal tumors of liver, nodular regenerative hyperplasia, benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymal harnartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis, Lipomas, Inflammatory pseudoturnor, Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerative hypetplasia)j, malignant liver tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, other tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittent porphyria, porphyria =
cutanea tarda), Zellweger syndrome).
[0822] Pancreatic diseases and/or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas, cystadenocarcinorna, insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-cell tumors, pancreoblastoma), and other pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).
[0823] Gallbladder diseases include gallstones (cholelithiasis and choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.
[0824] Diseases and/or disorders of the large intestine include antibiotic-associated colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g., vinous adenoma), colon carcinoma, colorectal cancer], colonic diverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease, toxic megacolon]; sigrnoid diseases [proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery), duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenal ulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ileal diseases (Heal neoplasms, ileitis), itnmunoproliferative small intestinal disease, inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis, balantidiasis, blastocystis infections, cryptcsporidiesis, dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal neoplasms (cecal neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferent loop syndrome, duodenal obstruction, impacted feces, intestinal pseudo-obstruction Vecal volvulusl, intussusception), intestinal perforation, intestinal polyps (colonic polyps, gardner syndrome, peutz-jeghers syndrome), jejunal diseases (jejunal neoplasms), malabsorption syndromes (blind loop syndrome, celiac disease, lactose intolerance, short bowl syndrome, tropical sprue, whipple's disease), mesenteric vascular occlusion, pneumatosis cystoides intestinalis, protein-losing enteropathies (intestinal lyrnphagiectasis), rectal diseases (anus diseases, fecal incontinence, hemorrhoids, proctitis, rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular ectasia, gastric fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis, stomach dilatation, stomach diverticulum, stomach .
neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis, visceroptosis, vomiting .(e.g., hematemesis, hyperemesis gravidarum, postoperative nausea and vomiting) and hemorrhagic colitis.
(08251 Further diseases and/or disorders of the gastrointestinal system include biliary tract diseases, such as, gastroschisis, fistula (e.g., biliary fistula, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary tract neoplasms, esophageal neoplasms, such as adenocarcinoma of the esophagus, esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the pancreas, pancreatic cystic neoplasms, panereatoblastoma, and peritoneal neoplasms), esophageal disease (e.g., bullous diseases, candidiasis, glycogenic acanthosis, ulceration, barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum), fistula (e.g., tracheoesophageal fistula), motility disorders (e.g., CREST syndrome, deglutition disorders, achalasia, spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia);
gastrointestinal diseased, such as, gastroenteritis (e.g., cholera morbus, norwalk virus infection), hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, ventral hernia), and intestinal diseases (e.g., cecal diseases (appendicitis, cccal neoplasms)).
Chemotaxis [0826] Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may have chemotaxis activity. A
chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.
[0827] Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may increase chemotaxic activity of particular cells.
These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.
[0828] it is also contemplated that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an inhibitor of chemotaxis.
Binding Activity [0829] Albumin fusion proteins of the invention may be used to screen for molecules that bind to the Therapeutic protein portion of the fusion protein or for molecules to which the Therapeutic protein portion of the fusion protein binds. The binding of the fusion protein and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the fusion protein or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
[08301 Preferably, the molecule is closely related to the natural ligand of the Therapeutic protein portion of the fusion protein of the invention, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic.
(See, Coligan et at., Current Protocols in Immunology 1(2):Chapter 5 (1991)). Similarly, the molecule can he closely related to the natural receptor to which the Therapeutic protein portion of an albumin fusion protein of the invention binds, or at least, a fragment of the receptor capable of being bound by the Therapeutic protein portion of an albumin fusion protein of the invention (e.g., active site). In either case, the molecule can be rationally designed using known techniques.
[08311 Preferably, the screening for these molecules involves producing appropriate cells which express the albumin fusion proteins of the invention. Preferred cells include cells from mammals, yeast, Drosophila, or E. coll.
[08321 The assay may simply test binding of a candidate compound to an albumin fusion protein of the invention, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the fusion protein.
[08331 Alternatively, the assay can be carried out using cell-free preparations, fusion protein/molecule affixed to a solid support, chemical libraries, or natural product mixtures.
The assay may also simply comprise the steps of mixing a candidate compound With a solution containing an albumin fusion protein, measuring fusion protein/molecule activity or binding, and comparing the fusion protein/molecule activity or binding to a standard.
108341 Preferably, an ELISA assay can measure fusion protein level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
The antibody can measure fusion protein level or activity by either binding, directly or indirectly, to the albumin fusion protein or by competing with the albumin fusion protein for a substrate.
108351 Additionally, the receptor to which a Therapeutic protein portion of an albumin fusion protein of the invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, in cases wherein the Therapeutic protein portion of the fusion protein corresponds to FGF, expression cloning may be employed wherein polyadenylated RNA is prepared from a cell responsive to the albumin fusion protein, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the albumin fusion protein. Transfected cells which are grown on glass slides are exposed to the albumin fusion protein of the present invention, after they have been labeled.
The albumin fusion proteins can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.
108361 Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.
108371 As an alternative approach for receptor identificaticn, a labeled albumin fusion protein can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule for the Therapeutoc protein component of an albumin fusion protein of the invention, the linked material may be resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the fusion protein can be excised, resolved into peptide fragments, and subjected to protein microse,quencing.
The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
108381 Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling") may be employed to modulate the activities of the fusion protein, and/or Therapeutic protein portion or albumin component of an albumin fusion protein of the present invention, thereby effectively generating agonists and antagonists of an albumin fusion protein of the present invention.
See generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997);
Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998)).
In one embodiment, alteration of polynucleotides encoding albumin fusion proteins of the invention and thus, the albumin fusion proteins encoded thereby, may be achieved by DNA shuffling. DNA
shuffling involves the assembly of two or more DNA segments into a desired molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides encoding albumin fusion proteins of the invention and thus, the albumin fusion proteins encoded thereby, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of an albumin fusion protein of the present invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-betal , TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).
[0839] Other preferred fragments are biologically active fragments of the Therapeutic protein portion and/or albumin component of the albumin fusion proteins of the present invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of a Therapeutic protein portion and/or albumin component of the albumin fusion proteins of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
[0840] Additionally, this invention provides a method of screening compounds to identify those which modulate the action of an albumin fusion protein of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, an albumin fusion protein of the present invention, and the compound to be screened and 3[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H]
thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[111 thymidine, Both agonist and antagonist compounds may be identified by this procedure.
[0841] In another method, a mammalian cell or membrane preparation expressing a receptor for the Therapeutic protien component of a fusion protine of the invention is incubated with a labeled fusion protein of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential fusion protein. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
[0842) All of these above assays can be used as diagnostic or prognostic markers.
The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the fusion protein/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the albumin fusion proteins of the invention from suitably manipulated cells or tissues.
108431 Therefore, the invention includes a method of identifying compounds which bind to an albumin fusion protein of the invention comprising the steps of:
(a) incubating a candidate binding compound with an albumin fusion protein of the present invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with an albumin fusion protein of the present invention, (b) assaying a biological activity, and (b) determining if a biological activity of the fusion protein has been altered.
, Targeted Delivery [0844] In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a component of an albumin fusion protein of the invention.
[0845) As discussed herein, fusion proteins of the invention may be associated with beterologous polypeptides, hcterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions a the invention to cells by administering fusion proteins of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a Therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

[0846] In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering an albumin fusion protein of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.
[0847] By "toxin" is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine endonuclease, RNAse, alpha toxin, ricin, abrin, Pseuclornonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By "cytotoxic prodrug" is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard ancylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, datmorubisin, and phenoxyacetamide derivatives of doxorubicin.
Drug Screening [08481 Further contemplated is the use of the albumin fusion proteins of the present invention, or the polynucleotides encoding these fusion proteins, to screen for molecules which modify the activities of the albumin fusion protein of the present invention or proteins corresponding to the Therapeutic protein portion of the albumin fusion protein. Such a method would include contacting the fusion protein with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of the fusion protein following binding.
108491 This invention is particularly useful for screening therapeutic compounds by using the albumin fusion proteins of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The albumin fusion protein employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the albumin fusion protein. Drugs are screened against such transformed cells or supernatants obtained from culturing such cells, in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and an albumin fusion protein of the present invention.
[08501 Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the albumin fusion proteins of the present invention. These methods comprise contacting such an agent with an albumin fusion protein of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the albumin fusion protein or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the albumin fusion protein of the present invention.
108511 Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to an albumin fusion protein of the present 'invention, and is described in great detail in European Patent Application 84/03564, published on September 13, 1984.
Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with an albumin fusion protein of the present invention and washed. Bound peptides are then detected by methods well known in the art. Purified albumin fusion protein may be coated directly onto plates for use in the aforementioned drag screening techniques.
In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.
10852] This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding an albumin fusion protein of the present invention specifically compete with a test compound for binding to the albumin fusion protein or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with an albumin fusion protein of the invention.

Binding Peptides and Other Molecules [0853] The invention also encompasses screening methods for identifying polypeptides and nonpolypeptides that bind albumin fusion proteins of the invention, and the binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists of the albumin fusion proteins of the invention. Such agonists and antagonists can be used, in accordance with the invention, in the therapeutic embodiments described in detail, below.
[0854] This method comprises the steps of:
contacting an albumin fusion protein of the invention with a plurality of molecules;
and identifying a molecule that binds the albumin fusion protein.
[0855] The step of contacting the albumin fusion protein of the invention with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the albumin fusion protein on a solid support and bringing a solution of the plurality of molecules in contact with the immobilized polypeptides. Such a procedure would be akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized albumin fusion protein of the invention.
The molecules having a selective affinity for the albumin fusion protein can then be purified by affinity selection. The nature of the solid support, process for attachment of the albumin fusion protein to the solid support, solvent, and conditions of the affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art.
[0856] Alternatively, one may also separate a plurality of polypeptides into substantially separate fractions comprising a subset of or individual polypeptides. For instance, one can separate the plurality of polypeptides by gel electrophoresis, column chromatography, or like method known to those of ordinary skill for the separation of polypeptides. The individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage).
Individual isolates can then be "probed" by an albumin fusion protein of the invention, optionally in the presence of an inducer should one be required for expression, to determine if any selective affinity interaction takes place between the albumin fusion protein and the individual clone. Prior to contacting the albumin fusion protein with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience. Such a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or nylon. In this manner, positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA
construct encoding a polypeptide having a selective affinity for an albumin fusion protein of the invention. Furthermore, the amino acid sequence of the polypeptide having a selective affinity for an albumin fusion protein of the invention can be determined directly by conventional means or the coding sequence of the DNA encoding the polypeptide can frequently be determined more conveniently. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined from the polypeptide itself, one may use micrasequencing techniques. The sequencing technique may include mass spectroscopy.
108571 In certain situations, it may be desirable to wash away any unbound polypeptides from a mixture of an albumin fusion protein of the invention and the plurality of polypeptides prior to attempting to determine or to detect the presence of a selective affinity interaction. Such a wash step may be particularly desirable when the albumin fusion protein of the invention or the plurality of polypeptides are bound to a solid support.
108581 The plurality of molecules provided according to this method inay be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind an albumin fusion protein of the invention. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries.
Examples of chemically synthesized libraries are described in Fodor et al., Science 251:767-773 (1991); Houghten et at., Nature 354:84-86 (1991); Lam et al., Nature 354:82-84 (1991);
Medynsld, Bio/Technology 12:709-710 (1994); Gallop et al., J. Medicinal Chemistry 37(9):1233-1251 (1994); Ohhneyer et al., Proc. Natl. Acad. Sci. USA 90:10922-(1993); Erb et al., Proc. Natl. Acad. Sci. USA 91:11422-11426 (1994); Houghten et al., Biotechniques 13:412 (1992); Jayawickreme et at., Proc. Natl. Acad. Sci. USA
91:1614-1618 (1994); Salmon et al., Proc. Natl. Acad. Sci. USA 90:11708-11712 (1993); PCT
Publication No. WO 93/20242; and Brenner and Lerner, Proc. Natl. Acad. Sci. USA 89:5381-(1992).
108591 Examples of phage display libraries are described in Scott et at., Science 249:386-390 (1990); Devlin et al., Science, 249:404-406 (1990); Christian et at., 1992, J.
Mol. Biol. 227:711-718 1992); Lenstra, J. Inununol. Meth. 152:149-157 (1992);
Kay et al., Gene 128:59-65 (1993); and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

108601 In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et at., Proc. Natl. Acad. Sci. USA 91:9022-9026 (1994).
[0861] By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., Proc. Natl. Acad. Sci. USA 91:4708-4712 (1994)) can be adapted for use.
Peptoid libraries (Simon et al., Proc. Natl. Acad. Sci. USA 89:9367-9371 (1992)) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been perrnethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (Proc. Natl. Acad. Sci. USA 91:11138-11142 (1994)).
[0862] The variety of non-peptide libraries that are useful in the present invention is great. For example, Ecker and Crooke (Bio/Technology 13:351-360 (1995) list benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminirnides, and oxazolones as among the chemical species that form the basis of various libraries.
[0863] Non-peptide libraries can be classified broadly into two types:
decorated monomers and oligomers. Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.
[0864] Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new shapes that depend on the order of the monomers.
Among the monomer units that have been used are carbamates, pyrrolinones, and morpholinos. Peptoids, peptide-like oligorners in which the side chain is attached to the alpha amino group rather than the alpha carbon, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer libraries utilized a single type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility.
[0865] Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley et al., Adv. Exp. Med. Biol. 251:215-218 (1989); Scott et al,. Science 249:386-390 (1990); Fowlkes et al., BioTechniques 13:422-427 (1992); Oldenburg et at., Proc. Natl. Acad. Sci. USA 89:5393-5397 (1992); Yu et al., Cell 76:933-945 (1994); Staudt et at., Science 241:577-580 (1988); Bock et at., Nature 355:564-566 (1992);
Tuerk et al., Proc. Natl. Acad. Sci. USA 89:6988-6992 (1992); Ellington et at., Nature 355:850-852 (1992); U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No.
5,198,346, all to Ladner et at.; Rebar et al., Science 263:671-673 (1993); and PCT Publication No. WO
94/18318.
[08661 In a specific embodiment, screening to identify a molecule that binds an albumin fusion protein of the invention can be carried out by contacting the library members with an albumin fusion protein of the invention immobilized on a solid phase and harvesting those library members that bind to the albumin fusion protein. Examples of such screening methods, termed "panning" techniques are described by way of example in Parinley et al., Gene 73:305-318 (1988); Fowlkes et at., BioTechniques 13:422-427 (1992); PCT
Publication No. WO 94/18318; and in references cited herein.
[08671 In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields et al., Nature 340:245-246 (1989); Chien et al., Proc. Natl. Acad.
Sci. USA 88:9578-9582 (1991) can be used to identify molecules that specifically bind to polypeptides of the invention.
[0868] Where the binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The term "biased" is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that govern the diversity of the resulting collection of molecules, in this case peptides.
[08691 Thus, a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position of the peptide is the same for all 20 amino acids. A bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a de.capeptide library be fixed to include only arginine. Clearly, many types of biases can be contemplated, and the present invention is not restricted to any particular bias.
Furthermore, the present invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and those that utilize a DNA construct comprising a lambda phage vector with a DNA insert.
[08701 As mentioned above, in the case of a binding molecule that is a polypeptide, the polypeptide may have about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids. In another embodiment, a binding polypeptide has in the range of 15-100 amino acids, or 20-50 amino acids.
[08711 The selected binding polypeptide can be obtained by chemical synthesis or recombinant expression.
Other Activities [08721 An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.
[0873] An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.
[0874] An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed stimulate neuronal growth and to treat and prevent neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.
[0875] An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.
[0876] An albumin .fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth.
Along the same lines, an albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.
108771 An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues. An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.
[0878] An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
[0879] An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, an albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be usectto modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
[0880] An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or hihibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
[0881] An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
[0882] The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.
108831 Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
[0884] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the alterations detected in the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
EXAMPLES
EXAMPLE 1: Generation of pSeNHSA and pSeCHSA. =
[0885] The vectors pScNHSA (ATCC Deposit No. PTA-3279) and pScCHSA
(ATCC Deposit No. PTA-3276) are derivatives of pPPC0005 (ATCC Deposit No. PTA-3278) and are used as cloning vectors into which polynucleotides encoding a therapeutic protein or fragment or variant thereof is inserted adjacent to and in translation frame with polynucleotides encoding human serum albumin "HSA". pScCHSA may be used for generating Therapeutic protein-HSA fusions, while pScNHSA may be used to generate HSA-Therapeutic protein fusions.
Generation of pSeCilS4.,, albionin fuston with the aktuttin moteOt C-terminal to the therapeutic portion.
[08861 A vector to facilitate cloning DNA encoding a Therapeutic protein N-terminal to DNA encoding the mature albumin protein was made by altering the nucleic acid sequence that encodes the chimeric HSA signal peptide in pPPC0005 to include the Xho I
and C1a I
restriction sites.
[0887] First, the Xho I and Cla I sites inherent to pPPC0005 (located 3' of the ADHI
terminator sequence) were eliminated by digesting pPPC0005 with Xho I and Cla 1, filling in the sticky ends with T4 DNA polymerase, and religating the blunt ends to create pPPC0006.
[0888] Second, the Alio I and Cla I restriction sites were engineered into the nucleic acid sequence that encodes the signal peptide of HSA (a chimera of the HSA
leader and a kex2 site from mating factor alpha, "MAF") in pPPC0006 using two rounds of PCR. In the first round of PCR, amplification with primers shown as SEQ ID NO:1039 and SEQ
ID
NO:1040 was performed. The primer whose sequence is shown as SEQ ID NO:1039 comprises a nucleic acid sequence that encodes part of the signal peptide sequence of HSA, a kex2 site from the mating factor alpha leader sequence, and part of the amino-terminus of the mature form of HSA. Four point mutations were introduced in the sequence, creating the Xho I and Cla I sites found at the junction of the chimeric signal peptide and the mature form of HSA. These four mutations are underlined in the sequence shown below. In pPPC0005 the nucleotides at these four positions from 5' to 3' are T, G, T, and G.
5'-GCCTCGAGAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCGATTTAAAGAT
TTGGG-3' (SEQ ID NO:1039) and 5'-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTCCTGGAA
TAAGC-3' (SEQ ID NO:1040). A second round of PCR was then performed with an upstream flanking primer, 5'-TACAAACTTAAGAGTCCAATTAGC-3' (SEQ ID NO:1041) and a downstream flanking primer 5'-CACTTCTCTAGAGTGGITTCATATGTCTT-3' (SEQ ID NO:1042). The resulting PCR product was then purified and digested with Aft II and Xba I and ligated into the same sites in pPPC0006 creating pScCHSA. The resulting plasmid has Xho land Ckt I
sites engineered into the signal sequence. The presence of the Xho I site creates a single amino acid change in the end of the signal sequence from LDKR to LEKR. The D to E
change will not be present. in the final albumin fusion protein expression plasmid when a nucleic acid sequence comprising a polynucleotide encoding the Therapeutic portion of the albumin fusion protein with a 5' Sal I site (which is compatible with the Xho I site) and a 3' Cla I site is ligated into the Xho I and Cla I sites of pScCHSA. Ligation of Sal Ito Xho I restores the original amino acid sequence of the signal peptide sequence. DNA encoding the Therapeutic portion of the albumin fusion protein may be inserted after the Kex2 site (Kex2 cleaves after the dibasic amino acid sequence KR at the end of the signal peptide) and prior to the Cla I
site.
GartOratkot ofp5cMJ34.õ
therapeutic portion.
108891 A vector to facilitate cloning DNA encoding a Therapeutic protein portion C-terminal to DNA encoding the mature albumin protein, was made by adding three, eight-base-pair restriction sites to pScCHSA. The Asc I, .Fse I, and Pme I
restriction sites were added in between the Bsu36 I and Hind ILI sites at the end of the nucleic acid sequence encoding the mature HSA protein. This was accomplished through the use of two complementary synthetic primers containing the Asc I, Fse I, and Pine I
restriction sites underlined (SEQ ID NO:1043 and SEQ ID
NO:1044).
5'-AAGCTGCCTTAGGCTTATAATAAGGCGCGCCGGCCGGCCGTTTAAACTAAGCT
TA ATTCT-T (SEQ ID NO:1043) and GCAGCTT-3' (SEQ ID NO:1044). These primers were annealed and digested with Bsu36 I
and Hind III and ligated into the same sites in pScCHSA creating pScNTISA.
EXAMPLE 2: General Construct Generation for Yeast Transformation.
108901 The vectors pScIsIHSA and pScCHSA may be used as cloning vectors into which polynucleotides encoding a therapeutic protein or fragment or variant thereof is inserted adjacent to polynucleotides encoding mature human serum albumin "HSA".
pScCHSA is used for generating Therapeutic protein-HSA fusions, while pScl\THSA may be used to generate HSA-Therapeutic protein fusions.
Generation of albumin Aston constructs comprLving AM-Therapeutic protein fusion products.
[08911 DNA encoding a Therapeutic protein (e.g., sequences shown in SEQ ID
NO:X
or known in the art) may be PCR amplified using the primers which facilitate the generation of a fusion construct (e.g., by adding restriction sites, encoding seamless fusions, encoding linker sequences, etc) For example, one skilled in the art could design a 5' primer that adds polynucleotides encoding the last four amino acids of the mature form of HSA
(and containing the Bsu36I site) onto the 5' end of DNA encoding a Therapeutic protein; and a 3' primer that adds a STOP codon and appropriate cloning sites onto the 3' end of the Therapeutic protein coding sequence. For instance, the forward primer used to amplify DNA
encoding a Therapeutic protein might have the sequence, 5'-aagctGCCTTAGGCTTA(N)15-3' (SEQ ID NO:1045) where the underlined sequence is a 11su36I site, the upper case nucleotides encode the last four amino acids of the mature HSA protein (ALGL), and (N)15 is identical to the first 15 nucleotides encoding the Therapetie protein of interest. Similarly, the reverse primer used to amplify DNA encoding a Therapeutic protein might have the sequence, 5'-GCGCGCGITTAAA A.Alfikt A_ = _At CCITATi3(N)15-3 (SEQ ID
NO:1046) where the italicized sequence is a Pine I site, the double underlined sequence is an Fse I site, the singly underlined sequence is an Asc I site, the boxed nucleotides are the reverse complement of two tandem stop codons, and (N)15 is identical to the reverse complement of the last IS nucleotides encoding the Therapeutic protein of interest. Once the PCR product is amplified it may be cut with Bsu36I and one of (Asc 1, Fse I, or Pine I) and ligated into pSeNHSA.
108921 The presence of the Xho I site in the HSA chimeric leader sequence creates a single amino acid change in the end of the chimeric signal sequence, i.e. the HSA-kex2 signal sequence, from LDKR (SEQ ID NO:2139) to LEKR. (SEQ ID NO:2140).
Generation of OfrepligaskrirggittLysis comprisbag gene-HSA fusiciktirotheits, 108931 Similar to the method described above, DNA encoding a Therapeutic protein may be PCR amplified using the following primers: A 5' primer that adds polynucleotides containing a Sall site and encoding the last three amino acids of the HSA
leader sequence, DKR; onto the 5' end of DNA encoding a Therapeutic protein; and a 3' primer that adds polynucleotides encoding the first few amino acids of the mature HSA
containing a Cia I site onto the 3' end of DNA encoding a Therapeutic protein. For instance, the forward primer used to amplify the DNA encoding a Therapeutic 'protein might have the sequence, 5'-aggagcgtcGACAAAAGA(N)i5-3' (SEQ ID NO:1047) where the underlined sequence is a Sal I site, the upper case nucleotides encode the last three amino acids of the HSA leader sequence =(DKR), and (MI5 is identical to the first 15 nucleotides encoding the Therapetic protein of interest. Similarly, the reverse primer used to amplify the DNA
encoding a Therapeutic protein might have the sequence, Y-CTITAAA TCGA TGAGCAACCTCACTCTTG'FGTGCATC(N)15-3t(SEQ ID NO:1048) where the italicized sequence is a Cia I site, the underlined nucleotides are the reverse complement of the DNA encoding the first 9 amino acids of the mature form of HSA
(DAHKSEVAH, SEQ ID NO:1106), and (N)15 is identical to the reverse complement of the last 15 nucleotides encoding the Therapeutic protein of interest. Once the PCR
product is amplified it may be cut with Sal I and Cla I and ligated into pScCHSA digested with X.ho I
and Cla I. A different signal or leader sequence may be desired, for example, invertase "INV" (Swiss-Prot Accession P00724), mating factor alpha "MAF" (Genbank Accession AAA18405), MPIF (Geneseq A.AF82936), Fibulin B (Swiss-Prot Accession P23142), Clustetin (Swiss-Prot Accession P10909), Insulin-Like Growth Factor- Binding Protein 4 (Swiss-Prot Accession P22692), and permutations of the HSA leader sequence can he subcloned into the appropriate vector by means of standard methods known in the art.
ge' 0,j sallmnf g is ig.,:g1:a0s Opi&t: ible for expreaston in yeast&
ceretiiiiag, [08941 The Not I fragment containing the DNA encoding either an N-terminal or C-terminal albumin fusion protein generated from pScNTISA or pScCHSA may then be cloned into the Not I site of pSAC35 which has a LEU2 selectable marker. The resulting vector is then used in transformation of a yeast S. cerevisiae expression system.
EXAMPLE 3: General Expression in Yeast S. cerevisiae.
108951 An expression vector compatible with yeast expression can be transformed into yeast S. cerevisiae by lithium acetate transformation, electropnration, or other methods known in the art and or as described in part in Sambrook, Fritsch, and Maniatis. 1989.
"Molecular Cloning: A Laboratory Manual, rd edition", volumes 1-3, and in Ausubel et al.
2000. Massachusetts General Hospital and Harvard Medical School. "Current Protocols in Molecular Biology", volumes 1-4. The expression vectors are introduced into S.
cerevisiae strains DXY1, D88, or wan by transformation, individual transfotmants can be grown, for example, for 3 days at 30 C in 10 rriL YEPD (1% w/v yeast extract, 2 % w/v, peptone, 2 %
w/v, dextrose), and cells can be collected at stationary phase after 60 hours of growth.
Supernatants are collected by clarifying cells at 3000g for 10 minutes.
[08961 pSAC35 (Sleep et al., 1990, Biotechnology 8:42 and see Figure 3) comprises, in addition to the LEU2 selectable marker, the entire yeast 2 pm plasmid to provide replication functions, the PRB1 promoter, and the ADH1 termination signal.
EXAMPIX 4:: General Purification of an Albumin Fusion Protein Expressed from an Albumin Fusion in Yeast S. cerevisiae.
108971 In preferred embodiments, albumin fusion proteins of the invention comprise the mature form of HSA fused to either the N- or C- terminus of the mature form of a therapeutic protein or portions thereof (e.g., the mature form of a therapeutic protein listed in Table 1, or the mature form of a therapeutic protein shown in Table 2 as SEQ
ID NO:Z). In one embodiment of the invention, albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the lAost used for expression. In a preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature albumin fusion protein is secreted directly into the culture medium, Albumin fusion proteins of the invention preferably comprise heterologous signal sequences (e.g., the non-native signal sequence of a particular therapeutic protein) including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art.
Especially preferred as those signal sequence listed in Table 2 and/or the signal sequence listed in the "Expression of Fusion Proteins" and/or "Additional Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins" section of the specification, above. In preferred embodiments, the fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
(08981 Albumin fusion proteins expressed in yeast as described above can be purified on a small-scale over a Dyax peptide affinity column as follows. Supernatants from yeast expressing an albumin fusion protein is diafiltrated against 3 TIM phosphate buffer pH 6.2,20 mM NaC1 and 0.01% Twee20 to reduce the volume and to remove the pigments. The solution is then filtered through a 0.22 p.m device. The filtrate is loaded onto a Dyax peptide affinity column. The column is eluted with 100 mM Tris/HCI, pH 8.2 buffer. The peak fractions ,containing protein are collected and analyzed on SDS-PAGE after concentrating 5-fold.
[08991 For large scale purification, the following method can be utilized.
The supernatant in excess of 2 L is diafiltered and concentrated to 500 mL in 20 mM Tris/HC1 pH
8Ø The concentrated protein solution is loaded onto a pre-equilibrated 50 mL
DEAE-SepharoaFast Flow column, the column is washed, and the protein is eluted with a linear gradient of NaCl from 0 to 0.4 M NaCI in 20 mM Tris/HCI, pH 8Ø Those fractions containing the protein are pooled, adjusted to pH 6.8 with 0.5 M sodium phosphate (NaH2PO4). A final concentration of 0.9 M (NH4)2SO4 is added to the protein solution and the whole solution is loaded onto a pre-equilibrated 50 mL Buty1650S column.
The protein is eluted with a linear gradient of ammonium sulfate (0.9 to 0 M (NH4)2SO4).
Those fractions with the albumin fusion are again pooled, diafiltered against 10 mM
Na2HPO4kitric acid buffer pH 5.75, and loaded onto a 50 mL pre-equilibrated SP-Sepharose Fast Flow column.
The protein is eluted with a NaCl linear gradient from 0 to 0.5 M. The fractions containing he protein of interest are combined, the buffer is changed to 10 mM
Na2HPO4/citric acid pH
6.25 with an Amicon concentrator, the conductivity is <2.5 mS/cm. This protein solution is loaded onto a 15 mL pre-equilibrated Q-Sepharoje" high performance column, the column is washed, and the protein is eluted with a Naa linear gradient from 0 to 0.15 M
NaCI. The purified protein can then be formulated into a specific buffer composition by buffer exchange.
EXAMPLE 5: General Construct Generation for Mammalian Cell Transfections Generation otalbuminaision consong compatible for expression in mananal' [0900) Albumin fusion constructs can be generated in expression vectors for use in mammalian cell culture systems. DNA encoding a therapeutic protein can be cloned N-terminus or C-terminus to HSA in a mammalian expression vector by standard methods known in the art (e.g., PCR amplification, restriction digestion, and ligation). Once the expression vector has been constructed, transfection into a mammalian expression system can proceed. Suitable vectors are known in the art including, but not limited to, for example, the pC4 vector, and/or vectors available from Lonza Biologics, Inc. (Portsmouth, NH).
[0901] The DNA encoding human serum albumin has been cloned into the pC4 vector which is suitable for mammalian culture systems, creating plasmid pC4:HSA (ATCC
Deposit # PTA-3277). This vector has a DiHydroFolate Reductase, "DHFR", gene that will allow for selection in the presence of methotrexate.
109021 The pC4:HSA vector is suitable for expression of albumin fusion proteins in CHO cells. For expression, in other mammalian cell culture systems, it may be desirable to subclone a fragment comprising, or alternatively consisting of, DNA which encodes for an albumin fusion protein into an alternative expression vector. For example, a fragment comprising, or alternatively consisting, of DNA which encodes for a mature albumin fusion protein may be subcloned into another expression vector including, but not limited to, any of the mammalian expression vectors described herein.
[0903) In a preferred embodiment, DNA encoding an albumin fusion construct is subcloned into vectors provided by Lonza Biologics, Inc. (Portsmouth, NH) by procedures known in the art for expression in NSO cells.
itc-1 ,n0.atbui, co A.: HSA- ;latetic j=n products.
[0904) Using pC4:HSA (ATCC Deposit it PTA-3277), albumin fusion constructs can be generated in which the Therapeutic protein portion is C terminal to the mature albumin sequence. For example, one can clone DNA encoding a Therapeutic protein of fragment or variant thereof between the Bsu 361 and Ase I restriction sites of the vector.
When cloning into the Bsu 361 and Ase I, the same primer design used to clone into the yeast vector system (SEQ ID NO:1045 and 1046) may be employed (see Example 2).
Generation of albumin fusion constructs= conprising gene-HSA fusion products.
109051 Using pC4:HSA (ATCC Deposit # PTA-3277), albumin fusion constructs can be generated in which a Therapeutic protein portion is cloned N terminal to the mature albumin sequence. For example, one can clone DNA encoding a Therapeutic protein that has its own signal sequence between the Barn HI (or Hind III) and Cla I sites of pC4:HSA. When cloning into either the Barn HI or Hind III site, it is preferrable to include a Kozak sequence (CCGCCACCATG, SEQ ID NO:1107) prior to the translational start codon of the DNA
encoding the Therapeutic protein. If a Therapeutic protein does not have a signal sequence, DNA encoding that Therapeutic protein may be cloned in between the Xho I and Cla I sites of pC4:HSA. When using the Xho I site, the following 5' (SEQ ID NO:1052) and 3' (SEQ
NO:1053) exemplary PCR primers may be used:
5'-CCqCCGCTCGAGOGGTOTGTTTCGTCGA(N)18-3' (SEQ ID NO: 1052) 5'-AGTCCCATCGATGAGCAACCTCACTCTFGTGTGCATC(N)18-3' (SEQ ID NO:1053) 109061 In the 5' primer (SEQ ID NO:1052), the underlined sequence is a Xho I site;
and the Xha I site and the DNA following the Xho I site code for the last seven amino acids of the leader sequence of natural human serum albumin. In SEQ ID NO:1052, "(N)s"
is DNA
identical to the first 18 nucleotides encoding the Therapeutic protein of interest. In the 3' primer (SEQ ID NO:1053), the underlined sequence is a Cla I site; and the Cla I site and the DNA following it are the reverse complement of the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO:1038). In SEQ ID NO:1053 "(N)18" is the reverse complement of DNA encoding the last 18 nucleotides encoding the Therapeutic protein of interest. Using these two primers, one may PCR amplify the Therapeutic protein of interest, purify the PCR product, digest it with Vic I and Cla I restriction enzymes and clone it into the Xho I and Cia I sites in the pC4:HSA vector.
109071 If an alternative leader sequence is desired, the native albumin leader sequence can be replaced with the chimeric albumin leader, i.e., the HSA-kex2 signal peptide, or an alternative leader by standard methods known in the art. (For example, one skilled in the art could routinely PCR amplify an alternate leader and subclone the PCR product into an albumin fusion construct in place of the albumin leader while maintaining the reading frame) EXAMPLE 6: General Expression in Mammalian Cell-Lines.
[0908) An albumin fusion construct generated in an expression vector compatible with expression in manunalian cell-lines can be transfected into appropriate cell-lines by calcium phosphate precipitation, lipofectamine, electroporation, or other transfection methods known in the art and/or as described in Sambrook. Fritsch, and Maniatis. 1989.
"Molecular Cloning: A Laboratory Manual, 2'd edition" and in Ausubel et al. 2000.
Massachusetts General Hospital and Harvard Medical School "Current Protocols in Molecular Biology", volumes 1-4. The transfected cells are then selected for by the presence of a selecting agent determined by the selectable marker in the expression vector.
[09091 The pC4 expression vector (ATCC Accession No. 209646) is a derivative of the plastnid pSV2-DHFR (ATCC Accession No. 37146)., pC4 contains the strong promoter Long Terminal Repeats "LT1t" of the Rous Sarcoma Viros (Cullen et at., March 1985, Molecular and Cellular Biology, 438-447) and a fragment of the CytoMegaloVirus "CMV"-enhancer (Boshart et al., 1985, Cell 41: 521-530). The vector also contains the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse MIR
gene under control of the SV40 early promoter.. Chinese hamster ovary "CHO"
cells or other cell-lines lacking an active MIER gene are used for transfecticin.
Transfection of an albumin fusion construct in pC4 into CHO cells by methods known in the art will allow for the expression of the albumin fusion protein in CHO cells, followed by leader sequence cleavage, and secretion into the supernatant. The albumin fusion protein is then further purified from the supernatant.
109101 The pEE12.1 expression vector is provided by Lonza Biologics, Inc.
(Portsmouth, NH) and is a derivative of pEE6 (Stephens and Cockett, 1989, Nucl. Acids Res.
17: 7110). This vector comprises a promoter, enhancer and complete 5'-untranslated region of the Major Immediate Early gene of the human CytoMegaloVirus, "hCMV-MIE"
(International Publication # W089/01036), upstream of a sequence of interest, and a Glutamine Synthetase gene (Murphy et al., 1991, Biochem J. 227: 277-279;
Bebbington et al., 1992, Bio/Technology 10:169-175; US patent US 5,122,464) for purposes of selection of transfected cells in selective methionine sulphoximine containing medium.
Transfection of albumin fusion constructs made in pEE12.1 into NSO cells (International Publication #
W086/05807) by methods known in the art will allow for the expression of the albumin fusion protein in NSO cells, followed by leader sequence cleavage, and secretion into the supernatant. The albumin fusion protein is then further purified from the supernatant using techniques described herein or otherwise known in the art.
(0911) Expression of an albumin fusion protein may be analyzed, for example, by SDS-PAGE and Western blot, reversed phase HPLC analysis, or other methods known in the art.
10912) Stable CHO and NSO cell-lines transfected with albumin fusion constructs are generated by methods known in the art (e.g., lipofectamine transfection) and selected, for example, with 100 nM methotrexate for vectors having the DiHydroFolate Reductase 'DEER' gene as a selectable marker or through growth in the absence of glutamine.
Expression levels can be examined for example, by hninunoblotting, primarily, with an anti-HSA serum as the primary antibody, or, secondarily, with serum containing antibodies directed to the Therapeutic protein portion of a given albumin fusion protein as the primary antibody.
[0913) Expression levels are examined by immunoblot detection with anti-HSA
serum as the primary antibody. The specific productivity rates are determined via ELISA in which the capture antibody can be a monoclonal antibody towards the therapeutic protein portion of the albumin fusion and the detecting antibody can be the monoclonal anti-HSA-biotinylated antibocly (or vice versa), followed by horseradish peroxidase/streptavidin binding and analysis according to the manufacturer's protocol.
EXAMPLE 'It General Purification of an Albumin Fusion Protein Expressed from an Albumin Fusion Construct in Mammalian Cell-lines.
10914] In preferred embodiments, albumin fusion proteins of the invention comprise the mature form of HSA fused to either the N- or C- terminus of the mature form of a therapeutic protein or portions thereof (e.g., the mature form of a therapeutic protein listed in Table 1, or the mature form of a therapeutic protein shown in Table 2 as SEQ
ID NO:Z). In one embodiment of the invention, albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature albumin fusion protein is secreted directly into the ulture medium. Albumin fusion proteins of the invention preferably comprise hcterologous signal sequences (e.g., the non-native signal sequence of a particular therapeutic protein) including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or= other heterologous signal sequences known in the art.
Especially preferred as those signal sequence listed in Table 2 and/or the signal sequence listed in the "Expression of Fusion Proteins" and/or "Additional Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins" section of the specification, above. In preferred embodiments, the fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
109151 Albumin fusion proteins from mammalian cell-line supernatants are purified according to different protocols depending on the expression system used.
Purifleationfrom CHO and 293T cell-iines.
[0916] Purification of an albumin fusion protein from CHO cell supernatant or from transiently transfected 293T cell supernatant may involve initial capture with an anionic HQ
resin using a sodium phosphate buffer and a phosphate gr _____________ dient elution, followed by affinity chromatography on a Blue Sepharos;m FF column using a salt gradient elution.
Blue SepharosemFF removes the main BSAJfetuin contaminants. Further purification over the Poros PI 50 resin with a phosphate gradient may remove and lower endotoxin contamination as well as concentrate the albumin fusion protein.
Purification from NSO cell-line.
109171 Purification of an albumin-fusion protein from NSO cell supernatant may involve Q-SepharosTemanion exchange chromatography, followed by SP-sepharosTpurification with a step elution, followed by Phenyl-650M purification with a step elution, and, ultimately, diafiltration.
109181 The purified protein may then be formulated by buffer exchange.
EXAMPLE 8: Construct ID 1966, EPO-HSA, Generation;
[0919] Construct ID 1966, pC4.EPO:MI-D192.HSA, encodes for an EPO-HSA
fusion protein which comprises the EPO native leader sequence as well as the mature EPO

protein with the exception of the final Arg residue, i.e., MI -D192, fused to the amino-terminus of the mature form of HSA cloned into the mammalian expression vector pC4.
Cloning of EPO cDNA for construct 1966 [09201 The DNA encoding EPO was amplified with primers EPO1 and EP02, described below, cut with Barn IIIICla I, and ligated into Barn 1111Cla I cut pC4:HSA.
Construct ID #1966 encodes an albumin fusion protein containing the leader sequence and the mature form of EPO, followed by the mature HSA protein (see SEQ NO:297 for construct 1966 in table 2).
109211 Two oligonucleotides suitable for PCR amplification of the polynucleotide encoding the full length EPO including the natural leader sequence (SEQ ID
NO:81, table 2), EPO1 and EP02, were synthesized.
EPO I : 5 ',-GACTGGA TCCGCCACCATGGGGGTGCACGAATGIVCTGCCMGCTGTGGCTTCT
CCTGTCCMCIVTOCCIreCTICTGGGCMCCACTCCEGGCCOCCCCACCACGCCIVATCT
GTGAC,3' (SEQ ID NO: 1122) EPO2 : 5AGIVCCATCOATGAGCAACCICACirriarGIMATCGTCCCCIVTICCMCACvGe CTCc-3' (SEQ ID NO: 804) 109221 EPO1 incorporates a Barn HI cloning site (shown in italics) and attaches a kozak sequence (shown double underlined) prior to the DNA encoding the first 35 amino acids of the ORF of the full-length EPO. In RP02, the underlined sequence is a Cla I site;
and the Cla I site and the DNA following it are the reverse complement of DNA
encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO:1038). In EP02, the bolded sequence is the reverse complement of the last 22 nucleotides encoding amino acid residues .
Glu-186 to Asp-192 of the full-length form of EPO, with the exception of the final Arg residue. Using these two primers, the MB-length EPO protein, with the exception of the final Arg residue, was PCR amplified. Annealing and extension temperatures and times must be empirically determined for each specific primer pair and template.
[09231 The PCR product was purified (for example, using Wizard PCR Preps DNA
Purification System (Promega Corp)) and then digested with Barn HI and Cla I.
After further purification of the Barn HI-Cla I fragment by gel electrophoresis, the product was cloned into BantillICla I digested pC4:HSA to produce construct ID # 1966.
[09241 Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing confirmed the presence of the expected EPO sequence (see below).

(0925] EPO albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of EPO lacking the final Arg residue, i.e., Ala-28 to Asp-192. In one embodiment of the invention, EPO albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature EPO albumin fusion protein is secreted directly into the culture medium. EPO albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAP, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, EPO albumin fusion proteins of the invention comprise the native EPO signal sequence. In further preferred embodiments, the EPO albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants.
are also encompassed by the invention.
Exsression and Purification of Construct 11)1966.
Expression in either 2937' or CHO cells.
[09261 Construct 1966 was transfected into either 293T cells or CHO cells by methods known in the art (e.g., lipofectamine transfection) and selected with 100 nM
methotrexate (see Example 6). Expression levels were examined by inmnmoblot detection with anti-HSA serum as the primary antibody, and the specific productivity rates were determined via ELISA using a monoclonal anti-human EPO antibody (Research Diagnostics, Inc.) for capture and a Biotrend monoclonal anti-HSA-biotinylated antibody for detection, followed by horseradish peroxidase/streptavidin binding and analysis.
Purification from 293T cell supernatant.
[09271 The 293T cell supernatant containing the secreted EPO-HSA fusion protein expressed from construct BD #1966 in 293T cells was purified as described in Example 7.
Specifically, initial capture was performed with an anionic HQ-50 resin at pH
7.2 using a step elution, followed by Blue sepharosTeFF chromatography again employing a step elution at pH
7.2. The pooled fractions were passed over the HQ-50 resin again using a step elution. The eluted sample was then loaded onto the Phenyl-650M column and eluted with a gradient elution at pH 7.2. The eluted sample was passed over the HQ-50 resin for a third time. The fractions of interest were diafiltrated into 50 mM Na2HPO4 + 200 mivl NaC1 pH
7.2. N-terminal sequencing generated the amino-terminus sequence (i.e., APPRL1) of the mature form of EPO. A protein of approximate MW of 90 kDa was obtained. A final yield of 0.42 mg protein per litre of 293T cell supernatant was obtained.
Purification from CHO cell supernatant.
109281 The cell supernatant containing the EPO-albumin fusion protein expressed from construct ID #1966 in CHO cells was purified as described in Example 7.
Specifically, initial cpture of a concentrated 1.4 L sample was performed with an anionic Poros HQ 50 resin using a sodium phosphate buffer and a phosphate gradient elution (0¨ 100 mM sodium phosphate, pH 7.2). Prior to loading the column, the sample was diluted with 3 =NI
phosphate until the conductivity was lower than 5.0 mS, as was the case for further column chromatography purifications. The HQ resin was equilibrated with 10 mM sodium phosphate, pH 7.2 prior to sample loading. EPO-HSA eluted at 20 mS, or 50 mM
sodium phosphate. The second purification step involved affinity chromatography. The combined fractions from the previous HQ resin elution, adjusted for a conductivity <5 mS using 3 mM
phosphate pH 7.2 buffer, were loaded onto a Blue SephardeFF column equilibrated with 125 1W NaC1, 15 mM sodium phosphate, pH 7.2. A salt gradient of ¨ 3 M NaCI
eluted EPO-HSA between 0.5 M and 1.0 M NaCi. Blue Sepharosem FY removes the main BSAifetuin contaminants. The conductivity of the desired fractions was again adjusted for, and the pooled fractions were loaded onto a third column containing Poros PI
50 resin which removes and lowers endotoxin contamination as well as concentrates the EPO-HSA
protein.
The resin was equilibrated with 25 mM NaC1, 10 mM sodium phosphate, pH 7.2.
EPO-HSA
was eluted with a 10 mM ¨ 100 mM phosphate gradient. The final buffer composition was 100 mM Na.C1, 20 mM Na2HPO4, pH 7.2. An approximate protein MW of 87.7 kDa was obtained. A final yield of 8.9 mg protein per liter of supernatant was obtained.. N-Terminal sequencing generated the sequence APPRL which corresponds to the amino-terminus of the mature form of EPO.
In vitro TF-1 cell proliferation assay.
Method [0929] The biological activity of an EPO albumin fusion protein can be measured in an in vitro TF-1 cell proliferation assay. The TF-1 cell-line was established by Kitamura et al. (Kitamura, T. et al., 1989, J. Cell. Physiol., 140: 323 -- 334). The TF-1 cells were derived from a heparinized bone man-ow aspiration sample from a 35 year old Japanese male with severe pancytopenia. The TF-1 cell-line provides a good system for investigating the proliferation and differentiation of myeloid progenitor cells as a result of its responsiveness to multiple cytokines.
(0930) TF-1 cell proliferation assay (Kitamura, T. et al., 1989, J. Cell.
Physiol., 140:
323 ¨ 334): Human TF-1 cells (ATCC # CRL-2003) are expanded in RPMI 1640 media containing 10% FBS, 1X pen-strep, IX L-glutarnine, and 2 ng/mL human GM-CSF to a maximum density of 1 x 106 cells/mL. Cells are passaged every 2-3 days by diluting 1:10 or 1:20 in fresh medium. On the day of the assay initiation, cells are washed in a 50 mL volume of RPM! 1640/10% FBS three times to remove GM-CSF and are resuspended at 1x105 cells/mL in RPIVL1 1640/10% FBS. Cells are plated at 10,000 cells/well in fiat-bottom TC-treated 96-well plates. Three-fold serial dilutions of control protein are made in RPMI
1640/10% FBS in a range of 10 U/mL to 0.001 UhriL (final concentration) and three-fold serial dilutions of an albumin fusion protein are made in RPMI 1640/10% FBS in a range of 100 ng/mL to 0.01 ng/mL (final concentration) where 1 U = 10 ng protein; 0.1 mL of each dilution is added to triplicate wells containing cells for a final volume of 0.2 mL in each well.
Cell proliferation response to the control protein and the albumin fusion protein is determined by measuring incorporation of 3H-thymidine (0.5 uCi/well). The assay is carried out at incubation times of 24, 48, or 72 hours prior to and for 4 ¨ 24 hours after the addition of 3H-thymidine. Since only a portion of the molar weight of an albumin fusion protein is actually a therapeutic protein molecule (i.e., the therapeutic protein portion of the fusion), in some cases dilutions may also be adjusted for the molar ratio.
In vitro MI cell proWeration assay for the gibigninAtion Pr_etein encode ke construct 1966.
Method 109311 IF-1 cell proliferation assay: Human IF-1 cells (ATCC # CRL-2003) were expanded in RPMI 1640 media containing 10% FBS, 1X pen-strep, 1X L-glutamine, and 2 ng/mL human GM-CSF to a maximum density of 1 x 106 cells/mL. Cells were passaged every 2-3 days by diluting 1:10 or 1:20 in fresh medium. On the day of the assay initiation, cells were washed in a 50 raL volume of RPMI 1640/10% FBS three times to remove GM-CSF and were resuspended at 1 x105 cells/mL in RPMI 1640/10% FBS. Cells were plated at 10,000 cells/well in flat-bottom TC-treated 96-well plates. Three-fold serial dilutions of hrEPO (R&D Systems; Research Diagnostics Inc., RIM) were made in RPMI 1640/10%
FES
in a range of 10 U/mL to 0.001 UlmL (final concentration) and three-fold serial dilutions of the EPO albumin fusion protein were made in RPMI 1640/10% FES in a range of 100 ng/mL
to 0.01 ng/mL (final concentration) where 1 U = 10 ng protein; 0.1 mL of each dilution was added to triplicate wells containing cells for a final volume of 0.2 inL in each well. Cell proliferation response to hrEPO and EPO albumin protein was determined by measuring incorporation of 3H-thymidine (0.5 laCi/well). The assay was carried out at incubation times of 24, 48, or 72 hours prior to and for 4-24 hours after the addition of 3H-thymidine. Since only 1/3 of the molar weight of the EPO albumin fusion protein is actually an EPO molecule, in some cases dilutions made were also to adjust for the molar ratio.
Results [0932] Supernatants from 293T cells expressing construct 1966 or >90%
purified EPO-HSA albumin fusion protein derived from CHO cells expressing construct 1966 were tested in the above assay for EPO activity. On average, an EC50 of greater than 5 fold of that of rhEPO was established (see Figure 4). = -In vivo Harlan mouse modelfor measuring hematocrit.
Methods [0933] This mouse model provides the means to measure the therapeutic activity of a protein in vivo by measuring its effect on the .hematocrit.
[0934] An in vivo mouse model, i.e., 6 ¨ 8 week old female DBA/2NHsd mice (Harlan), has been established to monitor the effect on hematocrit upon administration of a control protein at 2 ii,g/kg and at other concentrations or an albumin fusion protein at 30 Ag/kg and at other concentrations daily or every other day for 7 days either intravenously, intraperitoneally, or subcutaneously. Hematocrit is determined by sticking the tail vein with a needle, collecting the blood with a heparinized microcapillary tube, and then spinning the tubes throughout the experimental time-frame. Also, for certain experiments, the spleen is harvested and weighed. Other dosing schedules are blown within the art and can readily be adapted for use in this assay.
2Ag activity of the albumin fusion protein entkii hv corattrtict,k 66 can be assayed usinzan in vivo Harlan mouse model for measuring hernatocrit.

Methods 109351 An in vivo mouse model of 6 ¨ 8 week old female DBA/2NHsd mice (Harlan) was used to monitor the extent of EPO activity upon administration of rhEPO
(Research Diagnostics, Inc., cat # RDI-PB11965) at doses of 0.5, 1.5, 4.5, and 12 ig/kg on days 0, 2, 4 and 6 and upon administration of the purified EPO albumin fusion protein encoded by construct 1966 at concentrations of 2, 6, 18, and 54 lig/kg on days 0, 2, 4, and 6 subcutaneously, "SC". Hematocrit was determined by sticking the tail vein with a needle on days 0 and 7, collecting the blood with a heparinized microcapillary tube, and then spinning the tubes throughout the experimental time-frame. The higher doses of the EPO
albumin fusion protein is a rough equimolar comparison with the control recombinant human EPO, "rhEPO" (Research Diagnostics, Inc., cat # RDI-PB11965).
Results [0936] There was a significant increase in hematocrit (see Figure 5) from day 0 to day 7 for animals treated with either recombinant human EPO or EPO albumin fusion proteins.
However, the EPO albumin fusion protein encoded by- construct 1966 appeared to have a more drastic effect on hematocrit levels than the rhEPO control. Subcutaneous administration of 3 doses/week of 52 tig/kg, or 1 dose/week of 156 ggikg, of the EPO
albumin fusion protein encoded by construct 1966 caused a greater than or equal to 40%
change in hematocrit from day 0 to day 8 (see Figure 6). The % change in hematocrit was either maintained close to 40% for the triple dose or subdued to ¨20% for the single dose on =
day 14 as opposed to a decline from close to 30% to <10% for a 3 dose subcutaneous administration of 12 irg/kg of rhEPO in a week. The elevated hematocrit appears to be maintained with the EPO albumin fusion protein encoded by construct 1966 over a period of a week after the last subcutaneous administration in comparison with the hematocrit levels induced by the rhEPO protein which declines back to more normal levels.
[0937] DBA mice injected intravenously with a 150 ig/kg dose of the EPO
albumin fusion protein encoded by albumin fusion construct 1966 cleared this EPO
albumin fusion protein 7 times more slowly than rhEPO.
EXAMPLE 9: Construct ID 1981, HSA-EPO, Generation.
[09381 Construct ID 1981, pC4.HSA-EPO.A28-D192, comprises DNA encoding for an EPO albumin fusion protein which has the HSA full-length sequence, including the native HSA leader sequence, fused to the amino terminus of the mature form of EPO, with the exception of the final Arg residue, cloned into the mammalian expression vector pC4.
Cloning of EPO cDNA for construct 1981 [0939) The DNA encoding EPO was amplified with primers EP03 and EP04, described below, cut with Bsu 36IlAsc I, and ligated into Bsu 3611Asc I cut pC4:HSA.
Construct ID #1981 encodes an albumin fusion protein containing the native leader sequence and mature form of HSA and the mature form of EPO, Ala 28 to Asp 192 (Genbank Accession AAA52400).
[09401 Two oligonucleotides suitable for PCR amplification of the polynucleotide encoding the mature form of EPO (see SEQ ID NO:X for construct 1981 in Table 2), EPO3 and EP04, were synthesized:
EPO3: 5'. AAGCTGCCTTAGGCTTAGCCCCACCACGCCTCATCTGTGACAG -3' (SEQ ri3 NO: 805) EP04: 5'. GCGCGGCGCGCCGAATTCCTATT AGTCCCCTGTCCTGCAGGCCTCCCUIUTG -3' (SEQ ID
NO: 806) [09411 EPO3 incorporates a Bsu 361 cloning site (shown underlined) and nucleotides encoding 'the last four amino acid residues of the mature form of HSA, as well as 26 nucleotides, italicized, encoding the first 8 amino acid residues of the mature form of EPO.
In EP04, the Asc I site is underlined (SEQ ID NO:806) and the last 28 nucleotides, italicized, are the reverse complement of DNA encoding the last 9 amino acid residues of EPO (for general construct cloning see Example 5) , with the exception of the final Arg residue. The PCR amplimer generated using these primers was purified, digested with Bsu 361 and Asc I
restriction enzymes, and cloned into the Bsu 361 and )(Ise I sites of the pC4:HSA vector.
[09421 The PCR product was purified (for example, by using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested with Bsu36I and AscI. After further purification of the Bsu36I-AscI fragment by gel electrophoresis, the product was cloned into Bsu3611AscI digested pC4:HSA to give construct ID # 1981.
[09431 Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing confirmed the presence of the expected HSA sequence (see below).
109441 EPO albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of EPO lacking the final Arg residue, i.e., Ala-28 to Asp-192. In one embodiment of the invention, EPO albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature EPO albumin fusion protein is secreted directly into the culture medium. EPO albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAP, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HS.AJMAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, EPO albumin fusion proteins of the invention comprise the native EPO signal sequence. In further preferred embodiments, the EPO albumin fusion proteins of the invention further comprise an N-terminal rnethionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Purification of Construct ID 19j, Expression in CHO cells.
[09451 Construct 1981 was transfected into CHO cells as described in Examples 6 and 8. Expression levels and the specific productivity rates were determined as described in Example 8. =
Purification from CHO supernatant.
[09461 The cell supernatant containing the EPO albumin fusion protein expressed from construct ID #1981 in CHO cells was purified as described in Examples 7 and 8. N-terminal sequencing generated DAHKS, the sequence of the amino terminus of the mature form of HSA. For each litre of supernatant, 14 mg of protein was obtained. An approximate MW of 85.7 kDa was obtained.
In vitro TF-1 cell proliferation assay for construct 1981:
Method [09471 The in vitro TF-1 cell proliferation assay for the EPO albumin fusion protein encoded by construct 1981 was carried out as previously described in Example 8 under subsection heading "In vitro TF-1 cell proliferation assay for construct 1966".
Results 10948) Supernatants from CHO cells expressing construct 1981 were >90%
purified for the USA¨EPO albumin fusion protein and were tested in the assay, as described in Example 8. On average, an EC50 of greater than 5 fold of that of rhEPO was established (see Figures 4 and 7).
rhg activity of conskupt 198.1 can be. twayed using an in vivo Ifarign mouse model for measuring hematocrit.
Methods (0949) The in vivo Harlan mouse model was used to assay for hematocrit levels upon subcutaneous administration of either control rhEPO or EPO albumin fusion protein encoded by construct 1981. The assay was carried out as previously described in Example 8 under subsection heading "The activity of construct 1966 can be assayed using an in vivo Harlan mouse model for measuring hematocrit".
Results [09501 There was a significant increase in hematocrit (see Figure 5) from day 0 to day 7 for animals treated with either rhEPO or E1'0 albumin fusion proteins.
However, the EPO
albumin fusion protein encoded by construct 1981 appears to have a more drastic effect on hematocrit levels than the rhEPO control.
[0951) DBA mice injected intravenously with a 150 pg/kg dose of EPO
albumin fusion protein encoded by albumin fusion construct 1981 cleared this EPO
albumin fusion protein 7 times more slowly than rhEPO.
EXAMPLE 10: Construct ID 1997, EPO-11S4, Generation.
[0952] Construct ID 1997, pEE12.1:EPO MI-D192.11SA, comprises DNA encoding an EPO albumin fusion protein which has the full-length EPO protein (including the native leader sequence), i.e.. MI-D192, with the exception of the final Arg residue, fused to the amino-terminus of the mature form of HSA cloned into the mammalian expression vector pEE12.I.
Cloning of EPO cDNA for construct 1997.
[09531 The DNA encoding EPO was amplified with primers EP05 and EP06, described below, cut with Eco RIICla 1, and ligated into Eco RI]Cla I cut pcDNA3 (Invitrogen Corporation, 1600 Faraday Ave, Carlsbad, CA 92008). pcDNA3.EPO MI-D192.HSA was digested with Eco RI1Hind 111 to release the EPO M1 -D192.HSA
expression cassette fragment and cloned into Eco III
digested pEE12.1. Construct ID #1997 encodes an albumin fusion protein containing the leader sequence and the mature form of EPO, followed by the mature HSA protein (see SEQ ID NO:Y in Table 2 for construct 1997).
[09541 Two oligonucleotides suitable for PCR amplification of the polynucleotide encoding EPO (SEQ NO:X, Table 2 for construct 1997), EPO5 and EP06, were synthesized.
EP05:5'-GATCGAil TTCGCCACCATGGGGGTGCACGAATMCCMCCTOGCTGIGGCTTCTCCrGICC
CTGCMTCGC'TCCCTCTGGGCCIVCCAGTCCTGGGCGCCCCACCACGCCTCATCT'OTGAC- 3' (SEQ
ro NO: 775) EP06:51-CITTAAAMOVC34.0CAACCTCAtirrraCITOCATCOTCCCCIGTCCIVCAOGOCTCOC-3' (SEQ ID NO: 776) [09551 EPOS
incorporates an Eco RI site (shown in italics) and a kozak sequence (shown underlined) prior to the DNA encoding the first 35 amino acids of the ORF of the MI-length EPO. In EP06, the italicized sequence is a Cia I site, the underlined sequence is the reverse complement of the DNA encoding the first 9 amino acids of the mature form of USA protein (D.AIIICSEVAH, SEQ NO:1106), and the sequence following the =reverse complement of B[SA is the reverse complement of the last 23 nucleotides encoding the last 7 amino acids of EPO not including the final Arg-193 amino acid. Using these two primers, DNA encoding the full-length EPO protein was PCR amplified as in Example 8.
109561 The PCR
product was purified and then digested with Eco RI and Cla I. After further purification of the Eco. RI-Cla I fragment by gel electrophoresis, the product was cloned into Eco RI1Cla I digested pcDNA3. The Eco ILI
fragment containing the expression cassette was generated from pcDNA3.EPO.M1-D192.HSA and subcloned into the Eco RI/Hind III digested pEE12.1 to give construct ID # 1997.
[09571 Further, analysis of the N-terminus of the albumin fusion protein by amino acid sequencing confirmed the presence of the expected EPO sequence (see below).

albumin fusion proteins of the invention preferably comprise the mature form of USA, i.e., Asp-25 to Len-609, fused to either the N- or C- terminus of the mature form of EPO lacking the final Arg residue, i.e., Ala-28 to Asp-192. In one embodiment of the invention, EPO albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature EPO albumin fusion protein is secreted directly into the culture medium. EPO albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin 13, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, EPO albumin fusion proteins of the invention comprise the native EPO signal sequence. In further preferred embodiments, the EPO albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Purification of Construct ID 1997.
Expression in NSO cells.
[09591 Construct 1997 was transfected into NSO cells as described in Example 6.
Expression levels and specific productivity rates were determined as described in Example 8.
Purification from NSO cell supernatant.
[09601 Purification of the EPO albumin fusion protein from 500 rtiL cell supernatant from NSO cells transfected with construct 1997 involves Q-Sepharose anion exchange chromatography at pH 7.4 using a NaC1 gradient from 0 to 1 M in 20 mM Tris-HC1, followed by Poros P150 anion exchange chromatography at pH 6.5 with a sodium citrate gradient from to 40 mM, and diafilirating for 6 DV into 10 mM citrate, pH 6.5 and 140 mM
NaC1, the final buffer composition (see, Example 7). N-terminal sequencing yielded the sequence APPRLI which is the amino terminus of the mature form of EPO. The protein has an approximate MW of 87.7 kDa. A final yield of 52.2 mg protein per L of supernatant was obtained.
[0961] For larger scale purification, 50 L of NSO cell supernatant can be concentrated into ¨8 to 10 L. The concentrated sample can then be passed over the Q-Sepharosg4anion exchange column (10 x 19 cm, 1.5 L) at pH 7.5 using a step elution consisting of 50 mM
Na0Ac, pH 6.0 and 150 mM NaCI. The eluted sample can then be virally inactivated with 0.75% TritoirX 100 for 60 min at room temperature. SDR-Reverse Phase chromatography (10 cm x 10 cm, 0.8 L) can then be employed at pH 6.0 with 50 mM Na0Ac and 150 mM
NaC1, or alternatively, the sample can be passed over an SP-sepharosecoluron at pH 4.8 using a step elution of 50 mM Na0Ac, pll 6.0, and 150 mM NaCI. DV 50 filtration would follow to remove any viral content. Phenyl-650M chromatography (20 cm x 12 cm, 3.8 L) at pll 6.0 using a step elution consisting of 350 mM (N114)2SO4 and 50 mM Na0A.c, or alternatively consisting of 50 mM Na0Ac pH 6.0, can follow. Diafiltration for 6-8 DV will allow for buffer exchange into the desired final formulation buffer of either 10 mm Na2HPO4 + 58 mM sucrose + 120 mM NaCI, pH 7.2 or I 0 mM citrate, pH 6.5, and 140 mM
NaCI.
In viola TF-1 cell prolfersition assayfor conentet 109-1 Method 109621 The in vitro TF-1 cell proliferation assay for the EPO-HsA albumin fusion encoded by construct 1997 was carried out as previously described in Example 8 under subsection heading "In vitro TF-1 cell proliferation assay for construct 1966".
Results 109631 Supernatants from NSO cells expressing construct 1997 were >90%
purified for the EPO-HSA albumin fusion protein and were tested in the assay, as described in Example 8. On average, an EC50 of greater than 5 fold of that of rhEPO was established (see Figure 7).
The activity of construct 1997 can be assayed using an 4n vivo liarlan_moge_n_Loiletk measuring hematocrit.
Methods 109641 The in vivo Harlan mouse model was used to assay for hematocrit levels upon subcutaneous administration of either control rhEPO or EPO albumin fusion protein encoded by construct 1981 at various doses on days 0, 2, 4, and 6. The assay was carried out as previously described in Example 8 under subsection heading "The activity of construct 1966 can be assayed using an in vivo Harlan mouse model for measuring hematocrit".
Hematocrit was determined on days 0, 8, and 14.
Results 109651 There was a significant and similar increase in hematocrit (see Figure 8) from day 0 to day 8 for animals treated with either rhEPO or the EPO albumin fusion encoded by construct 1997. However, as was the case for the EPO albumin fusion protein encoded by construct 1966 but to a lesser extent, subcutaneous administration of 3 doses/week of 52 ug/kg of EPO albumin fusion encoded by construct 1997 caused close to 30%
change in hematocrit from day 0 to day 8 and subdued to ¨15% on day 14 (see Figure 6) as opposed to a decline from close to 30% to <10% for a triple dose of 12 ug/kg subcutaneous administration of rhEPO per week.

[09661 DBA mice injected intravenously with a 150 tg/kg dose of EPO-HSA
cleared this EPO albumin fusion 7 times more slowly than rhEPO.
EXAMPLE 11: Construct ID 2294, EPO-HSA, Generation.
[09671 Construct ID 2294, pC4.EPO.R140G.HSA, comprises DNA encoding an EPO-HSA fusion protein which has the full-length EPO protein including the native leader sequence of the EPO protein, with the exception of the final Arg residue, i.e., Ml-D192, with a point mutation mutating Arg-140 to Gly, fused to the amino-terminus of the mature form of HSA cloned into the mammalian expression vector pC4.
Cloninz of EPO cDNA for construct 2294.
[09681 Construct ID #2294 encodes an albumin fusion protein containing the leader sequence and the mature form of EPO, followed by the mature HSA protein.
Construct ID
#2294 was generated by using construct ID #1966, i.e., pC4:EPO.M1-D192.HSA) as a template in a two-step PCR method.
109691 Four oligonucleotides suitable for PCR amplification of the polynucleotide encoding EPO (SEQ ID NO:X for construct 2294, table 2), EPO7, EPOS, EP09, and EP010, were synthesized.
EPO7: __ GGA TCCGCCACCATGGGGGTGCACGAATGT(primer 82848)¨ 3' (SEQ ID NO: 915) EPOS: 5'-CCITCTGGGCTCCCAGAGCCCGAAG (primer 82847)-3' (SEQ ID NO: 1123) EP09: 5'-CATTATC,Q_ATGAGCAACCTCACTCTIGTGTGCATCGTCCC (primer 82849)-3' (SEQ ID
NO: 916) EP010: Y-CITCGGOCTCTGGGAGCCCAGAAGG (primer 82846)-3' (SEQ ED NO: 1124) [0970] In -the first round of PCR amplifications, the N-terminal and the C-terminal fragments of construct ID 1966 were independently amplified. The N-terminal fragment was generated using primers EPO7 and EPOS. EPO7 incorporates Barn HI (shown in italics) and has a kozak sequence (shown underlined) prior to the first 18 nucleotides encoding the first 6 amino acids of the ORF of the full-length EPO. The EPOS primer comprises the reverse complement of the sequence spanning amino acids 136 to 143 of the full-length form of EPO
with the exception that the codon CGA encoding the Arg residue at amino acid (highlighted in bold) is altered to the codon GGA which encodes a Gly residue.
The C-terminal fragment was generated using primers EPO9 and EP010. In EP09, the underlined sequence is a Cla I site; and the Cla I site and the DNA following it are the reverse complement of DNA encoding the first 10 amino acids of the mature USA protein (SEQ ID
NO:1038). In EP09, the last 5 nucleotides correspond to the reverse complement of the last nucleotides in the full-length EPO, which lacks the final Arg-193 residue. The primer comprises the nucleic acid sequence encoding amino acids 136 to 143 of the full-length form of EPO with the exception that the codon CGA encoding the Arg residue at amino acid 140 (highlighted in bold) is altered to the codon GGA which encodes a Gly residue. In the second round of PCR amplifications, primers EPO7 and EPO9 were used to amplify the full-length of EPO with the Arg-140 to Gly mutation in which the reaction mixture contained both the PCR amplified N-tenninal fragment and the PCR
amplified C-terminal fragment.
[09711 The PCR
product was purified and then digested with Barn HI and Cla I.
After further purification of the Barn I
fragment by gel electrophoresis, the product was cloned into Barn HUM I digested pC4:HSA to give construct ID # 2294.
(09721 Further, analysis of the N-terminus of the albumin fusion protein by amino acid sequencing can confirm the presence of the expected_EPO sequence (see below).

albumin fusion proteins of the invention preferably comprise the mature form of USA, i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of EPO lacking the final Arg residue, i.e., Ala-28 to Asp-192. In one embodiment of the invention, EPO albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, theµsignal peptide encoded by the signal sequence is removed, and the mature EPO albumin fusion protein is secreted directly into the culture medium. EPO albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAP, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant USA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, EPO albumin fusion proteins of the invention comprise the native EPO signal sequence. In further preferred embodiments, the EPO albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
pi ession wation oConm_L-1õuct ID 2294 Expression in CHO cells.
[09741 Construct 2294 can be tmnsfected into CHO cells as described in Examples 6 and 8. Expression levels and specific productivity rates can be determined as described in Example 8.
Purification from CHO supernatant.
109751 The cell supernatant containing the EPO-HSA fusion protein expressed from construct ID #2294 in CHO cells can be purified as in Examples 7 and 8. N-tenminal sequencing should yield the sequence AF'PRLI (SEQ ID NO:2141) which corresponds to the amino terminus of the mature form of EPO and should yield a protein of approximate MW of 87.7 IsDa.
In vitro 77-1 cell proliferation assay for construct 2294.
Method 109761 The in vitro TF-1 cell proliferation assay for the EPO-HSA albumin fusion encoded by construct 2294 can be carried out as previously deseribed in Example 8 under . subsection heading "In vitro TF-1 cell proliferation assay for construct 1966"..
The activi * construct 2391 can be assayed using an _in VIVO Harlan mouse model for measuring hematocrit.
[09771 The in vivo Harlan mouse model as previously described in Example 8 under subsection heading, "In vivo Harlan mouse model for measuring hematocrit", can be used to measure hematocrit levels for the EPO albumin fusion protein encoded by construct 2294.
EXAMPLE 12: Construct ID 2298., EPO-HSA, Generation.
[0978) Construct BT.) 2298, pEE12.1:EPO.R140G.HSA, comprises DNA encoding an EPO albumin fusion protein which has the full-length EPO protein (including the native leader sequence), with the exception of the final Arg residue, i.e., MI-D1.92, with a point mutation mutating Arg-140 to Gly, fused to the amino-terminus of the mature form of HSA
cloned into the mammalian expression vector pEE12.1.
CloninR of EPO cDNA for construct 2298 [09791 Construct ID #2298 encodes an albumin fusion protein containing the leader sequence and the mature form of EPO, followed by the mature HSA protein.
Construct ID

#2298 was generated by using construct ID #1997, i.e., pEE12.1:EPO.M1-D192.HSA) as a template for PCR mutagenesis.
[0980] Two oligonucleotides suitable for PCR amplification of template of construct ID #1997, EPO1 1 and EP012, were synthesized.
EPO 1 1 : 51-GOCITCCI rt:TOGGCMCcAGAOCOMAAGCAO-3* (SEQ ID NO: 924) EP012: 5-CTOCtretHICKIMEIGGAGCCCAGAAGOAAOCC4' (SEQ ID NO: 923) 109811 The EPO1 1 anti-sense primer comprises the reverse complement of the sequence spanning amino acids 135 to 145 of the full-length form of EPO with the exception that the codon CGA encoding the Arg residue at amino acid 140 (highlighted in hold) is altered to the clarion GGA which encodes a Gly residue. The EP012 sense primer comprises the nucleic acid sequence encoding amino acids 135 to 145 of the full-length form of EPO
with the exception that the codon CGA encoding the Arg residue at amino acid (highlighted in bold) is altered to the codon GGA which encodes a Gly residue.
Using the Site Directed Mutagenesis kit and protocol from Stratagene, the PCR reaction generated the whole template of construct ID #1997 with the exception of the Arg to Gly mutation. The PCR product was digested with Dpn I, transformed into competent XL1 Blue bacteria, and colonies were sequenced and confirmed. The Dpn I endonuclease is specific for methylated and hemimethylated DNA and targets the sequence 5'-GmATC-3'. Dpn I is used to digest the parental DNA template so as to select the mutation-containing synthesized DNA.
109821 Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected EPO sequence (see below).
[0983) EPO albumin fusion proteins of the invention preferably comprise the mature form of liSA i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of EPO lacking the final Arg residue, i.e., Ala-28 to Asp-192. In one embodiment of the invention, EPO albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature EPO albumin fusion protein is secreted directly into the culture medium. EPO albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, EPO albumin fusion proteins of the invention comprise the native EPO signal sequence. In further preferred embodiments, the EPO albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
=
Expression and Purification of Construct ID 2298 Expression in NSO cells.
[09841 Construct 2298 can be transfected into NSO cells as described in Examples 6 and 10. Expression levels and specific productivity rates can be determined as described in Example 8.
Purification from NSO cell supernatant.
[09851 The cell supernatant containing the EPO-HSA fusion protein expressed from ID #2298 in NSO cells can be purified as in Examples 7 and 10. N-terminal sequencing should yield the sequence APPRLI (SEQ ID NO:2141) which corresponds to the amino terminus of the mature form of EPO and should yield a protein of approximate MW of 87.7 kDa.
In vitro TF-1 cell proliferation assay for construct 2298.
Method [0986] The in vitro TF-1 cell proliferation assay for. the EPO-HSA albumin fusion protein encoded by construct 2298 can be carried out as previously described in Example 8 under subsection heading "In vitro TF-1 cell proliferation assay for the albumin-fusion protein encoded by construct 1966" and in Example 10 under subsection heading "In vitro TF-1 cell proliferation assay for construct 1997".
The iflOtiVily of eonst/vg 2298 can be amayed Wag an in vivo Ilartan mouse model for measuring hematocrit.
[0987] The in vivo Harlan mouse model as previously described in Example 8 under subsection heading, "In vivo Harlan mouse model for measuring hematocrit", and in Example 10 can be used to measure hematocrit levels for the EPO albumin fusion protein encoded by construct 2298.

EXAMPLE 13: onstruct ID 2325 EPO-IIS = Generatin 109881 Construct ID 2325, pC4.EPO:Ml-D192.11SA.codon optimized, comprises DNA encoding an EPO albumin fusion protein which has the full-length EPO
protein (including the native leader sequence), i.e., MI -D192 with the Arg-140 to Gly mutation, fused to the amino-terminus of the mature form of HSA cloned into the mammalian expression vector pC4.
Cloning ofEPO cDNA for construct 2325 [09891 DNA encoding the EPO open reading frame was codon optimized so as not to hybridize to the wild-type EPO gene sequence. The polynucleotide encoding EPO
was PCR
generated by 6 overlapping oligonucleotides and cloned into the TA vector.
Construct ID
#2325 encodes an albumin fusion protein containing the leader sequence and the mature form of EPO, followed by the mature HSA protein.
[09901 Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected EPO sequence (see below).
[09911 EPO albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of EPO lacking the final Arg residue, i.e., Ala-28 to Asp-192. In one embodiment of the invention, EPO albumin fusion proteins of the invention fiuther comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature EPO albumin fusion protein is secreted directly into the culture medium. EPO albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAP, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, EPO albumin fusion proteins of the invention comprise the native EPO signal sequence. In further preferred embodiments, the EPO albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
OfpresAtiAln.e*Puritkati011 of Construci ID 2325.

Expression in CHO cells, 109921 Construct 2325 can be transfected into CHO cells as described in Examples 6 and 8. Expression levels and specific productivity rates can be determined as describe in Example 8.
Purification from CHO supernatant.
[09931 The cell supernatant containing the EPO-HSA fusion protein expressed from construct ID #2325 in CHO cells can be purified by methods described in Examples 7 and 8.
N-terminal sequencing should yield the sequence APPRL1 (SEQ ID NO:2141) which corresponds to the amino terminus of the mature form of EPO and should yield a protein of approximate MW of 87.7 k.Da.
In vitro .TF-1 cell proliferation assay for construct 2325.
Method (09941 The in vitro TF-1 cell proliferation assay for the EPO-HSA albumin fusion encoded by construct 2325 can be carried out as previously described in Example 8 under subsection heading "In vitro TF-1 cell proliferation assay for construct 1966".
Pe agivity of construct 2321cqn be assayed using an in vivo Harlan mouse model for measuring hematocrit.
109951 The in vivo Harlan mouse model as previously described in Example .8 under subsection heading, "In vivo Harlan mouse model for measuring hematocrit", can be used to measure hematocrit levels for the EPO albumin fusion protein encoded by construct 2325_ EXAMPLE 14: Indications for EPO Albumin Fusion Proteins.
[09961 Results from in vitro and in viva assays described above indicate that EPO
albumin fusion proteins can be used in the treatment of bleeding disorders and anemia caused by a variety of conditions, including but not limited to: end-stage renal disease (dialysis patients), chronic renal failure in pre-dialysis, zidovudine-treated HIV
patients, cancer patients on chemotherapy, and premature infants. EPO albumin fusion proteins can also be used pre-surgery in anemic patients undergoing elective non-cardiac, non-vascular surgery to reduce the need for blood transfusions. Indications in development for these agents include:
aplastic and other refractory anemias, refractory anemia in Inflammatory Bowel Disease, and transfusion avoidance in elective orthopedic surgery. Anemia in renal disease and oncology are the two primary indications for EPO albumin fusion proteins encoded by constructs 1966, 1981, 1997, 2294, 2298, and 2325.
EXAMPLE 15: Construct ID 1812, IL2-1ISA, Generation.
(0997] Construct ID 1812, pSAC35:12.A21-T153.HSA, comprises DNA encoding an IL2 albumin fusion protein which has an HSA chimeric leader sequence, i.e., the HSA-kex2 signal peptide, the mature IL2 protein, i.e., A21-T153, fused to the amino-terminus of the mature form of HSA in the yeast S. cerevisiae expression vector pSAC35.
Cloning of11.2 cDNA
[0998J The polynucleotide encoding IL2 was PCR amplified using primers 1L2-1 and I1L2-2, described below. The amplimer was cut with Sal IICla I, and ligated into Xho IICla I
cut pScCHSA. Construct ID #1812 encodes an albumin fusion protein containing the chimeric leader sequence of HSA, the mature form of IL2, followed by the mature HSA
protein.
109991 Two oligonucleotides suitable for PCR amplification of the polynucleotide encoding the mature form of IL2, IL2-1 and IL2-2, were synthesized:
5'-AGGAGCGTCGACAAAAGAGCACCTACrICAAGTTCTACAAAG-3' (SEQ ID NO: 725) IL2-2: 5'4;1 I L AAATCGATGAGCAACCTCACTCTTGTGTGCATCAGTCAGTGTTG
AGATGATGCT1TG-3' (SEQ ID NO: 726) 110001 1L2-1 incorporates the Sal I cloning site (shown underlined), nucleotides encoding the last three amino acid residues of the HSA chimeric leader sequence, as well as 24 nucleotides encoding the first 8 amino acid residues of the mature form of IL2. In IL2-2, the Cla I site (shown underlined) and the DNA following it are the reverse complement of the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID
NO:1038) and the last 24 nucleotides are the reverse complement of DNA encoding the last 8 amino acid residues of IL2 (see Example 2). A PCR amplimer of IL2-1{SA was generated using these Primers, purified, digested with Sal I and Cla I restriction enzymes, and cloned into the Xho I
and Cia I sites of the pScCHSA vector. After the sequence was confirmed, the expression cassette encoding this 11.2 albumin fusion protein was subcloned into pSAC35 as a Not I
fragment.
[1001] Further, analysis of the N-terrninus of the expressed albumin fusion protein by anuno acid sequencing can confirm the presence of the expected IL2 sequence (see below).
110021 IL2 albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of IL2, i.e., Ala-21 to Thr-153. In one embodiment of the invention, IL2 albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion or' polypeptide in the secretory pathways of the host used for expression In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature ts- s EU albumin fusion protein is secreted directly into the culture medium. 112 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, IL2 albumin fusion proteins of the invention comprise the native IL2 signal sequence. In further preferred embodiments, the 112 albumin fusion proteins of the invention = further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention. -Expression and Purification of Construct ID 1812.
Expression in yeast S. cerevisiae.
[10031 Transfection of construct 1812 into . yeast S.
cerevisiae strain BXP10 was carried out by methods known in the art (see Example 3). Cells were collected at stationary phase after 72 hours of growth. Supernatants from yeast transfected by construct 1812 were collected by clarifying cells at 3000g for 10 rain. Expression levels were examined by itrununoblot detection with anti-HSA serum (Kent Laboratories) as the primary antibody. An HI albumin fusion protein of approximate molecular weight of 85 IcDa was obtained. The specific productivity rates were determined via ELLSA in which the capture antibody was the US Biological #A1327-35 monoclonal anti-NSA antibody or a monoclonal anti-human IL2 antibody (e.g., from Biosource #AHC0422, Pharrningen #555051, R&D Systems #MAI3202, or R&D Systems #MAJ3602), the detecting antibody was a monoclonal anti-human I1.2-biotinylatecl antibody (e.g., from Biosource #AHC069 or Endogen/Pierce #M-600-B) or a monoclonal anti-NSA antibody Biotrend #4T24, respectively, the conjugate was horseradish peroxidase/streptavidin (Vector Laboratories, #SA-5004), and the substrate was KPL TMB
Peroxidase Substrate (KPL #50-76-01). The analysis was carried out according to ,nanufacturers) protocol and/or by methods known in the art.
Purification from yeast S. cerevisiae cell supernatant.
[10041 The cell supernatant containing 1L2 albumin fusion protein expressed from construct 1D #1812 in yeast S. cerevisiae cells was purified either small scale over a Dyax peptide affinity column (see Example 4) or large scale by following 5 steps:
diafiltration, anion exchange chromatography using DEAE-Sepharosiem Fast Flow column, hydrophobic interaction chromatography (HIC) using Butyl 650S column, cation exchange chromatography using an SP-Sepharose'm Fast Flow column or a Blue-SepharoseTM
chromatography, and high performance chromatography using Q-sepharoile high performance column chromatography (see Example 4). The 1L2 albumin fusion protein eluted from the DEAE-SepharoaFast Flow column with 100 ¨ 250 niM NaC1, from the SP-SepharogFast Flow column with 150 ¨ 250 mM NaC1, and from the Q-Sepharosr High Performance column at 5 ¨ 7.5 mS/cm. N-terminal sequencing should yield the sequence APTSSST which corresponds to the amino terminus of the mature form of IL2.
The activi& ofIL2 can be assayed using an in vitro T and NK proliferation assay.
[10051 The murine CTLL T cell-line is used and is completely dependent on 112 for cell growth and survival. This cell-line expresses high levels of high affinity IL2 receptors and is extremely sensitive to very low doses of IL2.
Methods [10061 CTLL-2 cells (murine 1L2 dependent T cell-line) is grown in RPMI
10% FBS
containing 5 ng/mL recombinant human IL2 and BME. Prior to the assays, the cells are washed twice in PBS to remove 112. 1 x 104 cells/well are seeded in a 96-well plate, in a final volume of 200 iI of RPMI 10% PBS. The yeast and 293T supernatants are tested at final concentrations of: 10%, 5%, and 1%. In addition, recombinant human IL2, "rh1L2", is diluted in the negative control supernatant (BSA alone) to test for the effect of the medium on the stability of the recombinant protein. The cells are cultured at 37 C for 20 hours, then pulsed with 1 Ci 3H-thymidine for 6 hours. Proliferation is measured by thymidine incorporation, each sample is tested in triplicate.
The activity of the IL2 albuminjusion protein en oded by construct 1812 can be assayed usinz an in vitro T and NK cell-line proliferation assay.
Methods 110071 CTLL-2 cells (murine 112 dependent T cell-line) was grown in RPMI
10%
FBS containing 5 nghnl.. recombinant human 112 and BME. Prior to the assays, the cells were washed twice in PBS to remove 112. I x 104 cells/well were seeded in a 96-well plate, in a final volume of 200 p.1 of RPM! 10% FBS. The yeast and 293T supernatants were tested at final concentrations of 10%, 5%, and 1%. In addition, recombinant human IL2, "r101.2", was diluted in the negative control supernatant (LISA alone) to test for the effect of the medium on the stability of the recombinant protein. The cells were cultured at 37 C for 20 hours, then pulsed with 1 !lei 3H-thymidine for 6 hours. Proliferation was measured by thyniidine incorporation, each sample was tested in triplicate.
Results 110081 The IL2 albumin fusion construct ID #1812 stimulated CILL-2 cell proliferation in a dose-dependent manner (see Figure 9).
22te activity of the 112 albumin fiisfon protein encoded by construct 181,2 can be assayed using an in vivo BALB/c model: RENCA tumor response to therapy.
[1009! The mouse model employs the RENCA adenocarcinoma of BALM mice.
The RENCA tumor used in these studies arose spontaneously. The RENCA tumors were originally isolated by Dr. Sarah Stewart at the NCI (Bethesda, MD). RENCA
tumors grow progressively following transfer of as few as 50 viable cells and spontaneously metastasize from intrarenal implant to the regional lymph nodes, lungs, liver, and spleen, as well as other organs. The inunagenicity of RENCA has been determined to be low to moderate.
RENCA bearing mice routinely die within 35-40 days after intrarenal injection of 1 x 105 RENCA tumor cells. Mice given RENCA tumor cells intraperitoneally of a similar number of cells usually die within 30-50 days.
Methods (10101 BALB/c mice (6 ¨ 8 weeks of age) (n--,--10) were injected subcutaneously in mid-flank with 105 RENCA cells obtained from the fourth in vivo passage. After 10 days of daily (QD) or every other day (QM) injections with placebo (PBS), HSA, rhIL2 at a dose of 0.122 mg/kg/QD or at 200,000 or 300,000 Titmouse, or IL2 albumin fusion protein at 0.61 mg/kg, mice were monitored for change in tumor size at days 14, 17, 21, 25, 28, and 31 post tumor inoculation. The data are presented in dot-analysis where each dot respresents single animals. The horizontal line in each group represents MEAN value (see Figure 10).
Results [10111 IL2 albumin fusion protein encoded by construct ID#1812 was tested in the above assay.
[10121 Administration of IL2 albumin fusion protein expressed from construct ID#1812 everyday or every other day showed significant impact on tumor growth causing delay of growth and/or shrinkage of tumor size. Every other day administration was more beneficial since tolerance levels were greater (see Figure 10). By day 31 from the inoculation day, 3 mice receiving IL2 albumin fusion products out of 10 were tumor free, only 2 showed signs of reduced tumor, and 4 mice had small tumors that appeared to be shrinking. Only one mouse did not respond beneficially to this treatment. Daily treatment with 11,2 albumin fusion protein also caused a delay of growth or actual shrinkage of tumor (2 out of 10 mice were tumor free, 7 remaining mice had small tumors, and 2 had larger ones on the day of experiment termination). All animals receiving IL2 albumin fusion at 0.61 mg/kg were alive on the termination date, while only 40% of the mice receiving placebo (PBS) and 70% of mice receiving LISA were alive. The biological effect was far more pronounced than the recombinant human IL2 given daily either at 200,000 or 300,000 U/mouse.
Recombinant - human 112 had only mediocre effect on tumor growth (all mice that received rhIL2 developed tumors and the only effect observed was growth delay) Of the 10 mice receiving rhIL2 (200,000 or 300,000 U/mL), 3 were dead by day 31. The low dose of 0.122 mg/kg/day tested did not inhibit the tumor growth nor spare mice from tumor-related death. The 112 albumin fusion protein potently inhibited the in vivo RENCA growth and caused in several cases full recovery from tumors. .
EXAMPLE 16: Construct ID 2030. 1L2-HSA, Generation.
110131 Construct ID 2030, pSAC35:ycoli ,.A21-T153.HSA, comprises DNA
encoding an 1L2 albumin fusion protein which has the LISA chimeric leader sequence, i.e., the HSA-kex2 signal peptide, the mature form of the 1L2 protein, i.e., A21-T153, fused to the amino-terminus of the mature form of LISA in the yeast S. cerevisiae expression vector pSAC35.
Cloning ofIL2 cDNA
[10141 The 112 open reading frame "ORF" DNA was codon optimized so as not to hybridize to the wild-type IL2 gene. The polynucleotide encoding the codon optimized 1L2 was PCR generated by 6 overlapping oligonucleotides and cloned into a TA
vector. The polynucleotide encoding the codon optimized IL2 was PCR amplified from this clone using primers IL2-3 and IL2-4, described below, cut with Sal IICla 1, and ligated into Xho IICla I
cut pScCHSA. Construct ID #2030 encodes an albumin fusion protein containing the chimeric leader sequence of HSA and the mature form of IL2 fined to the amino terminus of the mature form of HSA.
110151 Two oligonucleotides suitable for PCR amplification of the codon optimized polynucleotide encoding the mature form of 1L2, 11,2-3 and IL2-4, were synthesized:
IL2-3: 5t- AGGAGCGTCGACAAAAGAGCTCCAACTTCTTCTTCTACTAAG-3' (SEQ
ID NO: 831) 112-4: 5"-CTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATCTGICAAAGTA
GAAATAATAGA TTGGCAG-3' (SEQ ID NO: 832) [10161 = 1L2-3 incorporates the Sal I cloning site (shown underlined) and encodes for the last three amino acid residues of the chimeric leader sequence of HSA, as well as the 24 nucleotides encoding the first 8 amino acid residues of the mature form of IL2. 1;1.112-4, the Cla 1 site (shown underlined) and the DNA following it are the reverse complement of the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID
NO:1038) and the last 24 nucleotides are the reverse complement of DNA encoding the last 8 amino acid residues of IL2 (see Example 2). A PCR amplimer was generated using these primers, purified, digested with Sal I and Cla I restriction enzymes, and cloned into the "Tito I and Cla sites of the pScCIISA vector. After the sequence was confirmed, the Not I
fragment containing the 1L2 albumin fusion protein expression cassette was subcloned into pSAC35 cut with Not I.
[10171 Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected 112 sequence (see below).
[1018) 1L2 albumin fusion proteins of the invention preferably comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of 112, i.e., Ala-21 to Thr-153. In one embodiment of the invention, 11.2 albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature 112 albumin fusion protein is secreted directly into the culture medium. IL2 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein variant HSA leader sequences including, but not limited to, a chimeric HSAMAF
leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, 11.2 albumin fusion proteins of the invention comprise the native 11,2 signal sequence. In further preferred embodiments, the IL2 albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Purification of Construct ID 2030.
Expression in yeast S. cerevisiae.
110191 Transfection into yeast S. cerevisiae strain BX.P10 can be carried out by methods known in the art (see Example 3) and as previously described for construct ID 1812 (see Example 15).
Purification from yeast S. cerevisiae cell supernatant.
[10201 The cell supernatant containing11.2-HSA expressed from construct ID
#2030 in yeast S. cerevisiae cells can be purified either small scale over a Dyax peptide ifimity column (see Example 4) or large scale by following 5 steps: diafiltration, anion exchange chromatography using DEAE-Sepharosr Fast Flow column, hydrophobic interaction chromatography (H1C) using Butyl 650S column, cation exchange chromatography using an SP-SepharoireFast Flow column or a Blue-Sepharoalcbromatography, and high performance chromatography using Q-sepharoiThigh performance column chromatography (see Example 4 and Example 15). N-terminal sequencing should yield the sequence APTSSST
(SEQ ID
NO:2142) which corresponds to the amino terminus of the mature form of IL2.
The activiV of the IP albumin s on otern en , ed ,,k osnstn t 2030 can be assaY4 usine the in vitro T and NK cell-line proliferation assay.
[10211 The activity of construct ID 2030 can be assayed using an in vitro T and NI( cell-line proliferation assay as in Example 15.
je activity of the 11,2 &ham* fiaion protein encoded by censt 2030 an be assetved using an in vivo BALE/a model: RENCel tumor response to therapy.
[10221 The activity of the 11,2 albumin fusion protein encoded by construct 2030 can be assayed using the in vivo BALM model as described in Example 15 in which the RENCA
tumor response to therapy is monitored.

EXAMPLE 17: Construct ID 2031, HSA-1L2, Generation.
[10231 Construct ID 2031, pSAC35:HSA.yco11.2.A21-T153, comprises DNA
encoding an IL2 albumin fusion protein which has the HSA full-length sequence that includes the HSA chimeric leader sequence, i.e., the HSA-kex2 signal peptide, fused to the amino-terminus of the mature form of IL2, A21-T153, in the yeast S. cerevisiae expression vector pSAC35.
Cloning of IL2 cDNA
[1024] The IL2 open reading frame "ORF" DNA was codon optimized so as not to hybridize to the wild-type IL2 gene. The polynucleotide encoding the codon optimized I12 was PCR generated by 6 overlapping oligonucleotides and cloned into a TA
vector. The polynucleotide encoding the codon optimized IL2 was PCR amplified from this clone using primers 1L2-5 and IL2-6, described below, cut with Bsu 361/Pine 1, and ligated into Bsu 361/Pine I cut pScNHSA. Construct ID #2031 encodes an albumin fusion protein containing the chimeric leader sequence and mature form of HSA and the mature form of I12.
[10251 Two oligonucleotides suitable for PCR amplification of the codon optimized polynucleotide encoding the mature form of IL2, IL2-5 and IL2-6, were synthesized:
112-5: 5'-AAGCTGCCTTAGGCTTAGCTCCAACTTCTICTTCTACFAAG-3' (SEQ ID
NO: 833) IL2-6: 5'-GCGCGCGTTTAAACGGTACCTTATGTCAAAGTAGAAATAATAGATTGG
CAG-3' (SEQ ID NO:834) [1026] 112-5 incorporates the Bsu 361 cloning site (shown underlined) and encodes for the last four amino acid residues of the mature form of HSA, as well as the 24 nucleotides encoding the first S amino acid residues of the mature form of ru. In IL2-6, the Pine I site is underlined (SEQ ID NO:834) and the last 24 nucleotides are the reverse complement of DNA
encoding the last 8 amino acid residues of 112 (see Example 2). A PCR amplimer was generated using these primers, purified, digested with Bsu 361 and Pine 1 restriction enzymes, and cloned into the Bsu 361 and Pine I sites of the pScNHSA vector. After the sequence was confirmed, the Not 1 fragment containing the IL2 albumin fusion protein expression cassette was subcloned into pSAC35 cut with Not I.
[1027] Further, analysis of the N-temiinus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected HSA sequence (see below).

10281 IL2 albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of IL2, i.e., Ala-21 to Thr-153. In one embodiment of the invention, 1L2 albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature 112 albumin fusion protein is secreted directly into the culture medium. EL2 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric USA/MAP leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, 1L2 albumin fusion proteins of the invention comprise the native IL2 signal sequence. In farther preferred embodiments, the IL2 albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Purification of Construct ID 2031.
Expression in yeast S. cerevisiae [10291 Transfection into yeast S. cerevisiae strain BXP10 can be carried out by methods known in the art (see Example 3) and as previously described for construct ED 1812 (see Example 15).
Purification from yeast S. cerevisiae cell supernatant.
[10301 The cell supernatant containing HSA41.2 expressed from construct ID
#2031 in yeast S. cerevisiae cells can be purified either small scale over a Dyax peptide affinity column (see Example 4) or large scale by following 5 steps: diafiltration, anion exchange chromatography using DEAE-Sepharose Fast Flow column, hydrophobic interaction chromatography (HIC) using Butyl 6505 column, cation exchange chromatography using an SP-SepharosIeFast Flow column or a Blue-Sepharosrchromatography, and high performance chromatography using Q-sepharosTemhigh performance column chromatography (see Example 4 and Example 15). N-terminal sequencing should yield the sequence DAHKS (SEQ
ID
NO:2143) which corresponds to the amino terminus of the mature form of USX
= activit o h L albumin figion protein encoded by constr_unLisci,,nasjuLd asing the hi vitro T and NK celUrne proliferation a.ry.
[10311 The activity of construct ID 2031 can be assayed using an in vitro T and NK
cell-line proliferation assay described in Example 15.
The activiv of ihcALalbut 'n ion v'n. = n ed ?met ;031 can be assayed using the in vivo BALB/c model: RENCA tumor response to therapy.
[1032j The activity of the IL2 albumin fusion protein encoded by construct 2031 can be assayed using the in vivo BALB/c model as described in Example 15 in which the RENCA
tumor response to therapy is monitored.
EXAMPLE 18: Indications for 11.2 Albumin Fusion Proteins.
[1033] Indications for IL2 albumin fusion proteins (including, but not limited to, those encoded by constructs 1812, 2030, and 2031) include, but are not limited to, solid tumors, metastatic renal cell carcinoma, metastatic melanoma, malignant melanoma, renal cell carcinoma, HIV infections treatment (AIDS), inflanamatory bowel disorders, Kaposi's sarcoma, leukemia, multiple sclerosis, rheumatoid arthritis, transplant rejection, type I
diabetes mellitus, lung _cancer, acute myeloid leukemia, hepatitis C, non-Hodgkin's Lymphoma, and ovarian cancer.
EXAMPLE 19: Construct ID 1642, GCSF-IISA, Generation.
[10341 Construct ID 1642, pSAC35:GCSF.T31-P204.HSA, comprises DNA encoding a GCSF albumin fusion protein which has the USA chimeric leader sequence, i.e., the }ISA-kex2 signal peptide, the mature form of the "short form" of Granulocyte Colony Stimulating Factor, "G-CSF", protein, i.e., T31-P204, fused to the amino-terminus of the mature form of IBA in the yeast S. cerevisiae expression vector pSAC35.
Cloning of GCSF cDNA
[1035] A polynucleotide encoding GCSF was PCR amplified using primers GCSF-and GCSF-2, described below. The amplimer was cut with Sal 11Cla 1, and ligated into xho IICla 1 cut pScCHSA. Construct ID #1642 comprises DNA which encodes an albumin fusion protein containing the chimeric leader sequence of USA, the mature form of GCSF, followed by the mature HSA protein.
[1036j Two oligonucleotides suitable for PCR amplification of a polynucleotide encoding the mature form of GCSF, GCSF-1 and GCSF-2, were synthesized:
GCSF-1: 5'- GAATTCGTCGACAAGAACCCCCCTGGGCCCTGCCAG -3' (SEQ ID
NO:665) GCS F -2 : 5`-AAGCTTATCGATGAGCAACCTCACTCTTGTGTGCATCGGGCTGGGC
AAGGTGGCGTAG-3' (SEQ ID NO:666) 110371 GCSF-1 incorporates the Sal I cloning site (shown underlined), nucleotides encoding the last three amino acid residues of the USA chimeric leader sequence, as well as 20 nucleotides encoding the first 6 amino acid residues of the mature form of GCSF. In GCSF-2, the Cla I site (shown underlined) and the DNA following it are the reverse complement of the DNA encoding the first 10 amino acids of the mature HSA
protein (SEQ
ID NO:1038) and the last 21 nucleotides are the reverse complement of DNA
encoding the last 7 amino acid residues of GCSF. Using these primers, a PCR amplimer was generated, purified, digested with Sail and Cla I restriction enzymes, and cloned into the Xho I and Cla sites of the pScCHSA vector. After the sequence was confirmed, the Not I
fragment containing the GCSF albumin fusion expression cassette was subeloned into pSAC35 cut with Not I.
[1038] Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing confirmed the presence of the expected GCSF sequence (see below).
110391 GCSF albumin fusion proteins of the invention preferably comprise the mature form of USA , i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of GCSF, Le., Thr-31 to Pro-204. In one embodiment nf the invention., GCSF albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent &skin polypeptide in the secretory pathways of the host used for expression.
In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature GCSF albumin fusion protein is secreted directly into the culture medium. GCSF
albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant USA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, GCSF albumin fusion proteins of the invention comprise the native GCSF signal sequence. In further preferred embodiments, the GCSF albumin fusion proteins of the invention further comprise an N-terminal rnethionine residue.
Polynucleatides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Purification of Construct ID 1642..
Expression in yeast S. cerevisiae.
110401 Transformation of construct 1642 into yeast S. cerevisiae strains D88, BX.P10, and DXY1 ¨ a YAP3 mutant, was carried out by methods known in the art (see Example 3).
A preliminary "Halo Assay" was carried out to assess if the transformed yeast are producing the proteins encoded by the fusion constructs. Secretion of USA fusion proteins into agar media containing anti-HSA antibodies will result in the formation of an insoluble "precipitin"
ring or halo. The size of the halo is proportional to the amount of IISA
protein being produced. LEU2 4. prototrophs were selected on synthetic complete leucine dropout medium containing dextrose, "SCD-Leu". Selected colonies as well as a positive contrel were gridded onto a Blvflv1D plate containing anti-HSA antibody. After growth, the plates were incubated at 4 C to allow for precipitin ring formation. Based on the "Halo Assay", colonies from transformation of construct 1642 produced protein. To establish the extent of secretion, transformed cells were collected at stationary phase after 48 hours of growth in suspension.
Supernatants were collected by clarifying cells at 3000g for 10 min.
Expression levels were examined by imrnunoblot detection with anti-HSA serum (Kent Laboratories) or with an antibody directed to the Therapeutic protein portion, i.e., GCSF, of the albumin fusion protein. The GCSF albumin fusion protein of approximate molecular weight of 88 lcDa was obtained. To obtain workable quantities for purification, the yeast transformants were inoculated in 1 L of BMM media at 150 rpm, 29.5 C. The culture was centrifuged and passed through a 0.45 m filter. The specific productivity rates can be determined via ELBA in which, for example, the capture antibody is the R&D Systems Clone 3316.111 monoclonal mouse anti-GCSF, the detecting antibody is the R&D Systems BAF2 14 (i.e., Clone ACNO30081) biotinylated goat anti-human GCSF antibody, the conjugate is horseradish peroxidase/streptavidin (Vector Laboratories, #SA-5004), and the substrate is KPL TMB
Peroxidase Substrate (KPL #50-76-01), where the analysis is carried out according to manufacturers' protocol and/or by methods known in the art.
Purification from yeast S. cerevisiae cell supernatant.
110411 A general purification procedure for albumin fusion proteins has been described in Example 4. The purification of GCSF albumin fusion protein is described specifically below. Another purification scheme is described in Example 20.

Step 1: Phenyl Fast Flow Chromatography (Amershant Pharmacia Biotech) 110421 The yeast culture supernatant (3 L) containing GCSF-IISA encoded by construct 1642 was loaded onto a phenyl fast flow column with 1 M of ammonium sulfate in 50 mM Tris, pH 7.2. The column was washed with 1 M of ammonium sulfate in 50 mM
Tris, pH 7.2, 0.2 M ammonium sulfate in 50 rriM Tris, pH 7.2, and then washed with the buffer. The GCSF-HSA fusion protein was eluted with water (Water For Injection distilled water WET).
Step 2: SP Fast Flow Chromatography (Atnersham Pharmacia Biotech) 110431 The eluate of Step 1 was mixed with an equal volume of a solution composed of 10.3 mM Na2HPO4 and 4.85 nilVI citric acid, pH 5Ø The mixture was loaded at 300 crn/hr onto a SP fast flow column and eluted with a solution composed of 0.5 M NaCl in 10.3 1W
Na2HPO4. and 4.85 mM citric acid, pH 5Ø The column was then stripped with a solution composed of 1M NaC1 in 10.3 mM Na2HPO4 and 4.85 mM citric acid, pH 5Ø
Step 3: Methyl HIC Chmmatography(BioRad) [10441 The eluate of Step 2 was litrated to a final concentration of 1. M
ammonium sulfate (143 mS) in 50 mM Tris, pH 7.2 and loaded onto methyl HIC column. The column was washed to a baseline, then washed with 0.6 M ammonium sulfate in 50 mM
Tris, pH 7.2.
A gradient from 0.6 M ammonium sulfate to 0 M ammonium sulfate was initiated, The column was finally stripped with WFI and 0.5 M NaOH. A lot of the impurities in the sample eluted at the lower ammonium sulfate concentrations thereby affording the GCSF-.
HSA fusion high purity.
Step 4: CM Fast Flow Chromatography (Antersham Pharmacia Biotech) [1045] The eluate of Step 3 was diluted with WFI to 5 mS, pH 5.5 and was loaded onto the CM column at 300 cm/hr. The column was eluted with 0.5 M NaC1 in 11 mM
Na21-11)04 and 4 mM citric acid, pH 5.5. The column was stripped with 1 M NaC1 in 11 RIM
Na2HPO4 and 4 mM citric acid, pH 5.5.
Step 5: Ultrafiltration/Diailltration (Amershant Pharmacia Biotech) 110461 The purified product was ultrafiltered and diafiltered into Phosphate Buffered Sal Me, "PBS", pH 7.2.
110471 The purified GCSF albumin fusion protein encoded by construct 1642 was analyzed for purity on SDS/PAGE. It was > 95% pure. The protein was sequenced confirmed and also showed 90% purity on N-terminal sequencing with an N-terminal sequence of "TPLGP" (SEQ ID NO:2144).

The activitval GCSFcan be azisaved using art in viiroiTS760 cejloalyiratiandsstiv, Method [10481 To assess GCSF activity, NSF-60 cells, a myeloid factor-dependent cell-line derived from Primary Lake Cascitus wild ecotropic virus-induced tumor of NFS
mice, are employed.
Cell growth and Preparation [10491 Cells are originally seeded in T-75 cm2 flasks at approximately 1.5 x 104 eells/mL in growth media (RPM' 1640 containing 10% Fetal Bovine Serum, "FBS", lx Penicillin/Streptomycin, lx L-Glutamine (final concentration of 2 mM), and recombinant murine interleukin-3, (1L3) at 30 ng/mL). Cells are split anywhere from 1:10 to 1:20 every 2 days and reseeded in fresh medium.
NFS-60 Bioassay [10501 The NFS-60 assay is performed as described in Weinstein et aL
(Weinstein et aL, 1986; Proc. Nati. Acad. S&L U S A, 83, pp5010-4). Briefly, the day before the assay is to be performed, Cells are reseeded to 1.0 x 105 in fresh assay growth medium containing 1L3.
The next day cells are transferred to 50 riaL conical tubes, centrifuged at low speeds, and ' washed twice in plain RPME without serum or growth factors. The pellet is resuspended in 25 nil, and the cells are subsequently counted. The cells are spun once more and resuspended at the working concentration in growth medium (described above) but lacking 1L3. The cells are plated in 96-well round-bottom TC-treated plates at 1 x 105 cells/well.
Increasing doses of GCSF are added to each well to a final volume of 0,1 mL. The assay is done in triplicate.
The cells arc cultured for 24 hours to determine the level of cell proliferation. 3114'hymidine (51.1.CihnL) is added 4 hours prior to the experiment termination. The cells are then harvested on glass fiber filters using a cell harvester and the amount of 3H-Thymidine labeled DNA is counted using TOP-Count.
The activity of OC,8F albumin firOint ethgadefi by camtroct )11) #160 an be .amayed usuzs aIf in ygro= AfFS-60 proifferatio# &sway.
Method [10511 GCSF albumin fusion protein encoded by construct 1642 was tested in the in vitro NFS-60 cell proliferation bioassay described above.

Cell growth and Preparation [10521 Cells were prepared as described above.
ATS-60 Bioassay [10531 The day before the assay was performed, cells were reseeded to 1.0 x 105 in fresh assay growth medium containing 1L3. The next day cells were transferred to 50 nil, conical tubes, centrifuged at low speeds, and washed twice in plain RPMI
without serum or growth factors. The pellet was resuspended in 25 rni- and the cells were subsequently counted. The cells were spun once more and resuspended at the working concentration in growth medium (described above) but lacking 11.3. The cells were plated in 96-well round-bottom TC-treated plates at 1 x 105 cells/well. Increasing doses either of HSA, recoinbinant human GCSF (rhGCSF), or a partially purified GCSF albumin fusion protein from the yeast supernatant, were added to individual wells to a final volume of 0.1 mL. The assay was done in triplicate. The cells were cultured for 24 hours to determine the level of cell proliferation.
3H-Thymidine (5 Ci/mL) was added 4 hours prior to the experiment termination, .The cells were then harvested on glass fiber filters using cell harvester and the amount of 3H-Thymidine labeled DNA was counted using TOP-Count.
Results [10541 Construct 1642 demonstrated NH-60 cell proliferation activity in a dose dependent manner, while the control supernatant from yeast expressing HSA
alone did not produce any activity (see Figure 11).
The activity of GCSF can bo assayed in vivo usjfig 57B1.16 mice:
GCSF as a Mobilizing Agent.
[10551 G-CSF is capable of mobilizing granulocytes to the periphery as well as increasing the total White Blood Cell, (WBC), count when administered to mice.

Recombinant human GCSF, (rhGCSF), cross-reacts with recombinant Murillo GCSF, (rmGCSF).
Methods [10561 Mice are ear tagged before the injections start. Mice are injected intraperitoneally with rhGCSF (Neupogen, AMEN) at either 5 g (n=5) or 10 g (n=5) twice a day for 7 consecutive days. The control mice (n=3) receive Hepes Buffered Sal inc Solution, (HBSS). At 24 hours after the last rhGCSF administration, peripheral blood is drawn from the tail and analysed for the granulocyte content and total WBC
count.
Results [1057] Both doses of rhGCSF efficiently increase both the frequency and the total number of granulocytes as well as the total WBC count (see Figure 12). This effect is apparent after 24 hours of the final rhGCSF intraperitoneal administration.
This effect is transient and the number of granulocytes return to mutat values by day 5.
The geilViti Pr GCST albutiiiii 'Won prot* rucoded by .09tatilitt"ID #
1612ta:be armeti in vivo uug C.57.1.3116 saw :GCSE-14d as q:MobiliziNt Ptttat.
Methods [10581 The GCSF albumin fusion protein encoded by construct 1642 can be assayed according to the procedure described above. Briefly, mice are to be ear tagged before the injections are to begin. Mice are to be injected intraperitoneally with either rhGCSF, as a control, or the GCSF albumin fusion protein at either 5 g (n=5) or 10 g (n=5) twice a day for 7 consecutive days. Additional control mice (n=3) are to receive Hepes Buffered saline Solution, "HBSS". At 24 hours after the last GCSF administration, peripheral blood can be drawn from the tail and analysed for the granulocyte content and total WBC
count.
EXAMPLE 20: Construct ID 1643, HSA-GCSF, Generation.
[1059] Construct ID 1643, pSAC35:1-1SA.GCSF.T31-P204, comprises DNA
encoding a GCSF albumin fusion protein which has the full-length HSA protein that includes the HSA
chimeric leader sequence, i.e., the HSA-kex2 signal peptide, fused to the amino-terminus of the mature form of the GCSF protein, i.e., A21-T153, in the yeast S.
cerevisine expression vector pSAC35.
Cloning of GCSE cDA1A
[10601 The polynucleotide encoding GCSF was PCR amplified using primers GCSF-3and GCSF-4, described below. The ampIimer was cut with Bsu 361/Else I, and ligated into Bsu 361.Msc I cut pScNIASA. Construct II) #1643 encodes an albumin fusion protein containing the chimeric leader sequence and mature form of HSA and the mature form of GCSF.
(10611 Two oligonucleotides suitable for PCR amplification of the polynucleotide encoding the mature form of GCSF, GCSF-3 and GCSF-4, were synthesized:
GCSF-3: 5!-AAGCTGCCTTAGGCTTAACCCCCCTGGGCCCTGCCAG -3' (SEQ ID
NO:667) GCSF-4: 5'-GCGCGCGGCGCGCCTCAGGGCTGGGCAAGGTGGCGTAG-3' (SEQ ID
NO:668) [10621 GCSF-3 incorporates the Bsu 361 cloning site (shown underlined) and nucleotides encoding the last four amino acid residues of the mature form of 'ISA, as well as 20 nucleotides encoding the first 6 amino acid residues of the mature form of GCSF. In GCSF-4, the Asc I site is underlined and the last 24 nucleotides are the reverse complement of DNA encoding the last 8 amino acid residues of GCSF. A PCR amplimer of HSA¨GCSF
was generated using these primers, purified, digested with Bsu 361 and Asc I
restriction enzymes, and cloned into the Bsu 361 and Asc I sites of the pSeNHSA vector.
After the sequence was confirmed, the expression cassette encoding this GCSF albumin fusion protein was subcloned into pSAC35 as a Not I fragment, 110631 Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing confirmed the presence of the expected HSA sequence (see below).
[1064] .GCSF albumin fusion proteins of the invention preferably comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of GCSF, i.e., Thr-31 to Pro-204. In one embodiment of the invention, GCSF albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression.
In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature GCSF albumin fusion protein is secreted directly into the culture medium. GCSF
albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, IvIAF, INV, 1g, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, GCSF albumin fusion proteins of the invention comprise the native GCSF signal sequence. In further preferred embodiments, the GCSF albumin fusion proteins of the invention further comprise an methionine residue. Polynucleotides -ncoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Purification of construct ID 1643.
Expression in yeast S. cerevisiae [10651 Transformation of construct 1643 into yeast S. cerevisiae was carried out by methods known in the art (see Example 3) and as previously described for construct ID 1642 (see Example 19).
PurOcation from yeast S. cerevisiae cell supernatant.
[10661 A general procedure for purification of albumin fusion proteins is described in Example 4. The cell supernatant containing GCSF albumin fusion protein expressed from construct ID #1643 in yeast S. cerevisiae cells was purified according to the following method. Another purification scheme is described in Example 19.
Step 1: Phenyl SepharosTemFast Flow (hs), pH 7.2 [10671 The fermentation supernatant (3.5 L) was adjusted to 139 mS and pH
7.2 with tuninonium sulfate to a final concentration of 1 M in 50 mM Tris, pH 7.2. The phenyl sepharose column was loaded at a flow rate of 300 cm/hr. The column was washed with 50 mM Tris-HC1, pH 7.2. A series of lower salt elutions were executed to remove contaminating proteins followed by a WF1 elution to elute the target protein.
A NaOH strip of the column revealed that a significant portion of the target protein was not removed by previous treatments.
Step 2: Mimetic Blue, pH 6.5 [10681 The eluted target protein was diafiltered with 20 mM citrate phosphate buffer, (CPB), pH 6.5 and then loaded onto a Mimetic Blue column previously equilibrated with 20 mM CPB, pH 6.5 buffer. The column was washed with equilibration buffer for 10 column volumes. The majority of the target protein was then eluted with a 0.2 M NaCI
wash. Higher salt concentration elution solutions (1 M and 2 M NaCl) revealed some target protein.
However, when HPLC-SEC was performed on these fractions the majority of the target protein was observed as aggregates. This purification step resulted in > 85%
purity of the target protein.
Step 3: Q HP, pH 6.5 [1069] The target protein was diluted with 20 mivi CPB, pH 6.5 (5-fold) to a conductivity of < 5 niS and loaded onto the Q HP resin. A series of elutions, 100 mM, 200 mM, 500 mM, and I M NaCI, were performed. The target protein eluted with 100 rnIVI NaCI.
Step 4: SP FF, pH" 5.5 [1070] The target protein was diluted with 20 mM CPB, pH 5.0, and adjusted to pH
5Ø The target protein was loaded onto SP Sepharose FF column. The column was washed with 5 column volumes of equilibration buffer. The 45 kDa contaminating protein, a proteolyzed fragment of HSA, did not bind to the resin and was observed in the load flow thru (LFT). The target protein was eluted in a shallow gradient from 0 ¨ 500 inivi NaCI. The target protein eluted at about 250 m.M NaCI. The target protein was diafiltered into the final storage buffer of 20 mM CPB, pH 6.5.
[1071] Analysis by SDS-PAGE identified an 88 kDa protein with > 95% purity.
N-terminal sequencing resulted in the major sequence being "DAHKS" (SEQ ID
NO:2143) which is the amino-terminus of the mature form of IISA. The final buffer composition is 20 mM CPB, pH 6.5. From 3.5 L of culture supernatant, 1.94 mg protein was purified.
The aetiyity of (iCSF au min fusion encoded by con,valief # 043 ea 4.assayett using an in vitro ATFS-60 cell proliferation assay.
Method 110721 The GCSF albumin fusion protein encoded by construct 1643 was tested in the in vitro NFS-60 cell proliferation bioassay previously des6ribed in Example 19 under subsection headings, "The activity of GCSF can be assayed using an in vitro NFS-60 cell proliferation assay" and "The activity of GCSF albumin fusion encoded by construct ID #
1642 can be assayed using an in vitro NFS-60 cell proliferation assay".
Results [1073] Construct 1643 demonstrated the ability to cause NFS-60 cell proliferation in a dose dependent manner, while the control supernatant with HSA alone did not produce any activity (see Figure 11).
;The activity of GCSF :albumin fusion encoded by construct 1D #1643 can be okayed in Yiva using C57B116 mice: GCSF-HSA a_s a Mobilizing Agent.
Methods [10741 The GCSF albumin fusion protein encoded by construct 1643 can be assayed according to the procedure as previously described in Example 19 under subsection headings, "The activity of GCSF can be assayed in vivo using C57131,/6 mice: GCSF-HSA as a Mobilizing Agent" and "The activity of GCSF albumin fusion encoded by construct ID #
1642 can be assayed in vivo using C5713E/6 mice: GCSILHSA as a Mobilizing Agent".
AMPLE 21: 1 dieations for :CM albumin Winn roteins, [10751 Based on the activity of GCSF albumin fusion proteins in the above assays, GCSF albumin fusion proteins are useful in chemoprotection, treating, preventing, and/or diagnosing inflammatory disorders, rnyelocytic leukemia, primary neutropenias (e.g., Kostmann syndrome), secondary neutropenia, prevention of neutropenia, prevention and treatment of neutropenia in HIV-infected patients, prevention and treatment of neutropenia associated with chemotherapy, infections associated with neutropenias, myelopysplasia, and autoimmune disorders, mobilization of hernatopoietic progenitor cells, bone marrow transplant, acute myelogeneous leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, Hodgkin's disease, accelerated myeloid recovery, and glycogen storage disease.
EXAMPLE22: Construct ID 2363, GGSV-IISA-EPO.A28-D192, Generation.
[10761 Construct ID 2363, pC4:GCSF.HSA.EPO.A28-D192, comprises DNA
encoding a GCSF-HSA¨EPO triple fusion protein having the full-length form of the Granulocyte Colony Stimulating Factor, (G-CSF), protein, fused to the amino-terminus of the mature form of IJSA, which is fused to the amino-terminus of the mature form of EPO, i.e., amino acids A28-D192, with the exception of the final Arg residue, in the CHO
mammalian cell-line expression vector pC4,.
Cloning Of GCSF-HS44-4P0 Construct JD # 1642, i.e., pSAC35:GCSF.T31-P204.HSA (Example 19), was used as a template to generate a part of construct 2363. The following polynueleotides were synthesized:
GCSF/EP 0-1: 5' -TGTOGCACAGTGC.ACTCTGGACAGTGCAGGAAGCCACCC
CCCTGGGCCCTGCCAGCTCCC-3' (primer 79388) (SEQ ID NO:1129) =
GCSFIEPO-2: 5 '-GGCACACTTGAGTCTCTGTTTGGCAGACG-3' (primer 79239) (SEQ
ID NO:1130) GC S F/EP 0-3 : 5 -ACCCA GAGCCCCATGAAGCTGATGG CCCTGCAGCTGCTGCTG
TGGCACAGTGCACTCTGG-3 (primer 79389) (SEQ ID NO:1131) GCSPEPO-4: 5- GGTTGGGATCCAAGCTTCCGCCACCATGGCTGGACCTGCCAC
CCAGAGCCCCATGAAGCT-3'(primer 79390) (SEQ ID NO:1132) (10781 The full-length sequence of GCSF was generated in a three-step overlapping PC12. reaction using combinations of primers GCSFIEPO-1, GCSFIEPO-2, GCSF/EPO-3, and GCSPEPO-4. Primers GCSF/EPO-1, GCSF/EPO-3, and GCSF/EPO-4 consist of sequences that span the amino-terminus of the full-length of GCSF. Primer GCSF/EPO-2 comprises of the reverse complement of the sequence that spans amino acids Ser-216 to Ala-225 of USA.
The first PCR reaction included construct 1642 as template and primers GCSF/EPO-1 and GCSF/EPO-2. The product obtained from the first PCR reaction was used as template in the second PCR reaction which included primers GCSF/EPO-3 and GCSREPO-2. The product obtained from the second PCR reaction was used as template in the third PCR
reaction which included primers GCSF/EPO-4 and GCSF/EPO-2, Primer GCSFTEPO-4 has a Barn HI
site (shown in italics) followed by the Kozak sequence (shown underlined). The final PCR
product contains a 5' Ban, HI restriction site followed by an appropriate Kozak sequence, the entire full-length GCSF coding sequence and part of the USA open reading frame from Asp-25 ¨ Ala-225. The Cia I site is inherent in the polynucleotide sequence of the mature form of EISA and is localized in close proximity to the 5'-end of the mature form of USA. ,The Barn HI-Cla I fragment was cloned into similarly digested pC4.HSA.EPO.A28-D192 construct ID
# 1981.
11079j . Construct ID #2363 encodes an albumin fusion protein containing.
the leader and mature forms Of GCSF, followed by the mature USA protein, followed by the mature fonn of EPO, [10801 Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected GCSF sequence (see below).
110811 GCSF/EPO albumin fusion proteins of the invention preferably comprise the mature form of 'ISA , i.e., Asp-25 to Len-609, fused to either the N- or C-terminus of the mature form of GCSF, i.e., Thr-31 to Pro-204, and fused to either the N- or C-terminus of the mature form of EPO, i.e,,Ala-28 to Asp-192. In one embodiment of the invention, GCSF/EPO albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature GCSF/EPO albumin fusion protein is secreted directly into the culture medium. GCSF/EPO albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, GCSREPO albumin fusion proteins of the invention comprise either the native GCSF or the native EPO
signal sequence.
In further preferred embodiments, the GCSWEPO albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments andlor variants, are also encompassed by the invention.
&pressjorl on e PurificatiOn of Coristnict Expression in CHO cells.
[1082] Construct 2363 can be transfected into CHO cells as previously described in Examples 6 and 8.
Purification from CHO supernatant:
(10831 A general purification procedure for albumin fusion proteins has been described in Example 7. The triple fusion protein GCSF-HSA-EPO encoded by construct 2363 can be purified as previously described in Examples 7 and 9. N-terminal sequencing should yield the sequence TPLGP (SEQ LD NO:2144) which corresponds to the mature form of GCSF.
The a iiv,ity qf fici8P-RS/1-0130Ø10ode4 by eontvtruet ID it 2343 cart be Lusa)* using an in vitro xr.,1 t;ell pt*feration assay and ell in vitro NEW cell proliferation assets Method [10841 The activity of the triple fusion protein GCSF-HSA-EPO encoded by construct 2363 was assayed in the in vitro TF-1 cell proliferation assay as previously described under , subsection heading, "In vitro TF-1 cell proliferation assay for construct 1981", in Example 9, as well as in the in vitro NFS-60 cell proliferation assay as previously described under subsection heading, "The activity of GCSF albumin fusion encoded by construct ID # 1642 can be assayed using an in vitro NFS-60 cell proliferation assay", in Example 19.
Result [1085] THE GCSF-HSA-EPO albumin fusion encoded by construct 2363 demonstrated proliferation of both TF-1 cells and NFS-60 cells.

The activity of GCSF-FISA-EPO albumin fusion encoded b_y construct ID #
2363 can be assayed iti vivo.
110861 The activity of the triple fusion protein GCSF-HSA-EPO encoded by construct 2363 can be assayed in the in viva Harlan mouse model to measure hematocrit levels as previously described in Example 9 under subsection heading, "The activity of construct 1981 can be assayed using an in vivo Harlan mouse model for measuring hematocrit", as well as in C57BL/6 mice where GCSF-HSA-EPO is a mobilizing agent as previously described in Example 19 under subsection heading, "The activity of GCSF albumin fusion encoded by construct ID # 1642 can be assayed in vivo using C57B1J6 mice: GCSF-HSA as a Mobilizing Agent".
EXAMPLE 23: Construct ID 2373, GCSF-IISA-EPO.A28-D192, Generation.
[10871 Construct ID 2373, pC4:GCSF.HSA.EPO.A28-D192.R140G, comprises DNA
encoding a GCSF-HSA--EPO triple fusion protein which has the full-length form of the Granulocyte Colony Stimulating Factor, "G-CSF", protein, fused to the amino-terminus of the mature form of HSA, which is fused to the amino-terminus of the mature form of EPO, i.e., A28-D192 which has the Arg-140 to Gly mutation, in the CHO mammalian cell-line expression vector pC4.
Cloning of EPO caVil for construct 2373 [10881 Construct ID #2373 encodes an albumin fusion protein containing the leader sequence and the mature form of GCSF, followed by the mature LISA protein followed by the mature form of EPO which has the Arg-140 to Gly mutation (SEQ ID NO:401).
Construct ID #2373 was generated by using construct ID #2363, i.e., pC4:GCSF.HSA.EPO.R140G as a template for PCR mutagenesis.
110891 Four oligonucleotides suitable for PCR amplification of template of construct ID #2363, GCSF/EPO-5, GCSFIEPO-6, GCSF/EPO-7, and GCSF/EPO-8, were synthesized.
GCSF/EPO-5: 5'-GTTGAAAGTAAGGATGTTTG-3' (primer 78219) (SEQ ID NO:1125) GCSF/EPO-6: 5'-CCTTCTGGGCTCCCAGAGCCCGAA.G-3' ( primer 82847) (SEQ ID
NO:1126) GCSREPO-7: 5'-CTTCGGGCTCTGGGAGCCCAGAAGG-3' ( primer 82846) (SEQ ID
NO:1127) GCSPEPO-8:5"-ACCAGGTAGAGAGCTTCCACC -3' (pC3') (SEQ ID NO:1128) 110901 Construct 2373 was generated by nested PCR amplification using construct 2363 as the template. In the first round of PCR amplifications, the N-terminal and the C-terminal fragments of construct [ID 2363 were independently amplified. The N-terminal fragment was generated using primers GCSF/EPO-5 and GCSF/EPO-6. The GCSF/EPO-5 corresponds to the nucleic acid sequence that encodes for amino acid residues 334 to 340 of the full-length form of HSA. The GCSF/EPO-6 primer comprises the reverse complement of the sequence spanning amino acids 136 to 143 of the full-length form of EPO
with the exception that the codon CGA encoding the Arg residue at amino acid 140 (highlighted in bold) is altered to the cod.on GGA which encodes a Gly residue. The C-terminal fragment was generated using primers GCSFIEPO-7 and GCSF/EPO-8. The GCSIVEPO-7 primer comprises the nucleic acid sequence encoding amino acids 136 to 143 of the full-length form of EPO with the exception that the codon CGA encoding the Axg residue at amino acid 140 (highlighted in bold) is altered to the codon GGA which encodes a Gly residue.
In GCSF/EPO-8, the sequence comprises nucleotides within the pC4 vector downstream of the stop codon. In the second round of PCR amplifications, primers GCSF/EPO-5 and GCSF/EPO-8 were used to amplify the GCSF-HSA-EPO triple fusion protein which has the full-length form of G-CSF fused to the amino-terminus of the mature form of 'ISA, which is fused to the amino-terminus of the mature form of EPO, i.e., A28-D192 which has the Arg-140 to Gly mutation. The reaction mixture contained both the PCR amplified N-terminal fragment and the PCR amplified C-terminalfragment.
[1091] The PCR product was purified and then digested with Bsu36I and Asa After further purification of the Bsu36I-Asel fragment by gel electrophoresis, the product was cloned into Bsu3611/4scl. digested construct 2363 to give Construct ID # 2373.
110921 Further, analysis of the N-terminus of the albumin fusion protein by amino acid sequencing can confirm the presence of the expected GCSF sequence (see below).
110931 GCSF/EPO albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e,, Asp-25 to Len-609, fused to either the N- or C-terminus of the mature fonn of GCSF, i.e., Thr-31 to Pro-204, and fused to either the N- or C-terminus of the mature form of EPO, i.e.,Ala-28 to Asp-192. In one embodiment of the invention, GCSF/EPO albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature GCSF/EPO albumin fusion protein is secreted directly into the culture medium. GCSF/EPO albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAP, INV, Ig, Fibulin B, .
Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric FISA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, GCSF/EPO albumin fusion proteins of the invention comprise either the native GCSF or the native EPO
signal sequence.
In further preferred embodiments, the GCSF/EPO albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and .Purification of Construct ID 2373.
Expression in CHO cells.
[1094]
Construct 2373 can be transfected into CHO cells as previously described in Examples 6 and 8.
Purification from CHO supernatant.
[1095] A
general purification procedure for albumin fusion proteins has been described .in Example 7. The triple fusion protein GCSF-HSA-EPO.R1400 encoded .by construct 2373 can be purified as previously described in Examples 7 and 8. N-terminal sequencing should yield the sequence TPLGP (SEQ ID NO:2144) which corresponds to the mature form of GCSF.
0 . A-EPO. 4; 40G by construct ID 2313. 4-vn be (waved using .an in vitro TF4, pferation assqv and. an in- vitro. N,FS-60. cell proliferation assay.
Method [1096] The activity of the triple fusion protein GCSF-HSA-EPO.R140G encoded by construct 2373 can be assayed in the in vitro TF-1 cell proliferation assay as previously described in Example 9 under subsection heading, "In vitro TF-1 cell proliferation assay for construct 1981", as well as in the in vitro NFS-60 cell proliferation assay as previously described in Example 19 under subsection heading, "The activity of GCSF
albumin fusion encoded by construct ID 4 1642 can be assayed using an in vitro NFS-60 cell proliferation assay".
Result [1097] THE GCSE-IISA-EPO.R140G albumin fusion encoded by construct 2373 demonstrated proliferation of both TF-1 cells and lelFS-60 cells.
The activity of,GC$F-144-EPa'R1100 albumin flak* encoded 4yataa$truct ID #
2373 04-0,;
be assayed in vivo.
Method [1098] The activity of the triple fusion protein GCSE-HSA-EPO.R140G encoded by construct 2373 can be assayed in the in vivo Harlan mouse model to measure hematocrit levels as previously described in Example 9 under subsection heading, "The activity of construct 1981 can be assayed using an in vivo Harlan mouse model for measuring hematocrit", as well as in C57BL/6 mice where GCSE-HSA-EPO.R1400 is a mobilizing agent as previously described in Example 19 under subsection heading, "The activity of GCSE albumin fusion encoded by construct ID # 1642 can be assayed in vivo using C57B116 mice: GCSF-HSA as a Mobilizing Agent".
EXAMPLE 24: Indications for the GCSFITSA-EPO Triple Fusion [1099] Indications for triple fusion proteins comprising GCSE, EPO and HSA, (including, but not limited to, those encoded by constructs 2363 and 2373) may include those indications specified for the EPO albumin fusion proteins and for the GCSE
albumin fusion proteins, including but not limited to, bleeding disorders and anemia caused by a variety Of conditions, including but not limited to end-stage renal disease (dialysis patients), chronic renal failure in pre-dialysis, zidovudine-treated HIV patients, cancer patients on cheniotherapy, and premature infants; pre-surgery in anemic patients undergoing elective non-cardiac, non-vascular surgery to reduce the need for blood transfusions;
aplastic and other refractory anemias, refractory anemia in inflammatory Bowel Disease, and transfusion avoidance in elective orthopedic surgerychemoprotection; treating, preventing, and/or diagnosing inflammatory disorders, m.yelocytic leukemia, primary neutropenias (e.g., Kostrnann syndrome), secondary neutropenia, prevention of neutropenia, prevention and treatment of neutropenia in HIV-infected patients, prevention and treatment of neutropenia associated with chemotherapy, infections associated with neutropenias, myelopysplasia, and autoinumine disorders, mobilization of hematopoietic progenitor cells, bone marrow transplant, acute myelogeneous leukemia, non-Hodgkin's lymphoma, acute lymphoblasfic leukemia, Hodgkin's disease, accelerated myeloid recovery, and glycogen storage disease.

EXAMPLE 25: Construct ID 2053, IFNb-HSA, Generation.
[1100] Construct ID 2053, pEEI2.1:IFNb.HSA, comprises DNA encoding an IFNb albumin fusion protein which has the full-length IFNb protein including the native IFNb leader sequence fused to the amino-terminus of the mature form of HSA in the NSO
expression vector pEE12.1.
Cloning of IFNb (DNA
[11011 The polynucleotide encoding IFNb was PCR amplified using primers IFNb-1 and IFNb-2, described below, cut with Barn HIICla I, and ligated into Bain 11110a I cut pC4:HSA, resulting in construct 2011. The Eco 1111Eco RI fragment from Construct ID ft 2011, was subcloned into the Eco RI site of pEE.12.1 generating construct ID
#2053 which which comprises DNA encoding an albumin fusion protein containing the leader sequence and the mature form of IFNb, followed by the mature HSA protein.
[1102] Two oligonucleotides suitable for PCR amplification of the polynucleotide encoding the full-length of IFNb, IFNb-1 and IFNb-2, were synthesized:
IFNb-1: 5'- GCGCGGATCCGAATTCCGCCGCCA TGACCAACAAGTGTCTCCTCCA
AATTGCTCTCCTGTTGTGCTTCTCCACTACAGCTCTTTCCATGAGCTACAACTTGC
TTGG-3' (SEQ ID NO:817) IFNb-2: 5'- GCGCGCATCGATGAGCAACCTCACTCTTGTGTGCATCGTTTCGGA
GGTAACCTGT-3' (SEQ ID NO:818) [1103] The IFNb-1 primer incorporates a Barn HI cloning site (shown underlined), an Eco RI cloning site, and a Kozak sequence (shown in italics), followed by 80 nucleotides encoding the first 27 amino acids of the full-length form of IFNb. In the Cla .1 site (shown underlined) and the DNA following it are the reverse complement of DNA
encoding the first 10 amino acids of the mature 'ISA protein (SEQ ID NO:1038) and the last 18 nucleotides are the reverse complement of DNA encoding the last 6 amino acid residues of IFNb, (see Example 2). A PCR amplimer was generated using these primers, purified, digested with Barn HI and Cla I restriction enzymes, and cloned into the Bain HI and Cla 1 sites of the .pC4:HSA vector. After the sequence was confirmed, an Eco RI
fragment containing the IFNb albumin fusion protein expression cassette was subcloned into Eco RI
digested pEE12.1.
[1104] Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected PFNb sequence (see below).

11051 IFNb albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of IFNb, i.e., Met-22 to Asn-187. In one embodiment of the invention, IFNb albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression.
In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature IFNb albumin fusion protein is secreted directly into the culture medium. IFNb albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAP, IN'!, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSAIMAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, IFNb albumin fusion proteins of the invention comprise the native IFNb. In further preferred embodiments, the IFNb albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Punfication of Construct ID 2053.
Expression in **urine myeloma NSO cell-lines.
[11061 Construct ID # 2053, pEE12.1:IFNb-HSA , was electroporated into NSO
cells by methods known in the art (see Example 6).
Purification from NSO cell supernatant.
[1107] Purification of IFNb-HSA from NSO cell supernatant may follow the methods described in Example 10 which involve Q-SepharosTemanion exchange chromatography at pH
7.4 using a NaC1 gradient from 0 to I M in 20 rriM Tris-HC1, followed by Poros P150 anion exchange chromatography at pH 6.5 with a sodium citrate gradient from 5 to 40 inM, and diafiltrating for 6 DV into 10 rtiM citrate, pH 6.5 and 140 naM NaC1, the final buffer composition. N-tenninal sequencing should yield the sequence MSYNLL which is the amino terminus of the mature form of IFNb. The protein has an approximate MW of 88.5 kDa.
[11081 For larger scale purification, e.g., 50 L of NSO cell supernatant can be concentrated into ¨8 to 10 L. The concentrated sample can then be passed over the Q-Sepharoseanion exchange column (10 x 19 cm, 1.5 L) at pH 7.5 using a step elution consisting of 50 triM Na0Ac, pH 6.0 and 150 trilvl NaCl. The eluted sample can then be virally inactivated with 0.75% Tritor=X 100 for 60 min at room temperature.
SDR-Reverse base chromatography (10 cm x 10 cm, 0.8 L) can then be employed at pH 6.0 with 50 mM
Na0Ac and 150 mM NaCI, or alternatively, the sample can be passed over an SP-sepharosTem column at pH 4.8 using a step elution of 50 mM Na0Ac, pH 6.0, and 150 mM NaCi.

filtration would follow to remove any viral content. Phenyl-650M
chromatography (20 cm x 12 cm, 3.8 L) at pH 6.0 using a step elution consisting of 350 mM (N114)2SO4 and 50 mM
Na0Ac, or alternatively consisting of 50 mM Na0Ac pH 6.0, can follow.
Diafiltration for 6-8 DV will allow for buffer exchange into the desired final formulation buffer of either 10 mM
Na2HPO4 +58 mM sucrose + 120 mM NaCI, pH 7.2 or 10 mM citrate, pH 6.5, and 140 mM
NaCI or 25 mM Nail-Rah 100 mM NaCI, pH 7.2.
7'he activity of IF.Nb can be assayed usinzan in vitro ISRE-SEAP assay.
[1109] All type I Interferon proteins signal through a common receptor complex and a similar Jak/STAT signaling pathway that culminates in the activation of Interferon, "IFN", responsive genes through the Interferon Sequence Responsive Element, "ISRE". A

convenient assay for type I IFN activity is a promoter-reporter based assay system that contains multiple copies of the ISRE element fused to a downstream reporter gene. A stable HEK293 cell-line can be generated .and contains a stably integrated copy of an ISRE-SEAP
reporter gene that is extremely sensitive to type I 1FNs and displays linearity over 5 logs of concentration.
Method of Screening of IFNb-IISA NSO stable clones.
111101 Construct 2053 was electroporated into NSO cells as described in Example 6.
The NSO cells transfected with construct ID # 2053 were screened for activity by testing conditioned growth media in the ISRE-SEAP assay. The ISRE-SEAP/293F reporter cells were plated at 3 x 104 cell/well in 96-well, poly-D-lysine coated, plates, one day prior to treatment. Reporter cells were treated with various dilutions (including but not limited to =
1:500 and 1:5000) of conditioned supernatant or purified preparations of IFNb albumin .
fusion protein encoded by construct ID 2053 or rhIFNb as a control. The reporter cells were then incubated for 24 hours prior to removing 40 L for use in the SEAP
Reporter Gene Cherniluminescent Assay (Roche catalog # 1779842). Recombinant human Interferon beta, "rbIFNb" (Biogen), was used as a positive control.
Result [11111 The purified preparation of MO expressed IFI\lb-IISA had a greater EC50 of 9.3 x 10-9 giml, than rill-171\Tb (Biogen) which had an EC50 of 1.8 x 10-1 g/mL (see Figure 13).
in vivo ifidtixti44 of 04$ by w boeifron.
Method [11121 The OAS
enzyme, 2'-5'- OligoAdenylate Synthetase, is activated at the transcriptional level by interferon in response to antiviral infection. The effect of interferon constructs can be measured by obtaining blood samples from treated monkeys and analyzing these samples for transcriptional activation of two OAS mRNA, p41 and p69. A
volume of 0.5 mi, of whole blood can be obtained from 4 animals per group at 7 different time points, day 0, day 1, day 2, day 4, day 8, day 10, and day 14 per animal. The various groups may include injection of vehicle control, intravenous and/or subcutaneous injection of either 30 g/kg and/or 300 g/kg TFN albumin fusion protein on day 1, and subcutaneous injection of 40 gikg of Interferon alpha (Schering-Plough) as a positive control on days 1, 3, and 5.
The levels of the p41 and the p69 mRNA transcripts can be determined by real-time quantitative PCR (Taqman) using probes specific for p41-OAS and p.69-OAS. OAS mRNA levels can be quantitated relative to 18S ribosomal RNA endogenous control.
&Alva indudifdn. It ad:by inteeemit Oeta.
4,x1Invkjigangg,(0.a.cions_k_titiaaZat Method [11131 The activity of the IISA-IFN b fusion protein encoded by construct 2053 can be assayed in the in vivo OAS assay as previously described above under subsection heading, "In vivo induction of OAS by an Interferon".
EXAMPLE 26: Indications for IFNb albumin fusion prOteins, [11141 IFN beta albumin fusion proteins (including, but not limited to, those encoded by construct 2053) can be used to treat, prevent, ameliorate and/or detect multiple sclerosis.
Other indications include, but are not limited to, melanoma, solid tumors, cancer, bacterial infections, chemoprotection, thrombocytopenia, HIV infections, prostate cancer, cancer, hematological malignancies, hematological disorders, preleukemia, gliorna, hepatitis B, hepatitis C, human papillomavirus, pulmonary fibrosis, age-related macular degeneration, brain cancer, glioblastoma multiforrne, liver cancer, malignant melanoma, colorectal cancer, Crohn's disease, neurological disorders, non-small cell lung cancer, rheumatoid arthritis, and ulcerative colitis.
EXAMPLE 27: Construct ID 1941, HSA-PT1184, Generation.
[11151 Construct ID 1941, pC4.HSA.PTH84, encodes for an HS.A-PT1184 fusion protein which comprises the full-length of HSA including the native HSA leader sequence, fused to the mature form of the human parathyroid hormone, "PTH84" Ser-1 to Gln-84, cloned into the mammalian expression vector pC4.
Cloning of PTI184 .cDNA for construct 1941 111161 The DNA encoding PTH84 was amplified with primers PTH84-1 and PTH84-2, described below, cut with Bsu 361/Not I, and ligated into Bsu 361/Not I cut pC4IISA.
Construct ID #1941 encodes an albumin fusion protein containing the full-length form of HSA that includes the native HSA leader sequence, followed by the mature PTH84 protein.
[11171 Two primers suitable for PCR amplification of the polynucleotide encoding the mature form of PTH84, P11184-1 and PTH84-2, were synthesized.
PTH84-I: 5'- GAGCGCGCCTTAGGCTCTGTGAGTGAAATACA.GCTTATGCATAAC-3' (SEQ ID NO:787) P11184-2: 5LCGGTGCGCGGCCGCITACTGGGATTTAGCTTTAGTTAATACATTCA
CATC-3' (SEQ ID NO:788) [11181 PTH84-1 incorporates a Bsu 361 cloning site (shown in italics) followed by the nucleic acid sequence encoding amino acid residues Ala-Leu-Gly corresponding to the end of the mature form of HSA (the last Leu is absent) in conjunction with amino acid residues Ser-1 to Asn-10 of the mature form of PTH84. In PT1184-2, the Not I site is shown in italics and the nucleic acid sequence that follows corresponds to the reverse complement of DNA
encoding the last II amino acids of the mature PTH84 protein. Using these two primers, the PTH84 protein was PCR amplified.
[1119] Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing confirmed the presence of the expected HSA sequence (see below).
[1120] PTH84 albumin fusion proteins of the invention preferably comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to either the N- or C-terminus of the mature form of PTH84, i.e., Ser-1 to Gln-84. In one embodiment of the invention, PTH84 albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature PT1184 albumin fusion protein is secreted directly into the culture medium.
PTH84 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, 1g, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSAJMAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, P11-184 albumin fusion proteins of the invention comprise the native PT1184. In further preferred embodiments, the PT1184 albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynueleotides encoding these polypeptides,, including fragments and/or variants, are also encompassed by the invention.
&press-ion and Parifitglion ,gf.ConstnliptID:1951.
Expression in 29.31" cells.
111211 Construct 1941. was transfected into 2931 cells cells by methods known in the art (e.g., lipofectamine transfection) and selected with 100 nM methotrexate (see Example 6).
Expression levels were examined by immunoblot detection with anti-HSA serum as the primary antibody.
Purification from 2937' cell supernatant.
111221 The 2931 cell supernatant containing the secreted HSA-PTH84 fusion protein expressed from construct ID #1941 in 293T cells was purified as described in Example 7.
Specifically, initial capture was performed with an anionic HQ-50 resin at pH
7_2 using a sodium phosphate buffer (25 mM Na21-1PO4 p11 7.2) and 16 column volumes of a salt gradient elution of 0 to 0.5 M NaC1, followed by Hydrophobic Interaction Chromatography, "H1C", with the Phenyl 650 M resin (from Tosohaas) using 36 column volumes of a salt gradient elution of 2.75 to 0 M NaC1 at pH 7.2 where the sample had a final conductivity of 180 mS.
The sample was concentrated using the HQ Poros 50 resin and a salt step elution of 0.15 M
NaCI increments. The final buffer composition consisted of 25 rnM Na2HPO4 150 rnM
NaC1 pH 7.2. N-terminal sequencing generated the amino-terminus sequence (i.e., DAIIICS, SEQ ID NO:2143) of the mature forth of HSA. A protein of approximate MW of 78 kDa was obtained. A final yield of 038 mg protein per litre of 293T cell supernatant was obtained.

In 'vitro inductiOnSofevdkiAMP in:402:44s;
Method [1123] The biological activity of a PTH84 albumin fusion protein can be measured in an in vitro assay in which Sa0S-2, an osteosarcorna ce1171ine, is used. PTH
activates adenylate cyclase thereby increasing intracellular cyclic AMP levels.
Induction of cAMP in Sa0S-2 cells:
[1124] The Sa0S-2 .cells are subcultured at a density of 8.0 x 104...cells/well 24 hours prior to the start of the experiment. On the day of the experiment, the cells are serum starved for 2 hours and then treated for 10 minutes with positive controls (e.g., forskolin at 5 mg/rxiL), recombinant PTH, or the PTH albumin fusion proteins. Following treatment, the cells are then rinsed and the intracellular cyclic AMP is extracted with cold ethanol. The ethanol extracts can be lyophilized and stored at ¨80 C for further use. The amount of cyclic AMP present in the samples is quantitated by ELISA as per the manufacturer's protocol (Amersham Life Sciences, Inc.).
fr vitro :induction &los& 440ii in Sit0g2 cells. by the albumin jiaiOn proem. 016)&4 11 construct 1941 Method [1125] The in vitro assay to measure the induction of cyclic AMP in Sa0S2 cells by the PTH albumin fusion protein encoded by construct 1941 can be carried out as previously described above.
In vivo: Induced release of calcium in TPTX animals..
Methods [1126) PTH activity is tested by monitoring the PTH albumin fusion proteins ability to reduce the demineralization of bone following ThyroParaThyroidectomy, "TPTX", administration of a low calcium diet, and parathyroid hormone treatment. .
[1127] The animals display a variability in pharmacological response as suggested by Votta, et al., 1997, J. Bone and Mineral Res., 12: 1396 ¨ 1406; Millest, et al., 1997, Bone, 20: 465 ¨ 471; and Iwata, et al., 1997, Arthritis and Rheumatism, 40: 499 ¨
509. Therefore, between 5 and 8 thyloparathyroidectomized animals (purchased from an outside vendor) per group are used. The animals receive replacement injections of thyroxine every other day.

Each experiment will include several groups: (1) placebo and parathyroid hormone (PTH 1-34) injected groups which correspond to the negative and positive controls, respectively; (2-5) PTH albumin fusion proteins, at various concentrations ranging from 0.1 to 12 p.g./kg, injected intravenously, intraperitoneally, subcutaneously, and intramuscular, either before, = = during, or after parathyroid hormone treatment; (6) for some experiments, a cysteine protease inhibitor is tested.
[11281 Under isofluorane anesthesia, the left femoral vein and either the left femoral artery or carotid artery is cannulated with PE-10 tubing fused to PE-50 polyethylene tubing filled with heparinized saline. The catheters are tunneled subcutaneously, exteriorized at the nape and secured to the skin. The animals are allowed torecover for approximately 18 hours prior to being used for experimentation during which time they are given a calcium free diet.
During the course of the experinient, 3 blood samples (200 mL each) are taken via the carotid or femoral catheter following 2, 4, and 6 hours of infusion. Longer time points, e.g., 18 hours, may also be desirable.
[11291 A dose relationship between human PTH 1 ¨ 34, the positive control, and the appearance of ionized calcium levels in whole blood was established (data not shown).
-Tire ac_ILIA,pflIA,fiSysiLLADjeas.t_o_e_thr s o zs___gukNggygt_i co *Or/jet /941. = 1. .. rg TPTX animals.
[11301 The activity of the PTH albumin fusion protein encoded by construct 1941 can be measured using TPTX animals and the in viva assay described above under the heading, "In viva: Induced release of calcium in TPTX animals".
An. 0. vivo. ovartgotOmfte.dfcmakt. .tva.modot .
Methods [11311 PTH activity is tested by monitoring the ability to induce bone formation in ovariectomized female Lewis or Sprague Dawley rats.
[11321 Surgery is performed on female Lewis or Sprague Dawley rats 8 ¨
9 weeks of age and experiments are not initiated until 7 to 10 days after the surgery.
Samples from blood, urine, and left tibia are obtained weekly from 9 to 12 animals per group. The various groups can include a sham control injected with saline everyday for four weeks, ovariectomized rats injected with saline everyday for four weeks, and ovariectomized rats injected with rat PTH peptide 1-34 at 10 g/kg subcutaneously five times per week.

Following the fourth and final week of tissue collection, the tibias are sent to Skeletech for bone densitometry analysis.
[11331 The parameters tested are body weight, bone densitometry on left tibia in 70%
ethanol, serum pyridinoline from blood, and urine deoxypyridinoline and alpha helical protein.. Urine samples are taken in the morning. Blood is obtained from bleeding the heart and the serum is saved for ELISA analysis. Bone densitometry is conducted on the proximal tibia. The left femur can be cut with bone shears just above the knee. The paw can also be removed by cutting the distal tibia. The skin is slit laterally to allow in ethanol and the remainder of the limb is put in a 50 cc tube filled with 70% ethanol. The tube is stored at room temperature until shipped. The rat tibial specimens are allowed to thaw to room temperature the day of the testing. Excised rat tibiae are subjected to bone mineral density determinations using peripheral quantitative computed tomography (pQCT, XCT-RM, Norland/Stratec). The scan is performed at a proximal tibia site (12% of the total length away from the proximal end). One 0.5 mm slice is taken. Scans are analyzed as a whole (total.
bone) or using a threshold delineation of external and internal boundaries (cortical bone) or an area that is 45% of the total bone tissue by peeling from the outer edge (cancellous bone).
Bone mineral density, area and content are then determined by system software.
The differences between sham and ovariectomized animals, at, each different time point, are determined by two-tailed t-test using SAS statistical software (SAS Institute, Cory, NC). The student t test is used for statistical comparison of means. P values .of less than 0.05 are considered statistically significant.
Th: trgY 0 the 0 told jai mt. 10 teen - ztcod COLiJ. d .94 f.
so__ -me in vtypcovariecioniized female rat ineside4 [11341 The activity of the PTH albumin fusion protein encoded by construct 1941 can be measured using the in vivo, assay described above under the heading, "An in vivo ovariectomized female rat model".
ANAIV,IPLE.28: Construct ID 1949õ PTII$4-11S.Atatuer4tion.
[11351 Construct ID 1949, pC4.PT}1.84.S1-Q84..HSA, encodes a PTH84-HSA
fusion protein which comprises the MPIF leader sequence, followed by the mature form of PTH8.4, i.e., Ser-I to Gln-84, fused to the amino-terminus of the mature form of HSA
cloned into the mammalian expression vector pC4.

C19/1ing qfPrIal eDIVA fik coWnict 1949 [1136] The DNA encoding PTH84 was amplified with primers PTH84-3 and PTH84-4, described below, cut with Barn Hi/Spel, and ligated into Barn HlabaI cut pC411SA.
Construct ID #1949 encodes an albumin fusion protein comprising the mature PTH84 protein followed by the mature form of HSA.
[11371 Two primers suitable for PCR amplification of the polynucleotide encoding the Mature form of PTH84, PTH84-3 and PTH84-4, were synthesized..
P1 H84-3 : 5',GACIC.,GC6G/1 TCCOCCATCATOAAGGIVICCGTGGCMCCCTcrec TCieCTCATGGITGITACTGCCCMIGATCTCAGGM,TC1 c;WAGTGAAATACAG
CTTATGC-3' (SEQ NO:793) PTH84-4: 5'- GTCGTCACTAGTCTOGGATTTAGCTTTAGTTA:ATACATTCAC-3' (SEQ II) NO:794) [11381 ' PTH84-3 IricorpOrates a Barn HI cloning site (shown in italics) followed by a nucleic acid sequence that encbdeS the MPIF signal peptide (shown underlined) and amino acid residues 5er-1 to Met-8 (shown in bold) of the mature form of PTH84. In PTH84-4, the Spel site is shown in italics and the nucleic acid sequence that follows corresponds to the -reverse complement of DNA encoding the last 10 amino acids of the mature PTI184 protein (shown hi bold). Using these two primers, the PTH84 protein was PCR amplified.
The PCR
amplimer was purified, digested with Barn HI and Spel and ligated into Barn HI/XbaI cut pC4:HSA.
[1139] There are two additional amino acid residues, i.e., Thr and Ser, between PTH84 and HSA as a result of the introduction of the Spar Cloning site into the PTI-184-4 primer.
[1140] Further analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing confirmed the presence of the expected PTH84 sequence (see below).
[1141] PTH84 albumin fusion proteins of the invention preferably comprise the mature form of LISA, i.e., Asp-25 to Leu-609, fused to either the N- or C-terminus of the mature form of PTH84, i.e., Ser-1 to Gln-84. In one embodiment of the invention, PTH84 albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature PTH84 albumin fusion protein is secreted directly into the culture medium.

PTH84 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAP. INV, 1g, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric IIS/VMAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, PTH84 albumin fusion proteins of the invention comprise the native PTH84. In further preferred embodiments, the PTH84 albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Purification of Construct ID 1949.
Expression in 293T cells.
(11421 Construct 1949 was transfected into 293T cells cells by methods known in the art (e.g., lipofectamine transfection) and selected with 100 riM
methotrexate.(see Example 6), Expression levels were examined by immunoblot detection with anti-HSA serum as the primary antibody. .
Purification from 2937' cell supernatant.
[1143] The 293T cell supernatant containing the secreted PTH84-HSA fusion protein expressed from construct II) #1949 in 293T cells was purified as described in Example 7.
Specifically, initial capture was performed with an anionic HQ-50 resin at pH
7.2 using a sodium phosphate buffer (25 mM Na2HPO4 pH 7.2) and 16 column volumes of a salt gradient elution of 0 to 0.5 M NaC1, followed by Hydrophobic Interaction Chromatography, "HIC", with the Phenyl 650 M resin (from Tosohaa.$) using 36 column volumes of a salt gradient elution of 2.75 to 0 M NaCI at pH 7.2 where the sample had a final conductivity of 180 mS.
The sample was concentrated using the HQ Poros 50 resin and a salt step elution of 0.15 M
MCI increments. The final buffer composition consisted of 25 mM Na2HPO4 + 150 mM
NaC1 pH 7.2. N-terminal sequencing generated the amino-terminus sequence (i.e., SVSEI, SEQ ID NO:2145) of the mature form of PTH84. A protein of approximate MW of 78 kDa was obtained. A final yield of 0.32 mg protein per litre of 293T cell supernatant was obtained.
P-vitro wduczu*- 0fçydicA 2 Al by albumin fu.m.proteuz consttjjct1942 Result 111401J A purified HSA-PTH84 albumin fusion protein derived from 293T cells expressing construct 1949 was tested in the in vitro assay described in Example 27 under subsection heading, "In vitro induction of cyclic AMP in Sa0S2 cells". HSA-induced an increase in intracellular cyclic AMP levels.
Th_e gt_g_Cremin lesion prpiein encOesi.by eamrintet 1949 can be assawci itsing TPTX animals.
[1145] The activity of the PTH albumin fusion protein encoded by construct 1949 can be measured using TPTX animals and the in vivo assay described in Example 27 under the subsection heading, "In viva: Induced release of calcium in TPTX animals".
The activity 9f the albmidgiajtmotein encod;eti yettkittowet 1949 can be 4rssayed usin7 the in viva ovarieetomized female rat model.
[11461 The activity of the PTH albumin fusion protein encoded by construct 1949 can be measured using the in vivo assay described in Example 27 under the subsection heading, "An in vivo_ovatiectomized female rat model".
EXAMPLE 291 Cunstruet ID 2021, PT11844ISA, Generation [11471 Construct ID 2021, pC4.PTH84.S1-Q84,HSA, encodes for an PTH84-HSA
fusion protein which comprises the native HSA leader, followed by the mature form of PTH84, i.e., Ser-1 to Gln-84, fused to the amino-terminus of the matitre form of HSA cloned into the mammalian expression vector pC4.
epAr ,enA.Ljaggibst2M.
[1148] The DNA encoding PTH84 was amplified with primers PTH84-5 and PTH84 6, described below, cut with Alio I/C1O I, and liga.ted into Xlzo IICla I cut pC4:HSA.
Construct #2021' encodes an albumin fusion protein containing the mature PTH84 protein followed by the mature form of HSA (see Example 5).
[1149] Two primers suitable for PCR amplification of the polynucleotide encoding the mature form of PTH84, PTH84-5 and PTI184-6, were synthesized.
PTH84-5: 5'- CCGCCGCTCGAGGGGTGTGTTTCGTCGATCTGTGAGTGAAATAC
AGCTTATGCATAAC-3' (SEQ ID NO:823) PTH34-6! 5'- A GT CCCA TCGATGAG'CAACCTC A Cit.711PTGTOCATCCIVGGATT
TACCTTTAGTTAATACATTCACATC-3' (SEQ 11) NO:824) 111501 PTH84-5 incorporates a Xho I cloning site (shown in italics). The Xho I site combined with the nucleic acid sequence that follows (shown underlined) encodes for the last four amino acid residues of the chimeric signal peptide of IISA. The nucleic acid sequence in bold encodes for amino acid residues Ser-1 to Asn-10 of the mature form of PTH84. In PTH84-6, the Cla I site is shown in italics and the nucleic acid sequence that follows (shown underlined) corresponds to the reverse complement of DNA encoding the first 10 amino acids of the mature form of HSA. The nucleic acid sequence highlighted in bold in corresponds to the reverse complement of DNA encoding the last 11 amino acids of the mature form of PTH84. Using these two primers, the PTH84 protein was PCR
amplified.
The PCR amplimer was purified, digested with Alto I and Cla I and ligated into Xho 11Cla I
cut pC4IISA.
[1151] Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected PTH84 sequence (see below).
[1152] PTH84 albumin fusion proteins of the invention preferably comprise ,the mature form of HSA, i.e., Asp-25 to Leu-609, fused to either the N- or C-terminus of the mature form of PTH84, i.e., Ser-1 to Gln-84. In one embodiment of the invention, PTH84 albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature PTH84 albumin fusion protein is secreted directly into the culture medium.
PTH84 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, 1g, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric IISAftv1AF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, PTH84 albumin fusion proteins of the invention comprise the native PTH84. In further preferred embodiments, the PTH84 albumin fusion proteins of the invention further comprise an N-terminal rnethionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.

ftptession::andPureekition-01Coostruct 2021..
Expression in 2937' cells.
11153] Construct 2021 can be transfected into 293T cells cells by methods known in the art (e.g., lipofectamine transfection) and selected with 100 nM
methotrexate (see Example 6). Expression levels can be examined by immunoblot detection with anti-HSA
serum as the primary antibody.
Purification from 2937' cell supernatant.
El 154] The 293T cell supernatant containing the secreted PTII84-HSA
fusion protein expressed from construct ID #202I in 293T cells Can be purified as described in Example 7.
Specifically, initial capture can be performed with an anionic HQ-50 resin at pll 7.2 using a sodium phosphate buffer (25 niM Na41PO4 pH 7:2) and 16 column volumes of a salt 'gradient elution of 0 to 0.5 M NaC1, followed by Hydrophobic Interaction Chromatography, "HIC", with the Phenyl 650 M resin (from Tosohaas) using 36 column volumes of a salt gradient elution of 2.75 to 0 M NaC1 at pH 7.2 where the sample has a final conductivity of 180 mS.
The sample can be concentrated using the HQ Poros 50 resin and a salt step elution of 0.15 M
NaC1 increments. The final buffer composition may consist of 25 rnM Na2HPO4 +
150 mM
Naa pH 7.2. N-terminal sequencing should generate the amino-terminus sequence (i.e., SVSEI) of the mature form of PTH84. A protein of approximate MW of 78 kDa should be obtained.
. ' __________________ Odic fait in AtQSZ-ceill.liv.Me -albumin filsion protein encoded by construct 2021.
11155] HSA-PTI-184 albumin fusion protein derived from 293T cells expressing = construct 2021 can be tested in the in vitro assay described in Example 27 under subsection heading, "In vitro induction of cyclic AMP in Sa0S2 cells".
lite..activitypf tie cdinpnin *ion proteht encoded brcono-uct 2924 On. be tISSaY0.1:1 tai4g:
TPTX animals.
11156] The activity of the PTH albumin fusion protein encoded by construct 2021 can be measured using 1TTX animals and the in vivo assay described in Example 27 under the subsection heading, "In vivo: Induced release of calcium in TPTX animals".
..attivitv c.if the alpimin fusion peotein encoded by oftstetict. 2Ø7.4 can be assayed taing µ, the in vivo ovarieciont#edigmale rat model.
111571 The activity of the PTH albumin fusion protein encoded by construct 2021 can be measured using the in vivo assay described in Example 27 under the subsection heading, "An in vivo ovariectomized female rat model".
EXAMPLE 30: Indications for PTI184 Albumin Fusion Proteins.
111581 Results from in vitro and in vivo assays described above indicate that PTHael albumin fusion proteins are useful for the treatment, prevention, and/or diagnosis of osteoporosis, malignant hypercalcaemia, and Paget's disease.
EXAMPLE 31: Construct ID 2249, IFNa2-HSA, Generation.
[1159] . Construct ID 2249, pSAC35:1FNa2.11SA, comprises DNA encoding an IFNa2 albumin fusion protein which has the HSA chimeric leader sequence, followed by the mature form of IFNa2 protein, i.e., Cl-E165, fused to the amino-terminus of the mature form of HSA
in the yeast S. cerevisiae, expression vector`pSAC35.
Cloning of IFNa2 cDNA
111601 The polynucleotide encoding IFNa2 was PCR amplified using primers IFNa2-1 and IFNa2-2, described below. The PCR amplimer was cut with Sal IICla I, and ligated into Xho I/Cla I cut pScCHSA. Construct ID #2249 encodes an albumin fusion protein containing the chimeric leader sequence of HSA, the mature form of IFNa2, followed by the mature HSA protein.
[1161] Two oligonucleotides suitable for PCR amplification of the polynucleotide encoding the mature form of IFNa2, IFNa2-1 and IFNa2-2, were synthesized:
IFNa2-1: S'-CGCGCGCGTCGACAAAAGATGTGATCTGCCTCAAACCCACA-3' (SEQ
11.) NO:887) IFNa2-2: 51-GCGCGCA.TCGATGAGCAACCTCACTCTTGTGTGCATCTTCCTTAC
TTCTTAAACTTTCT-3' (SEQ ID NO:888) 111621 The IFNa24 primer incorporates a Sal I cloning site (shown underlined), nucleotides encoding the last three amino acid residues of the chimeric HSA
leader sequence, as well as 22 nucleotides (shown in bold) encoding the first 7 amino acid residues of the mature form of IFNa2. In 1FN'a2-2, the Cia I site (shown underlined) and the DNA following it are the reverse complement of DNA encoding the first lp amino acids of the mature HSA
protein and the last 22 nucleotides (shown in bold) are the reverse complement of DNA
encoding the last 7 amino acid residues of I7Na2 (see Example 2). A PCR
arnplimer of IFNa2-1ISA was generated using these primers, purified, digested with Sal I
and Cia I
restriction enzymes, and cloned into the .17io I and Cla I sites of the pScCHSA vector. After the sequence was confirmed, the expression cassette encoding this IFNa2 albumin fusion protein was subcloned into Not I digested pSAC35.
(1163) Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected IFNa2 sequence (see below).
111641 Other IFNa2 albumin fusion proteins using different leader sequences have been constructed by methods known in the art (see Example 2). Examples of the various leader sequences include, but are not limited to, invertase "INV" (constructs 2343 and 2410) and mating alpha factor "MAI"' (construct 2366). These IFNa2 albumin fusion proteins can be suhcloned into mammalian expression vectors such as pC4 (constructs 2382) and pEE1.2.1 as described previously (see Example 5). IFNa2 albumin fusion proteins with the therapeutic [11651 fFNa2 albumin fusion proteins of the invention preferably comprise the mature form:of USA , i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the mature form of IFNa2, i.e, Cys-I to Gin-165. In one embodiment of the invention, IFNa2 albumin fusion proteins of the invention farther comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression.
In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature IFNa2 albumin fusion protein is secreted directly into the culture medium. IFNa2 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, 1g, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant FISA leader sequences including, but not limited to, a chimeric LISA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, IFNa2 albumin fusion proteins of the invention comprise the native IFNa.2. In further preferred embodiments, the IFNa2 albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments andior variants, are also encompassed by the invention.
.giprAysion OttPur_kirj*Asit 4703-i-tr,,,,_,J___)22t ,..xpression in yeast S. cerevisiae.
[11661 Transformation of construct 2249 into yeast S. cerevisiae strain BXP10 was carried out by methods known in the art (see Example 3). Cells can be collected at stationary phase after 72 hours of growth. Supernatants are collected by clarifying cells at 3000g for 10 min. Expression levels are examined by immunoblot detection with anti-HSA
serum (Kent Laboratories) or as the primary antibody. The IFNa2 albumin fusion protein of approximate molecular weight of 88.5 IrDa can be obtained.
Purification from yeast S. cerevisiae cell supernatant.
[11671 The cell supernatant containing IFNa2 albumin fusion protein expressed from construct ID #2249 in yeast S. cerevisiae cells can be purified either small scale over a Dyax peptide affinity column (see Example 4) or large scale by following 5 steps:
diafiltration, anion exchange chromatography using DEAE-Sepharosim Fast Flow column, hydrophobic interaction chromatography (H1C) using Butyl 6505 column, 'cation exchange chromatography using an SP-Sepharosr Fast Flow column or a Blue-Sepharose chromatography, and high performance chromatography using Q-sepharoSW high performance column chromatography (see Example 4). The IFNa2 albumin fusion protein may elute from the DEABSepharog Fast Flow column with 100 ¨ 250 mM NaCI, from the SP-Sepharose Fast Flow column with 150 ¨ 250 mIVI NaC1, and from the Q-SepharosTemHigh Performance column at 5 ¨ 7.5 mS/cm. N-terminal sequencing should yield the sequence CDLPQ
(SEQ
ID NO:2146) which corresponds to the mature form of TENa2.
The activity of IFNa2 can be assayed using an in vitro ISRE-SEAP assay.
Method [11681 The IFNa2 albumin fusion protein encoded by construct ID # 2249 can be tested for activity in the ISRE-SEAP assay as previously described in Example 25. Briefly, conditioned yeast supernatants were tested at a 1:1000 dilution for their ability to direct 1SRE
signal transduction on the ISRE-SEAP/293F reporter cell-line. The ISRE-reporter cells were plated at 3 x 104 celliwell in 96-well, poly-D-lysine coated, plates, one day prior to treatment. The reporter cells were then incubated for 18 or 24 hours prior to removing 40 itL for use in the SEAP Reporter Gene Chemiluminescent Assay (Roche catalog # 1779842). Recombinant human Interferon beta, "rhIFNb" (Biogen), was used as a positive control.
Result 11169] The purified preparation of IFNa2-I1SA demonstrated a relatively linear increase in the ISRE-SEAP assay over concentrations ranging from 10-1 to 101 ngiml., (see Figure 15) or 10-i0 to 10-8 ng/mle (see Figure 16).
fir vivo Wird' on of OAS by iirterlimm: aiphajitsion..encqfkdhjiethistrt0t ID

Method 111701 The OAS enzyme, 2'-5'- OligoAdenylate Synthetase, is activated at the transcriptional level by interferon in response to antiviral infection. The effect of interferon constructs can be measured by obtaining blood samples from treated monkeys and analyzing these samples for transcriptional activation of two OAS m.RNA, p41 and p69. A
volume of 0.5 nile of whole blood was obtained from 4 animals per group at 7 different time points, day 0, day 1, day 2, day 4, day 8, day 10, and day 14 per animal. The various groups include vehicle control, intravenous injection of 30 ug/kg HSA-IFN on day 1, subcutaneous injection of 30 p.g/kg of FISA-1FN on day 1, subcutaneous injection of 300 jig/kg of HSA-WN on day 1, and subcutaneous injection of 40 rig/kg of Interferon alpha (Schering-Plough) as a positive control on days 1, 3, and 5. The levels of the p4-1 and the p69 mRNA
transcripts were determined by real-time quantitative PCR (Taqman) using probes specific for p41-OAS and p69-OAS. OAS in.RNA levels were quantitated relative to 18S ribosomal RNA
endogenous control. The albumin fusion encoded by construct 2249 can be subjected to similar experimentation.
Results 111711 A significant increase in rnRNA transcript levels for both p41 and p69 OAS
was observed in HSA-interferon treated monkeys in contrast to IFNa treated monkeys (see Figure 17 for p41 data). The effect lasted nearly 10 days.
EX.A.MPLE 32: Indications for IFNa2 Albumin Fusion Proteins 111721 IFN alpha albumin fusion protein (including, but not limited to, those encoded by constructs 2249, 2343, 2410, 2366, 2382, and 2381) can be used to treat, prevent, ameliorate, andior detect multiple sclerosis. Other indications include, but are not limited to, Hepatitis C, oncology uses, cancer, hepatitis, human papilloma virus, fibrorriyalgia, Sjogren's syndrome, hairy cell leukemia, chronic myelogeonus leukemia, AIDS-related Kaposi's sarcoma, chronic hepatitis B, malignant melanoma, non-Hodgkin's lymphoma, external condylomata acumin.ata, HIV infection, small cell lung cancer, hematological malignancies, herpes simplex virus infections, multiple sclerosis, viral hemorrhagic fevers, solid tumors, renal cancer, bone marrow disorders, bone disorders, bladder cancer, gastric cancer, hepatitis D, multiple myelorna, type I diabetes mellitus, viral infections, cutaneous T-cell lymphoma, cervical dysplasia, chronic fatigue syndrome, and renal cancer.
[1173] Preferably, the EFNct-albumin fusion protein or IFN hybrid fusion protein is administered in combination with a CCR5 antagonist, further in association with at least one of ribavirin, 1L-2, IL-12, pentafuside alone or in combination with an anti-IHV drug therapy, e.g.. HAA.RT, for preparation of a medicament for the treatment of HIV-I
infections, HCV, or HIV-1 and HO/ co-infections in treatment-naïve as well as treatment-experienced adult and pediatric patients.
EXAMPLE 33: Construct In 2250, HSA-Insulin (GYG), Generation.
111741 Construct ID 2250, pSAC35.HSA.T.NSULIN(GYG).F1-N62, encodes for an HSA-INSULIN (GYG) fusion protein which comprises full length HSA, including the native HSA leader sequence, fused to the amino-terminus of the synthetic single-chain long-acting insulin analog (INSULIN (GY32G)) with a Tyr at position 32, cloned into the yeast S.
cerevisiae expression vector pSAC35.
CloninR of INSULIN (GYG) cDNA for construct 2250.
111751 The DNA encoding the synthetic single-chain form of INSULIN (GYG) was PCR generated using four overlapping primers. The sequence corresponding to the C-peptide in the middle region of the proinsulin cDNA was replaced by the c-domain of Insulin Growth Factor I, "IGF-1" (GY32GSSSRRAPQT, SEQ ID NO:2147), to avoid the need for proinsulin processing and to ensure proper folding of the single-chain protein. The sequence was codon optimized for expression in yeast S. cerevisiae. The PCR fragment was digested and subcloned into Bsu 361//lsc I digested pScNTISA. A Not 1 fragment was then subcloned into the pSAC35 plasmid. Construct ID #2250 encodes for full length HSA, including the native HSA leader sequence, fused to the amino-terminus of the synthetic single-chain form of INSULIN (GYG).
[11761 The 5' and 3' primers of the four overlapping oligonucleotides suitable for PCR amplification of the polynucleotide encoding the synthetic single-chain form of INSULIN (GYG), INSULIN (GYG)-1 and INSULIN (GYG)-2, were synthesized:
INSULIN (GYG)-1: 5'-GICAAGCMCCn'A GGCTTATTCGTTAACCAACACTTGTGTGGTT
CTCACTTGGITGAAGCTITGTACITGGTTTGTGGTGAA-3 ' (SEQ ID NO:889) INSULIN (GYG)-2: 5'-ATCGCATATG GCG CC' CCCT ATTAGTTACAGTAGTMCCAATTG
GIACAAAGAACAAATAGAAGTACAA -3' (SEQ ID NO:890) 111771 INSULIN (GYG)-1 incorporates a Bs u 361 cloning site (shown in italics) and encodes the first 21 amino acids (shown in bold) of the ORF of the synthetic single-chain form of INSULIN (GYG). In iNSULIN (GYG)-2, the italicized sequence is an Asc I
site. In INSULIN (GYG)-2, the bolded sequence is the reverse complement of the last 49 nucleotides encoding amino acid residues Cys-49 to Asn-63 of the synthetic single-chain form of INSULIN (GYG). With these two primers, the synthetic Single-chain form of INSULIN
(GYG) was PCR amplified. Annealing and extension temperatures and times must be empirically determined for each specific primer pair and template.
111781 The PCR product was purified (for example, using Wizard PCR Preps DNA
Purification System (Promega Corp)) and then digested with 13su36I and Awl.
After further purification of the Bsu361-Ascl fragment by gel electropharesis, the product was cloned into Bsit361IAsd 'digested 'pSeNHSA. A Not I fragment was further subcloned into pSAC35 to give construct ID # 2250.
111791 Further analysis of the N-temiinus of the expressed albumin fusion protein by amino acid sequencing should confirm the presence of the expected Mature HSA
sequence (see below).
[11801 INSULIN albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e., Asp-25 to Leu-609, fused to either the N- or C-terminus of the synthetic single-chain analog of INSULIN, i.e., Phe-1 to Asn-62; the sequence corresponding to the C-peptide in the middle region of the proinsulin cDNA was replaced by the C-domain of Insulin Growth Factor 1, "IGF-1" (GY32GSSSRRAPQT, SEQ ID
NO:2147).
In one embodiment of the invention, INSULIN albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature INSULIN albumin fusion protein is secreted directly into the culture medium. INSULIN albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant EISA leader sequences including, but not limited to, a chimeric EISA/MAF
leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, INSULIN albumin fusion proteins of the invention comprise the native INSULIN.
In further preferred embodiments, the INSULIN albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragrnents and/or variants, are also encompassed by the invention.
Expression and Purification of Construct ID 2250..
- Expression in yeast S. cerevisiae.
[11811 Construct 2250 can be transformed into yeast S. cerevisiae by methods known in the art (see Example 3). Expression levels can be examined by irnmunoblot detection with anti-HSA serum as the primary antibody.
Purification from yeast S. cerevisiae cell supernatant.
[11821 The cell supernatant containing the secreted INSULIN (GYG) albumin fusion protein expressed from construct ID #2250 in yeast S. cerevisiae can be purified as described in Example 4. N-terminal sequencing of the albumin fusion protein should result in the sequence DAHKS (SEQ ID NO:2143) which corresponds to the amino .terminus of the mature form of HSA.
In: vitro fiffi,.2_Deoki,g1t.s. Uptake Assay 14 the prognce :Of the.-141bOtin fitSidg protein encoded by construct 2250.
Method [11831 The in vitro assay to measure. the glucose uptake in 3T3-LI
adipocytes in the presence of the INSULIN (GYG) albumin fusion protein encoded by construct 2250, was carried out as described below in Example 41. Other assays known in the art that may be used to test INSULIN (GYG) albumin fusion proteins' include, but are not limited to, L6 Rat Myoblast Proliferation Assay via glycogen synthase kinase-3 (GSK-3) and II411e reporter assays (see Example 48) including the rat Malic Enzyme Promoter (rMEP)-SEAP, Sterol Regulatory Element Binding Protein (SREBP)-SEAP, Fatty Acid Synthetase .(FAS)-SEAP, and PhosphoEnoiPyruvate CarboxyKinase (PEPCK)-SEAP reporters.
Result [11841 The supernatant derived from transformed yeast S. cerevisiae expressing insulin albumin fusion encoded by construct 2250 demonstrated glucose uptake/transport activity in 3T3-L1 adipocytes (see Figure 18).
vitro PanCreatfr Cep-litcltibt_LAgel)L.iiljaggsstioisf_; me, 10- irdWLMne idtt:

A
Prtgebt encoded .by-coniimet 22A
Method [11851 The in vitro assay to measure the differentiation and proliferation of ductal epithelium pancreatic ARIP cell-line into insulin-producing beta cells and/or to measure the proliferation of the insulin-producing RIN-M beta cell-line in the presence of the INSULIN
(GYG) albumin fusion protein encoded by construct 2250 can be carried out as described below under heading: "Example 42: In vitro Assay of 13H1-Thysid:ne Incorporation into Pancreatic Cell-lines".
The.atdv!pypieLt .elibetethiefitsion protein-tweed-4d by .construct 2250 can be ZSsyed in viVa:
MOS_dirdietic NOD and/Or MOMS mouse models.
[11861 The activity of the INSULIN (GYG) albumin fusion protein encoded by construct 2250 can be measured using NOD and/or NIDDM Mouse Models described below under the headings, "Example 44: Occurrence of Diabetes in NOD Mice", "Example 45:
Histological Examination of NOD Mice", and "Example 47: In vivo Mouse Model of NEDDM".
EXAMPLE 34: Construct ID 2255, Insulin (GYG)-HSA, Generation.
[11871 Construct ID 2255, pSAC35.INSULIN(GYG).FI-N62.11SA, encodes for an INSULIN (GYG)-HSA fusion protein which comprises the HSA chimeric leader sequence of HSA fused to the amino-terminus of the synthetic single-chain long-acting insulin analog (INSULIN (GY32G)) with a Tyr in position 32, which is, in turn, fused to the matnre form of HSA, cloned into the yeast S. cerevisiae expression vector pSAC35, donlizz 01.124tIL1N (GM): eDNAJO etinntua 2355.
[11881 The DNA encoding the synthetic single-chain form of INSULIN (GYG) was PCR generated using four overlapping primers. The sequence corresponding to the C-peptide in the middle region of the proinsulin eDNA was replaced by the C-domain of Insulin Growth Factor I, "IGF-1" (GY32GSSSREAPQT, SEQ ID NO:2147), to avoid the need for proinsulin processing and to ensure proper folding of the single-chain protein. The sequence was codon optimized for expression in yeast S. cerevisiae. The PCR fragment was digested with Sal 11 Cla I and subcloned into Kilo IICIa I digested pScCHSA. A Not I
fragment was then subcloned into the pSAC35 plasmid. Construct ID #2255 encodes for the chimeric leader sequence of HSA fused to the amino-terminus of the synthetic single-chain form of INSULIN (GYG) followed by the mature form of HSA.
[1189] The 5' and 3' primers of the four overlapping oligonucleotides suitable for PCR amplification of the polpucleotide encoding the synthetic single-chain form of INSULIN (GYG), INSULIN (GYG)-3 and INSULIN (GYG)-4, were synthesized:
INSULIN (GYG)-3: S'-'17CCAGGAGCGTCGA CAAAAGATTCGTTAACCAACACTTGTGTGG
TTCTCACTTCGTVGAAGCTTTGTACTTGGTTIGTGGTGAA -3' (SEQ ID NO:895) INSULIN (GYG)-4: 5'-AGAGrUI AAA TCGA 7GAGCAACCTCACTCHGTGTGCATCGTTAC
AGTAGTTTTCCAATTGGTACAAAGA.ACAAATAGAAGTACAA-3 ' (SEQ ID NO: 896) INSULIN (GYG)-3 incorporates a Sal I cloning site (shown in italics) and the DNA encoding the first 21 amino acids (shown in bold) of the ORF of the synthetic single-chain form of INSULUAGYG). In INSULIN (GYG)-4, the italicized sequence is a C/a I site; and the Cla I
site and the DNA following it are the reverse complement of DNA encoding the first 10 amino acids of the mature HSA protein. The bolded sequence is the reverse complement of the 46 nucleotides encoding the last 15 amino acid residues Cys-49 to Asn-63 of the synthetic single-chain form of INSULIN (GYG). With these two primers, the synthetic single-chain INSULIN (GYG) protein was generated by annealing, extension of the annealed primers, digestion with Sall and C/a I, and subcloning into Xho IICla I digested pScCHSA. The Not fragment from this clone was then ligated into the Not I site of pSAC35 to generate construct ID 2255., Construct ID #2255 encodes an albumin fusion protein containing the chimeric leader sequence, the synthetic single-chain form of INSULIN (GYG), and the mature form of HSA.
[1190] Further analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing should confirm the presence of the expected INSULIN
(GYG) sequence (see below).
111911 INSULIN albumin fusion proteins of the invention preferably comprise the mature form of IISA , i.e., Asp-25 to Leu-609, fused to either the N- or C-terminus of the synthetic single-chain analog of INSULIN, i.e., Phe-1 to Asn-62; the sequence corresponding to the C-peptide in the middle region of the proinsulin cDNA was replaced by the C-domain of insulin Growth Factor 1, "IGF-1" (GY32GSSSRRANT, SEQ ID
NO:2147).
In one embodiment of the invention, INSULIN albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature INSULIN albumin fusion protein is secreted directly into the culture medium. INSULIN albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin 13, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric IISA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, INSULIN albumin fusion proteins of the invention comprise the native INSULIN.
In further preferred embodiments, the INSULIN albumin fusion proteins of the invention further comprise an N-terminal rnethionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
'Expression and Purification oltonatrueiV1251 Expression in yeast S. cerevisiae.
(11921 Construct 2255 can be transformed into yeast S. cerevisiae by methods known in the art (see Exainple 3). Expression levels can be examined by immimoblot detection with anti-HS.A: serum as the primary antibody.
Purification from yeast S. cerevi.siae cell supernatant.
[1193] The cell supernatant containin the secreted INSULIN (GYG) albumin fusion protein expressed from construct ID #2255 in yeast S. cerevisiae can be purified as described in Example 4. N-terminal sequencing of the expressed and purified albumin fusion protein should generate FNINQII which corresponds to the amino terminus of the synthetic single-chain long-acting insulin analog (INSULIN (0Y.32G)).
In- -vitro elik-2-Deoxviitroye.tipiake.1 encoded by construct 2255.
Method 111941 The in vitro assay to measure, the glucose uptake in 3T3-L1 adipocytes in the presence of the INSULIN (GYG) albumin fusion protein encoded by construct 2255 can be carried out as described below in Example 41. Other assays -known in the art that may be used to test INSULIN (GYG) albumin, fusion proteins' include, but are not limited to, L6 Rat Myoblast Proliferation Assay via glycogen synthase kinase-3 (GSK-3) and H411e reporter assays (see Example 48) including the rat Malic Enzyme Promoter (rMEP)-SEAP, Sterol Regulatory Element Binding Protein (SREBP)-SEAP, Fatty Acid Synthetase (FAS)-SEAP, and PhosphoEnolPyruvate CarboxyKinase (PEPCK)-SEAP reporters.
In vitro Pancreatic Cell-lines Proliferation Assay in the presence of the albumin fusion protein encoded by construct 2255, Method [1195] The in vitro assay to measure the differentiation and proliferation of ductal epithelium pancreatic ARIP cell-line into insulin-producing beta cells and/or to measure the proliferation of the insulin-producing RIN-M beta cell-line in the presence of the INSULIN
(GYG) albumin fusion protein encoded by construct 2255 can be carried out as described below under heading: "Example 42: In vitro Assay of [31-1]-Thyrnidine Incorporation into Pancreatic Cell-lines".
_ e aetivi o ieµabumin is', !ratan - coded. , gruel 2 5 can, be a s ed in using diabetic NOD and/or NIDDM mouse ntodels.
[1196] The activity, of the INSULIN (GYG) albumin fusion protein encoded by construct 2255 can be measured using NOD and/or NI-1)DM mouse models described below under the headings, "Example 44: Occurrence of Diabetes in NOD Mice", "Example 45:
Histological Examination of NOD Mice", and "Example 47: In vivo Mouse Model of NIDDM".
EXAMPLE 35: Construct II) 2276, IISA-Insulin (GGG), Generation.
[1.197] Construct ID 2276, pSAC35.11SAINSULIN(GGG).F1-N58, encodes for an HSA-INSULIN (GGG) fusion protein which comprises full length HSA, including the native HSA leader sequence fused to the amino-terminus of the synthetic single-chain long-acting insulin analog (INSULIN (GG32G)) with a Gly at position 32, cloned into the yeast S.
cerevisiae expression vector pSAC35.
Cloning of.N$ULINIGGO cDIVA for construct 2276, [1198] The DNA encoding the synthetic single-chain form of INSULIN (GGG) was PCR. generated using four overlapping primers. The sequence corresponding to the C-peptide in the middle region of the proinsulin cDNA was replaced by the synthetic linker "GG32GPGKR" (SEQ ID NO:2148) to avoid the need for proinsulin processing and to ensure proper folding of the single-chain protein. The sequence was codon optimized for expression in yeast S. cerevisiae. The PCR fragment was digested and subcloned into BSZI
361/Ase digested pSeNHSA. A Not I fragment was then subcloned into the pSAC35 plasmid.

Construct ID #2276 encodes for full length FISA, including the native HSA
leader sequence fused to the amino-terminus of the synthetic single-chain form of INSULIN
(GGG).
(1199) The 5' and 3' primers of the fOur overlapping oligonucleotides suitable for PCR amplification of the polynucleotide encoding the synthetic single-chain form of INSULIN (GGG), INSULIN (GGG)-1 and INSULIN (GGG)-2, were synthesized:
INSULIN (GGG)-5: 5 '-GTCAAGCTGCC7TA GGCrIATTCGTTAACCAACACTTGTGTGGTT
CICACTIGGTTGAAGMTGIACTTGGITTGTGGTGAA-3 ' (SEQ ID NO:90 1) INSULIN (GGG)-6: 5'-ATCCiCATATOGCGCGCCCTATTAGTTACAGTAGTTTTCCAATTG
GTACAAAGAACAAATAGAAGTACAA -3' (SEQ ID NO:902) [1200] INSULIN (GGG)-5 incorporates a Bszt 361 cloning site (shown in italics) and encodes the first 21 amino acids (shown in bold) of the ORF of the synthetic single-chain , form of INSULIN (GGG). In INSULIN (GGG)-6, the italicized sequence is an Asc.
I site. In INSULIN (GGG)-6, the bolded sequence is the reverse complement of the last 49 nucleotides encoding amino acid residues Cys-44 to Asn-58 of the synthetic single-chain form of INSULIN (GGG).' With these two primers, the synthetic single-chain form of INSULIN
(GGG) was PCR. amplified. Annealing and extension temperatures and times must be empirically determined for each specific primer pair and template.
[12011 The PCR product was purified (for example, using Wizard PCR Preps DNA
Purification System Oromega Corp)) and then digested with Bsti36I and Asa:
After further purification of the -Bsu361-AscI fragment by gel electrophoresis, the product was cloned into Bsu361/21sel digested pScNHSA; A Not 1 fragment was further subcloned into pSAC35 to give construct ID it 2276.
[1202] Further, analysis of the N-teiniinus of the expressed albumin fusion protein by amino acid sequencing should confirm the presence of the expected mature IISA
sequence (see below).
[1203] iNsuuN albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e., Asp-25 to Leu-609, fused to either the N- or C-terminus of the synthetic single-chain analog of INSULIN, i.e., Phe-1 to Asn-58; the sequence corresponding to the C-peptide in the middle region of the proinsulin cDNA was replaced by the synthetic linker "GG32GPGKR" (SEQ ID NO:2148). In one embodiment Of the invention, INSULIN albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature INSULIN albumin fusion protein is secreted directly into the culture medium. INSULIN albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant BSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, INSULIN albumin fusion proteins of the invention comprise the native LNSULIN In further preferred embodiments, the INSULIN albumin fusion proteins of the invention further comprise an N-tenninal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Purification of Construct ID 2276.
Expression in yeast S. cerevisiae.
112041 Construct 2276 can be transformed into yeast S. cerevisiae by methods known in the art (see Example 3). Expression levels can be examined by immunoblot detection with anti-HSA serum as the primary antibody. -Purification from yeast S. cerevisiae cell supernatant.
[12051 , The cell supernatant containing the secreted INSUT,IN (GGG) albumin fusion protein, expressed from construct ID #2276 in yeast S. cerevisiae can be purified as described in Example 4. N-terminal sequencing should yield DAIIK.S (SEQ ID NO:2143) which corresponds to the amino terminus of the mature form of FISA.
in *lira P111-2-Deoxyg1nceiso ,Uptake AseOy in the pivtlice of the albumin fithion protein encoded by construct 2276.
Method 112061 The in vitro assay to measure the glucose uptake in 3T3-L1 adipocytes in the presence of the INSULIN (GGG) albumin fusion protein encoded by construct 2276 was carried out as described below in Example 41. Other assays known in the art that may be used to test INSULIN (GGG) albumin fusion proteins' include, but are not limited to, L6 Rat Myoblast Proliferation Assay via glycogen synthase kinase-3 (GSK-3) and H4fle reporter assays (see Example 48) including the rat Malic Enzyme Promoter (rMEP)-SEAP, Sterol Regulatory Element Binding Protein (SREBP)-SEAP, Fatty Acid Synthetase (FAS)-SEAP, and PhosphoEnolPyruvate CarboxyKinase (PEPCK)-SEAP reporters.
Result [1207] The supernatant derived from transformed yeast S. cerevisiae expressing insulin albumin fusion encoded by construct 2276 demonstrated glucose uptake/transport activity in 3T3-L1 adipocytes (see Figure 18).
vitto Pcmpreatic .C.:41-iines Proliferation Assay in the prewice of the albumin fUsion protein encoded by construct 2276.
Method [12081 The in vitro assay to measure the differentiation and proliferation of ductal epithelium pancreatic ARIP cell-line into insulin-producing beta cells and/or to measure the proliferation of the insulin-producing RIN-M beta cell-line in the presence of the INSULIN
(GGG) albumin fusion protein encoded by construct 2276 can be carried out as described below under heading: "Example 42: In vitro Assay of [3M-ThYrnidine Incorporation into Pancreatic Cell-lines".
The activittof the damn* *ion protein ottani:thy construct 227( can be osavOilfn vivq usinz diabetic NOD and/or NIDDM mouse models.
[12091 The activity of the INSULIN (GGG) albumin fusion protein encoded by construct 2276 can be measured using NOD and/or NIDDM mouse models described below under the headings, "Example 44: Occurrence of Diabetes in NOD Mice", "Example 45:
Histological Examination of NOD Mice", and "Example 47: In vivo Mouse Model of NIDDM".
'EXAMPLE 3Cflst net I 22 8 Insight GCG -USA Generation.
[1210] Construct ID 2278, pSAC35.INSULIN(GGG).HSA, encodes for an INSULIN
(060)-HSA fusion protein which comprises the USA chimeric leader sequence of USA
fused to the amino-terminus of the synthetic single-chain long-acting insulin analog (INSULIN (GG32G)) with a Gly in position 32, which is, in turn, fused to the mature form of IBA, cloned into the yeast S. cerevisiae expression vector pSAC35.

Clqgkgi5LPISV, (GGG) cDNA for mnstnict 2278.
[12111 The DNA encoding the synthetic single-chain form of INSULIN (GGG) was PCR generated using four overlapping primers. The sequence corresponding to the C-peptide in the middle region of the proinsulin cDNA was replaced by the synthetic linker "GG32GPGKR" (SEQ ID NO:2148) to avoid the need for proinsulin processing and to ensure proper folding of the single-chain protein. The sequence was codon optimized for expression in yeast S. cerevisiae. The PCR fragment was digested with Sal IICla I and subcloned into Xho I/Chz I digested pScCHSA. A Not I fragment was then subcloned into the pSAC35 plasmid. Construct ID #2278 encodes for the chimeric leader sequence of HSA
fused to the amino-terminus of the synthetic single-chain form of INSULIN (GGG) followed by the mature form of HSA.
[12121 The 5' and 3' primers of the four overlapping oligonucleotides suitable for PCR amplification of the polynucleotide encoding the synthetic' single-chain form of INSULIN (GGG), INSULIN (GGG)-7 and INSULIN (GGG)-8, were synthesized: =
INSULIN (GGG)-7: 5'-TCCAGGAGCGTCGACAAAAGATTCGTTAACCAACACTTG
=
TGTGGTTCTCACTTGGTTGAAGCTTTGTACTTGGTTTG TGGTGAA -3' (SEQ ID NO:903) INSULIN (GGG)-8: T-AGACTITAMTCGATGAGCAACCTCACTCTTGTGTC.ICATCG
TTACAGTAGTTTTCCAATTGGTACAAAGAACAAATAGAAG TACAA-3' (SEQ ID, NO:904) [12131 INSULIN (GGG)-7 incorporates a Sal I cloning site (shown in italics) and the DNA encoding the first 21 amino acids (shown in bold) of the ORE of the synthetic single-chain form of INSULIN(GGG). In INSULIN (GGG)-8, the italicized sequence is a Cla I
site; and the Cla I site and the DNA following it are the reverse complement of DNA
encoding the first 10 amino acids of the mature HSA protein. The bolded sequence is the reverse complement of the 46 nucleotides encoding the last 15 amino acid residues Cys-44 to Asn-58 of the synthetic single-chain form of INSULIN (GGG). With these two primers, the synthetic single-chain ENSULIN (GGG) protein was generated by annealing, extension of the annealed primers, digestion with Sal I and Cla I, and subcloning into Xho IJCla I digested pSeCHSA. The Not I fragment from this clone was then ligated into the Not I
site of pSAC35 to generate construct ID 2278. Construct ID #2278 encodes an albumin fusion protein containing the chimeric leader sequence, the synthetic single-chain form of INSULIN (GGG), and the mature form of HSA.

[12141 Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing should confirm the presence of the expected INSULIN
(GGG) sequence (see below).
112151 INSULIN albumin fusion proteins of the invention preferably comprise the mature form of USA , i.e., Asp-25 to Leu-609, fused to either the N- or C-terminus of the synthetic single-chain analog of INSULIN, i.e., Phe-1 to Asn-58; the sequence corresponding to the C-peptide in the middle region of the preinsulin cDNA was replaced by the synthetic linker "GG32GPGKR" (SEQ ID NO:2148). In one embodiment of the invention, INSULIN albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature INSULIN albumin fusion protein is secreted directly into the culture medium. INSULIN albumin fusion proteins of the invention may comprise laeterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin 13, Cluster-in, Insulin-Like Growth Factor Binding Protein 4, variant HSA
leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, INSULIN albumin fusion proteins of the invention comprise the native INSULIN. In further preferred embodiments, the INSULIN albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Zrprenion and Piµnficatient titomnicti.1) 2278.
Expression in yeast S. cerevisiae.
[12161 Construct 2278 can be transformed into yeast S. cerevisiae by methods known in the art (see Example 3). Expression levels can be examined by irnmunoblot detection with anti-USA serum as the primary antibody.
Purification from yeast S. cerevisiae cell supernatant.
[1217i The cell supernatant containing the secreted INSULIN (GGG) albumin fusion protein expressed from construct JD #2278 in yeast S. cerevisiae can be purified as described in Example 4. N-terminal sequencing of the expressed and purified albumin fusion protein should generate FVNQH (SEQ ID NO:2149) which corresponds to the amino terminus of the synthetic single-chain long-acting insulin analog (INSULIN (GG32G)).

In vitro [3111-2-Deoxvg1ucoSe Uptake. Assay ilvthe presen.. of the aibitniinjusion protein ;acodgfry construct 2278.
Method [1218] The in vitro assay to measure the glucose uptake in 313-Li adipocytes in the presence of the INSULIN (GGG) albumin fusion protein encoded by construct 2278 can be carried out as described below in Example 41. Other assays known in the art that may be used to test INSULIN (GGG) albumin fusion proteins' include, but are not limited to, L6 Rat Myob last Proliferation Assay via glycogen synthase Icinase-3 (GSK-3) and H411e reporter assays (see Example 48) including the rat Malic Enzyme Promoter (rMEP)-SEAP, Sterol Regulatory Element Binding Protein (SREBP)-SEAP, Fatty Acid Synthetase (FAS)-SEAP, and PhosphoEnolPynivate CarboxyKinase (PEPCK)-SEAP reporters.
In vitro Pancreaiic Proliferation Assay in the _presence of the albumin *ion protein encoded by construct 2278.
Method [1219] The in vitro assay to measure the differentiation and proliferation of ductal epithelium pancreatic ARIP cell-line into insulin-producing beta cells and/or to measure the proliferation of the insulin-producing RIN-M beta cell-line in the presence of the INSULIN
(GGG) albumin fusion protein encoded by construct 2278. can be carried out as described below under heading: "Example 42: In vitro Assay of [311]-Thymidine Incorporation into Pancreatic Cell-lines".
The activity of the ali2mtninfiat roeinencadetmx_iC..them,m,2m2_,..121..majz.õ_pcoteassadinvivq using diabetic NOD and/or NIDDM mouse models, [1220] The activity of the INSULIN (GGG) albumin fusion protein encoded by construct 2278 can be measured using NOD and/or NIDDM mouse models described below under the headings, "Example 44: Occurrence of Diabetes in NOD Mice", "Example 45:
Histological Examination of NOD Mice", and "Example 47: In vivo Mouse Model of NIDDM".
Eta oink 37: Indications for Instal% albumin fusion protein s.
112211 Results from in vitro assays described above indicate that insulin albumin .?+
fusion proteins are useful for the treatment, prevention, and/or diagnosis of hyperglycemia, insulin resistance, insulin deficiency, hyperlipidemia, hyperketonemia, and diabetes mellitus, Type I and Type 2 diabetes.
Exam k3& Prte ..tira don vf -ISAACH . Alston= P Wirt [12221 An HSA-hGH fusion protein was prepared as follows:
Cloning of hGH clIVA
[1223] The hGH cDNA was obtained from a human pituitary gland cDNA library (catalogue number HLI097v, Clontech Laboratories, Inc) by PCR amplification.
Two oligonucleotides suitable for PCR amplification of the hGH cDNA, HGH1 and HGH2, were synthesized using an Applied Riosystems 38013 Oligonucleotide Synthesizer.
HGH I : - CCCAAGAATTCCCTTATCCAGGC -3' (SEQ ID NO:1020) HGH2: 5' GGGAAGCTTAGAAGCCACAGGATCCCTCCACAG -3' (SEQ ID NO:1021) [1224] HGII I and HGH2 differed from the equivalent portion of the hGH
cDNA
sequence (Martial et. al., 1979) by two and three nucleotides, respectively, such that after PCR amplification an EcoRT site would be introduced to the 5 end of the cDNA
and a 1?amH1 site would be introduced into the 3' end of the cDNA. In addition, HGH2 contained a Hinditi site immediately downstream of the hGH sequence.
[1225] PCR amplification using a Perkin-Elmer-Cetus Thermal Cycler 9600 and a =
Perkin-ElmerrCetus PCR kit, was performed using single-stranded DNA template isolated from the phage particles of the cDNA library as follows: 101AL phage particles were lysed by the addition of 10 pL phage Iysis buffer (280 proteinase K in TE buffer) and incubation at 55 C for 15 min followed by 85 C for 15 min. After a 1 mm.
incubation on ice, phage debris was pelleted by centrifugation at 14,000 rpm for 3 min. The PCR
mixture contained 6 uL of this DNA template, 0.1 uM of each primer and 200 !..tM of each deoxyribonucleotide. PCR was carried out for 30 cycles, denaturing at 94 C for 30 s, annealing at 65 C for 30 s and extending at 72 C for 30 s, increasing the extension time by 1 s per cycle.
[1226] Analysis of the reaction by gel electrophoresis showed a single product of the expected size (589 base pairs).
[1227] The PCR product was purified using Wizard PCR Preps DNA
Purification System (Promega Corp) and then digested with EcoR1 and HindilI. After further purification of the EcoRl-Hindill fragment by gel electrophoresis, the product was cloned into pUC19 (GIBCO BRL) digested with EcoRI and HindiII, to give pHall. DNA sequencing of the EcoR1 Hindill region showed that the PCR product was identical in sequence to the hGH
sequence (Martial et al., 1979), except at the 5' and 3' ends, where the EcoR1 and BamHI
sites had been introduced, respectively.
Expression of the hGH cDNA.
[1228] The polylinker sequence of the phagemid pBluescribe (+) (Stratagene) was replaced by inserting an oligonucleotide linker, formed by annealing two 75-mer oligonucleotides, between the EcoR1 and Hindlll sites to form pBST(+). The new polylinker included a unique Notl site.
112291 The Nail IISA expression cassette of pAYE309 (EP 431 880) comprising the PRBI promoter, DNA encoding the HSA/Mfa-1 hybrid leader sequence, DNA encoding HSA and the ADH1 terminator, was transferred to pBST(+) to form pHSA1. The HSA

coding sequence = was removed from this plasmid by digestion with Hind ,111 followed by religation to form pHSA2.
[1230] Cloning of the liGH cDNA provided the hGH coding region lacking the pro-hGH sequence and the first 8 base pairs (bp) of the mature hGH sequence.
In order, to construct an expression plasmid for secretion of hGH from yeast, a yeast promoter, signal peptide and the first 8 bp of the hGH sequence were attached to the 5" end of the cloned hGH
sequence as follows: The Hindift-SfaNI fragment from pHSA 1 was attached to the 5' end of the EcoRlIllindlII fragment from pHGHI via two synthetic oligonucleotides, HGH.3 and HG114 (which can anneal to one another in such a way as to generate a double stranded fragment of DNA with sticky ends that can anneal with SfaNIand EcoR1 sticky ends):
HaI3: 5' - GATAAAGATTCCCAAC -3' (SEQ.ED NO:1023) HGI-14: 5' - AATTGTTGGGAATCTTT- 3' (SEQ ID NO:1024) [1231] The Hind 111 fragment so formed was cloned into HindIII-digested pHSA2 to make pfiGH2, such that the hGH cDNA was positioned downstream of the PRBI
promoter and HSA/MFa-1 fusion leader sequence (see, International Publication No. WO
90/01063).
The Nod expression cassette contained in pHGH2, which included the ADH1 terminator downstream of the hGH cDNA, was cloned into Nod-digested pSAC35 (Sleep et al., BioTechnology 8:42 (1990)) to make pHGH12. This plasmid comprised the entire 2 p,rn plasmid to provide replication functions and the LEU2 gene for selection of transfomiants.

[12321 p1101112 was introduced into S. eerevisiae D88 by transformation and individual transfonnants were grown for 3 days at 30 C in 10 mL YEPD (1% wiv yeast extract, 2 % w/v, peptone, 2 % wiv, dextrose).
[12331 After centrifugation of the cells, the supernatants were examined by SDS-polyacrylarnide gel electrophoresis (SDS-PAGE) and were found to contain protein which was of the expected size and which was recognized by anti-hGH antiserum (Sigma, Poole, UK) on Western blots.
cloning and expression of an liSA-hG11 fusion protein.
[12341 In order to fuse the RSA cDNA to the 5' end of the hal cDNA., the pHSA1 hrindIII-Bsu361 fragrient .(containing most of the NSA cDNA) was joined to the pHGH1 EcoRI-Hind111 fragment (containing most of the hGH cDNA) via two =
oligonucleotides, -110117 and IIGH8 II0H7: 5' - TTAGGCTTATTCCCAAC 3' (SEQ ID NO:1025) Hal& 5' - AATTGI'IGGGAATAAGCC 3' (SEQ ID NO:1026) [12351 ThelfindlII fragment so forined was cloned into pHSA2 digested with HindflI
to make pHGH10, and the Notl expression cassette of this plasmid was cloned into Notl-digested pSAC35 to make pHGII16.
[12361 ' PFIGHI 6 was used to transform S. eerevisiae D88 and supernatants of cultures were analyzed as described above. A predominant band was observed that had a molecular weight of approximately 88 kr), corresponding to the combined masses of HSA
and hGH.
Western blotting using anti-HSA and anti-hGH antisera ,(Sigma)-4confirmed the presence of the two constituent parts of the albumin fusion protein.
[12371 The albumin fusion protein was purified from culture supernatant by cation exchange chromatography, followed by anion exchange and gel permeation chromatography.
Analysis of the N-terminus of the protein by amino acid Sequencing confirmed the presence of the expected albumin sequence.
[12381 An in vitro growth hormone activity assay (Ealey et al., Growth Regulation 5:36 (1995)) indicated that the albumin fusion protein possessed full hGH
activity: In a hypophysectomised rat weight gain model, performed essentially as described in the European Pharmacopoeia (1987, monograph 556), the fusion molecule was more potent than hGH when the same number of units of .activity (based on the above in vitro assay) were administered daily. Further experiments in which the albumin fusion protein was administered once every four days showed a similar overall growth response to a daily administration of hal. Pharmacokinetic experiments in which 1251- labeled protein was administered to rats indicated an approximately ten-fold increase in circulatory half-life for the albumin fusion protein compared to hGH.
[12391 A similar plasmid was constructed in which DNA encoding the S.
cerevisiae invertase (SUC2) leader sequence replaced the sequence for the hybrid leader, such that the encoded leader and the junction (4, ) with the HSA sequence were as follows:
MiLLQAFLITLAGFAAKISA DAIIKS ........ (SEQ ID NO:1027) Invertasc leader HSA sequence [12401 On introduction into S. cerevisiae DB1, this plasmid directed the expression and secretion of the albumin fusion protein at a level similar to that obtained with pHGH16.
Analysis of the N-terminus of the albumin fusion protein indicated precise and efficient cleavage of the leader sequence from the mature protein.
Cloning and expression of an hGH-HSA fusion protein.
[12411 In order to fuse the hGH cDNA to the 5' end of the HSA cDNA, the USA
cDNA was first altered by site-directed mutagenesis to introduce an EcoNisite near the 5' end of the coding region. This was done by the method of Kunkel et al. (Methods in Enzymol.
154:367 (1987)) using single-stranded DNA template prepared from pHSAI and a synthetic oligonucleotide, LEU4:
LEU4: 5' - GAGATGCACACCTGAGTGAGG -3' (SEQ ID NO:1028) Site-directed mutagenesis using this oligonucleotide changed the coding sequence of the FIS.A.
cDNA from Lys4 to Leu4 (K4L). However, this change was repaired when the hGH
cDNA
was subsequently joined at the 5' end by linking the pHGH2 Notl-BamH1 fragment to the EcoNI-Nott fragment of the mutated pHSAI, via the two oligonucleotides HGH5 and H0116:
HGH5: 5'- GATCCTGTGGCTICGAIGCACACAAGA -3' (SEQ ID NO:1029) HGH6: 5' - CTCTTGTGTGCATCGAAGCCACAG -3' (SEQ JD NO:1030) [1242] The Notl fragment so formed was cloned into Notl-digested pSAC35 to make pHGII14. pHGH14 was used to transform S. cerevisiae D88 and supernatants of culture were analyzed as above. A predominant band was observed that had a molecular weight of approximately 88 kD, corresponding to the combined masses of hGH and HSA.
Western blotting using anti-HSA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion protein.
[12431 The albumin fusion protein was purified from culture supernatant by cation exchange chromatography, followed by anion exchange and gel permeation chromatography.
Analysis of the N-terminus of the protein by amino acid sequencing confirmed the presence of the expected liGH sequence.
(12441 in ,vitro studies showed that the albumin fusion protein retained hGH activity, but was significantly less potent than an albumin fusion protein comprising full length HSA
(1-585) as the N-terminal portion and hGH as the C-terminal portion, as described above.
Construction of plasmids for the expression of hal fusions to domains of NSA.
[12451 Fusion polypeptides were made in which the hGH molecule was fused to the first two domains of LISA (residues 1 to 387). Fusion to the N terminus of hGH
was achieved by joining the pHSA1 Ilindlil-Sapl fragment, which contained most of the coding sequence for domains 1 and 2 of HSA, to the pliGH1 EcoRI-Hindill fragment, via the oligonucleotides HGH 11 and HGF1 12:
FIGH11: 5' - TGIGGAA.GAGCCTCAGAATTIATTCCCAAC -3' (SEQ 111 NO:1031) HGFI12: 5 - AATTGTTGGGAATAAATTCTGAGGCTCTTCC 3' (SEQ ID NO:1032) f124.151 The Hindill fragment so formed was cloned into Hindi:II-digested pHSA2 to make pHGH37 and the ATM expression cassette of this plasitid was cloned. into Nod-digested .pSAC35.
[1247] The resulting plasmid, pHGH38, contained an expression cassette that was found to direct secretion of the fusion polypeptide into the supernatant when transformed into S. cerevisiae DB I. Western blotting using anti-NSA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion protein. . r =
112481 The albumin fusion protein was purified from culture supernatant by 'cation exchange chromatography followed by gel permeation chromatography.
[1249] In vivo studies with purified protein indicated that the circulatory half-life was longer than that of hGH, and similar to that of an albumin fusion protein comprising HSA (1-585) as the N-terminal portion and liGH as the C-terminal portion, as described above. in vitro studies showed that the albumin fusion protein retained hG11 activity.
[12501 Using a similar strategy as detailed above, an albumin fusion protein comprising the first domain of HSA (residues 1-194) as the N-terminal portion and hGH as the C-terminal portion, was cloned and expressed in g cerevisiae DBL. Western blotting of culture supernatant using anti-NSA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion protein.

Fusion of HSA to IrGH using d.flexible linker sequence 112511 Flexible linkers, comprising repeating units of [Gly-Gly-Gly-Gly-Ser], (SEQ
ID NO:2150) where n was either 2 or 3, were introduced between the HSA and hGH
albumin fusion protein by cloning of the oligonucleotides HG1416, HGII17, HGH18 and HGH19:
IIGH16:5'-TTAGGCTTAGGTGGCGGTGGATCCGGCGGTGGTGGATCTTTCCCAAC-31 (SEQ ID NO:1133) HGH17:5"-AATTGTTGGGA.AAGATCCACCACCGCCGGATCCACCGCCACCTAAGCC
-3" (SEQ ID NO:1134) , HGH18:5"-TTAGGCTTAGGCGGTGCITGGATCTGGTGGCGGCGGATCTGGTGGCG-GT
GGA'TCCTTCCCAAC-3' (SEQ ID NO:1135) IIGH19:5'-AATTGTTGGGAAGGATCCACCGCCACCAGATCCGCCGCCACCAGATCC
ACCACCGCCTAAGCC-3' (SEQ ID NO:1136) 112521 Annealing of HGH16 with HGH17 resulted in n=2, while HGH18 annealed to HOH19.resulted in n=3. After annealing, the double- stranded oligonucleotides were cloned with: the EcoR1-Bsu361 fragment isolated from pHGH1 into Bsu361-digested pliGH10 to make pHGH56 (where n=2) and pHGH57 (where n=3). The Notl expression cassettes from these plasmids were cloned into Notl-digested pSAC35 to make pHGH58 and pHGH59, respectively.
112531 Cloning of the oligonucleotides to make pHGH56 and pHGH57 introduced,a BamHI site in the linker sequences. It was therefore possible to construct linker sequences in which n=1 and n = 4, by joining either the Hindlit-BamH1 fragment from pHGH56:
to the -BamHI-Hindill fragment from pHGI157 (making n = 1), or the Hindlil-Bamill fragment from pIIGH57 to the Batn111-Hindill fragment from pI1GH56 (making n=2). Cloning of these fragments into the Hind 111 site of pHSA2 resulted in pHG1-160 (n= 1) and plIGH61 (n=4).
The Nati expression cassettes fromlpHGH60 and piiGH61 were cloned into Nod-digested pSAC35 to make pHGH62 and pHGH63, respectively.
112541 Transformation of S. cerevisiae with pHGH58, pHGH59, pHG1162 and pHGEI63 resulted in transformants that secreted the fusion polypeptides into the supernatant.
Western blotting using anti-HSA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion proteins.
[12551 The albumin fusion proteins were purified from culture supernatant by cation exchange chromatography, followed by anion exchange and gel permeation chromatography.
Analysis of the N-termini of the proteins by amino acid sequencing confirmed the presence of the expected albumin sequence. AnalysiS of the purified proteins by electrospray mass spectrometry confirmed an increase in mass of 315 D (n=1), 630 D (n=2), 945 D
(n=3) and 1260 D (n=4) compared to the IISA-hGH fusion protein described above. The purified protein was found to be active in vitro.
112561 hGH albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e., Asp-25 to Len-609, fused to either the N- or C- terminus of the mature form of hGH. In one embodiment of the invention, hG1I albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, .
the signal peptide encoded by the signal sequence is removed, and the mature hG11, albumin fusion protein is secreted directly into the culture medium. hGH albumin fusion proteins of, the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, 1g, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence;
or other heterologous signal- sequences known in the art. In a preferred embodiment, hGH
albumin fusion proteins of the invention comprise the nativeliGIL mV further preferred embodiments, the hGli albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
= ,= .
õracroued'Sfielfliife f 1114-hGgfitsion prole*: Methods.
[12571 HSA-hGH and hGH were separately diluted in cell culture media containing 5% horse serum to final concentrations of 100-200 p.g/m1 and incubated at 4, 37 or 50 C. At time zero and at weekly intervals thereafter, aliquots of the samples were tested for their biological activity in the Nb2 cell proliferation assay, and the data normalized to the biological activity of the control (hGH solution at time zero). In other assays hGH and HSA-hal: were incubated in phosphate buffer saline in at 4, 37 and 50 degree C.
112581 Nb2 cell proliferation assay: The growth of these cells is dependent on hGH or other lactogenic hormones. In a typical experiment 104 cells /well are plated in 96-well plate in the presence of different concentration of hGH or HSA-hGH in media such as DMEM
containing 5-10% horse serum for 24-48 lus in the incubator. After the incubation period, 1:10 volume of MIT (5mg/m1 in H20) is added to each well and the plate is incubated for a further 6-16 hrs. The growing cells convert mTT to insoluble fonnazan. The formazan is solublized by acidic isopropanol, and the color produced is measured at 570 run on microtiter plate reader. The extent of fomiazan formation reflects the level of cellular proliferation.
Increased shelf of IISA-hGII fusion proteins: Results 112591 The fusion of Therapeutic proteins to albumin confers stability in aqueous or other solution. The shelf-life of an HSA fusion protein is extended in terms of the biological activity of IISA-hGH remaining after storage in cell culture media for up to 5 weeks at 37 C.
A solution of 200 ug/m1 HSA-hGH was prepared in tissue culture media containing 5%
horse serum, and the solution incubated at 37 C starting at time zero. At the indicated times, a sample was removed and tested for its biological activity in the Nb2 cell assay, at 2 ngtml final concentration. The biological activity of HSA-hGH remains essentially intact (within, experimental variation) after 5 weeks of incubation at 37 C. The recombinant hGH used as control for this experiment lost its biological activity in the first week of the experiment, , [1260]
After storage in cell culture media for up to 3 weeks at 4, 37, or 50 C, HSA-hG1-1 vas Stable. At time zero, usolution of HSA-hGH was prepared in tissue culture media - containing 5% horse serum, and incubated at 4, 37, and 50 C. At the indicated periods a , sample was removed and assayed for its biological activity in the Nb2 cell proliferation assay, at 60 ngirril filial concentration. HSA-hGH retains over 90% of its initial activity at all temperatures tested for at least 3 weeks after incubation while hGH loses its biological activity within the first week. This level of activity is further retained for at least 7 weeks at 37 C and 5 weeks at 50 C. These results indicate that HSA-hGH is highly stable in aqueous solution even under temperature stress.
[1261] The biological activity of HSA-hGH was stable compared to hGH in the Nb2 dell proliferation assay. Nb2 cells were grown in the presence of increasing concentrations of recombinant hGH or HSA-hGH, added at time zero. The cells were incubated for 24 or 48 hours before measuring the extent of proliferation by the MTT method. The increased stability of HSA-hGH in the assay results in essentially the same proliferative activity at 24 hours as at 48 hours while hGH shows a significant reduction in its proliferative activity after 48 hours of incubation. Compared to hGH, the HSA-hGIT has lower biological potency after I. day; the albumin fusion protein is about 5 fold less potent than hGH.
However, after 2 days the HSA-hGH shows essentially the same potency as hGH due to the short life of hGH in the assay. This increase in the stability of the hGH as an albumin fusion protein has a major unexpected impact on the biological activity of the protein.
11,,tampl639:.indicatittos for h(41 Albumin Fusion Proteins:
112621 Results from in vitro and in vivo assays indicate that hGH albumin fusion proteins can be used to treat, prevent, detect, diagnose, andior ameliorate acrornegaly, growth failure, growth failure and endogenous growth hormone replacement, growth hormone deficiency, growth failure or growth retardation Prader-Willi syndrome in children 2 years or older, growth deficiencies, growth failure associated with chronic renal insufficieney, postmenopausal osteoporosis, bums, cachexia, cancer cachexia; dwarfism, metabolic disorders, obesity, renal failure, Turner's Syndrome (pediatric and adult), fibromyalgia, fracture treatment, frailty, or AIDS wasting.
gxarrinle 40: isolation of a Selected cDNA. Clone From the Deposited Sault&
112631 , Many of the albumin, fusion constructs of the invention ,have been deposited with the ATCC as shown in Table 3. The albumin fusion constructs may compris,e, any one of the following expression vectors: the yeast S. cerevisiae expression vector pSAC35, the mammalian ,expression vector pC4, or the mammalian expression vector pEE12.1.
(1264) PSAC35 (Sleep et al., 1990, Biotechnology 8:42), pc4 (ATCC
Accession No. 209646; Cullen et al., ,Molecular and Cellular Biology, 438-447, (1985);
Boshart et al, Cell 41: 521-530 (1985)), and pEE12.1 (Lonza Biologics, Inc.;
Stephens and Cockett, Nucl. Acids Res. 17: 7110 (1989); International Publication #W089/01036; Murphy et al., Biochern J. 227: 277-279 (1991); Bebbington et aL, Rio/Technology 10:169-175 (1992); US patent US 5,122,464; International Publication #W086/05807) vectors comprise an ampicillin resistance gene for growth in bacterial cells. These vectors and/or an albumin fusion construct comprising them can be transformed into an E. coil strain such as Stratagene XL-1 Blue (Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, CA, 92037) using techniques described in the art such as H.anahan, spread onto Luria-Broth agar plates containing 100 ug/triL ampicillin, and grown overnight at 37 C.
[1265] The deposited material, in the sample assigned the ATCC Deposit Number cited in Table 3 for any given albumin fusion construct also may contain one or more additional albumin fusion constructs, each encoding different albumin fusion proteins. Thus, deposits sharing the same ATCC Deposit Number contain at least an albumin fusion construct identified in the corresponding row of Table 3.
[1266] Two approaches can be used to isolate a particular albumin fusion construct from the deposited sample of plasmid DNAs cited for that albumin fusion construct in Table 3.
Method 1: Screening 112671 First, an albumin fusion construct may be directly isolated by screening the sample of deposited plasmid DNAs using a polynucleotide probe corresponding to SEQ ID
NO:X for an individual construct ID number in Table 1, using methods known in the art For example, a specific polynucleotide with 30-40 nucleotides may be synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide can be labeled, for instance, with 32P-y-ATP using T4 polynucleotide kinase and purified according to routine methods, (E.g., Maniatis et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY (1982)). The albumin fusion construct from a given ATCC deposit is transformed into a suitable host, as indicated , above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above.
The transfomiants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transfonnants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g.; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.
Method 2: PCR
112681 Alternatively, DNA encoding a given albumin fusion protein may be amplified from a sample of a deposited albumin fusion construct with SEQ ID NO:X, for example, by using two primers of 17-20 nucleotides that hybridize to the deposited albumin fusion construct 5' and 3' to the DNA encoding a given albumin fusion protein. The polyrnerase chain reaction is carried out under routine conditions, for instance, = in 25 p.1 of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 rnM IvIgC12, 0.01% (w/v) gelatin, 20 p.M each of dATP, deTP, (IGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Tag polymerase. Thirty five cycles of PCR
(denaturation at 94 C for I min; annealing at 55 C for 1 min; elongation at 72 C for I min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.
112691 Several methods are available for the identification of the 5' or 3' non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5' and 3' "RACE" protocols which are known in the art.
For instance, a method similar to 5' RACE is available for generating the missing 5' end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993)).
[1270] Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA presumably containing full-length gene RNA transcripts. A
primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5' portion of the desired MI-length gene. This amplified product may then be sequenced and used to generate the full length gene.
112711 This above method starts with total RNA isolated from the desired source,.
'although poly-A+ RNA can 'be used, The RNA preparation can then be treated with phosphatase if 'necessary to eliminate 5 phosphate groups on degraded or damaged RNA
which may interfere' with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatdse in order to remove the cap structure present at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA

oligonucleotide using T4 RNA. ligase.
112721 This modified RNA preparation is used as a template for first strand cDNA
synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest.
The resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the desired gene.
110mm le 41: 311 -2-Daa lame. Uotake..ASSa.
[12731 Adipose, skeletal muscle, and liver are insulin-sensitive tissues.
Insulin can stimulate glucose uptake/transport into these tissues. In the case of adipose and skeletal muscle, insulin initiates the signal transduction that eventually leads to the translocation of the glucose transporter 4 molecule, GLUT4, from a specialized intracellular compartment to the cell surface. Once on the cell surface, GLUT4 allows for glucose uptake/transport.
141-11-2-Deo..xyglzicose Uptake [1274] A number of adipose and muscle related cell-lines can be used to test for glucose uptake/transport activity in the absence or presence of a combination of any one or more of the therapeutic drugs listed for the treatment of diabetes mellitus.
In particular, the 3T3-L1 murine fibroblast cells and the L6 interine skeletal muscle cells can be differentiated into 3T3-L1 adipocytes and into myottibes, respectively, to serve as appropriate in vitro models for the [31-1]-2-deoxyglucose uptake assay (Urso et al., J Biol Chem, 274(43): 30864-73 (1999); Wang et al., I Mol.Endocrinol, 19(3): 241-8 (1997); Haspel et al., J /vierribr Biol, 169 (1): 45,53 (1999); Tsakiridis et al., Endocrinology, 136(10): 4315-22 (1995)). Briefly, 2 x 105 cells/100 !.LL of adipocytes or differentiated L6 cells are transferred to 96-well Tissue-Culture, "TC", treated, i.e., coated with 50 pg/rriL of poly-L-lysine, .plates in post-differentiation medium and are incubated overnight at 37 C in 5% CO2. The cells are first washed once with serum free low glucose DMEM medium and are then starved with p.L/well of the same medium and with 100 uUwell of either buffer or of a combination of any one or more of the therapeutic drugs listed for the treatment of diabetes mellitus, for example, increasing concentrations of 1 nM, 10 nM, and 100 aM of the therapeutics of the subject invention (e.g, specific fusions disclosed as SEQ ID NO:Y and fragments and variants thereof) for 16 hours at 37 ?C in the absence or presence of 1 nM insulin. The plates are washed three times with 100 gL/well of HEPES buffered saline. Insulin is added at 1 nM in HEPES buffered saline for 30 min at 37 C in the presence of 10 iM labeled deoxyglueose (Arnersham, #TRK672) and 10 AM unlabeled 2-deoxyglucose (SIGMA, D-3179). As control, the same conditions are carried out except in the absence of insulin. A
final concentration Of 10 uM cytochalasin B (SIGMA, C6762) is added at 100 pt/well in a separate well to meaStire the non-specific uptake. The cells are washed three times with HEPES buffered saline. Labeled, i.e., 10 p.lvi of [3H]-2-deoxyglucose, and unlabeled, i.e., 10 0/1 of 2-deoxyglucose, are added for 10 minutes at room temperature. The cells are washed three times with cold Phosphate Buffered Sal Me, "PBS". The cells are lysed upon the addition of 150 A/well of 0.2 N NaOH and subsequent incubation with shaking for 20 minutes at room temperature. Samples are then transferred to a scintillation vial to which is added 5 ride of scintillation fluid. The vials are counted in a Beta-Scintillation counter.
Uptake in duplicate conditions, the difference being the absence or presence of insulin, is determined with the following equation: [(Insulin counts per minute "cpm" ¨
Non-Specific cprn)/(No Insulin cpm ¨ Non-Specific cpm)]. Average responses fall within the limits of about 5-fold and 3-fold that of controls for adipocytes and myotubes, respectively.
Differentiation of Cells [12751 The cells are allowed to become fully confluent in a T-75 crn2 flask. The medium is removed and replaced with 25 rriL of pre-differentiation medium for 48 hours.
The cells are incubated at 37 C, in 5% CO2, 85% humidity. After 48 hours, the pre-differentiation medium is removed and replaced with 25 rriL differentiation medium for 48 hours, The cells are again incubated at 37 T, in 5% CO2, 85% humidity. After 48 hours, the medium is removed and replaced with 30 niL post-differentiation medium. Post-differentiation medium is. maintained for 14-20 days or until complete differentiation is achieved. The medium is changed every 2-3 days. Human =adipocytes can be purchased from Zen-Bio, INC (# SA-1096).
Exam le 4 an itro Asa of 311 -Th anidine Into $ oration into Pancreatic Cell-lines [12761 It has recently been shown that GLP-1 induces differentiation of the rat pancreatic ductal epithelial dell-line ARIP in a time- and dose-dependent manner which is associated with an increase in Islet Duodenal Homeoboxl (IDX-1) and insulin mRNA levels (Hui et al., 2001, Diabetes, 50(4): 785-96). The IDX-1 in turn increases niRNA
levels of the GLP-I receptor.
Cells Types Tested [12771 RIK-m cells: These cells are available from the American Type Tissue Culture Collection (ATCC Cell Line Number CRL-2057 ). The R1N-M cell line was derived from a radiation induced transplantable rat islet cell tumor. The line was established from a nude mouse xenograft of the tumor. The cells produce and secrete islet polypeptide hormones, and produce L-dopa decarboxylase (a marker for cells having amine precursor uptake and decarboxylation, or .APUD, activity).
112781 ARP cells: These are pancreatic exocrine cells of epithelial morphology available from the American Type Tissue Culture Collection (ATCC Cell Line Number CRL-1674). See also, references: Jessop, N.W. and Hay, R.3., "Characteristics of two rat pancreatic exocrine cell lines derived from transplantable tumors," In Vitro 16: 212, (1980);
Cockell, M. et al., "Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas," Mol.
Cell. Biol. 9: 2464-2476, (1989); Roux, E., et al. "The cell-specific transcription factor PTI-71 contains two different subunits that interact with the DNA" Genes Dev. 3: 1613-1624, (1989); arid, Hui, H., et al., "Glucagon-like peptide 1 induces differentiation of islet duodenal homeobox- 1-positive pancreatic ductal cells into insulin-secreting cells," Diabetes 50;
785-796 (2001).
Preparation of Cells [1279) The R1N-M cell-line is grown in RPM1 1640 medium (Hyclone, #SH300027.01) with 10% fetal bovine serum (HyClone, #SH30088.03) and is subcultured every 6 to 8 days at a ratio of 1:3 to 1:6. The medium is changed every 3 to 4 days.
[1280) The ARIL' (ATCC #CRL-1674) cell-line is grown in Ham's Fl2K medium (ATCC, #30-2004) with 2 niM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 10% fetal bovine serum. The ARIP cell-line is subcultured at a ratio of 1:3 to 1:6 twice per week. The medium is changed every 3 to 4 days.
Assay Protocol [12811 The cells are seeded at 4000 cells/well in 96-well plates and cultured for 48 to 72 hours to 50% confluence. The cells are switched to serum-free media at 100 IA/well.
After incubation for 48-72 hours, serum and/or the therapeutics of the subject invention (e.g., albumin fusion proteins of the invention and fragments and. variants thereof) are added to the well. Incubation persists for an additional 36 hours. [311J-Thymidine (5-20 Ci/mtnol) (Amersham Pharmacia, #TRK120) is diluted to 1 microCuries/5 microliters. After the 36 hour incubation, 5 microliters is added per well for a further 24 hours. The reaction is terminated by washing the cells gently with cold Phosphate-Buffered Sal in;
"PBS", once.
The cells are then fixed with 100 microliters of 10% ice cold TCA for 15 min at 4 C. The PBS is removed and 200 microliters of 0.2 N NaOH is added. The plates are incubated for 1 hour at room temperature with shaking. The solution is transferred to a scintillation vial and nil, of scintillation fluid compatible with aqueous solutions is added and mixed vigorously.
The vials are counted in a beta scintillation counter. As negative control, only buffer is used.
As a positive control fetal calf serum is used.

Example 43: Assaying for Glycosuria.
112821 Glycosuria (i.e., excess sugar in the urine), can be readily assayed to provide an index of the disease state of diabetes mellitus. Excess urine in a patient sample as compared with a normal patient sample is symptomatic of IDDM and N1DDM.
Efficacy of treatment of such a patient having IDDM and N1DDM is indicated by a resulting decrease in the amount of excess glucose in the urine. In a preferred embodiment for IDDM
and N1DDM
monitoring, urine samples from patients are assayed for the presence of glucose using techniques known in the art. Glycosuria in humans is defined by a urinary glucose concentration exceeding 100 mg per 100 ml. Excess sugar levels in those patients exhibiting glycosuria can be measured even more precisely by obtaining blood samples and assaying serum glucose.
ExaMple 44: Occurrence of Diabetes in NOD Mice.
(1.2831 Female NOD (non-obese diabetic) mice are characterized by displaying IDDM
with a course which is similar to that found in humans, although the disease is more pronounced in female than male NOD mice. Hereinafter, unless otherwise stated, the term "NOD mouse" refers to a female NOD mouse. NOD mice have a progressive destruction of beta cells which is caused by a chronic autoimmune disease. Thus, NOD mice begin life with euglycemia, or normal blood glucose levels: By about 15 to 16 weeks of age, however, NOD
mice start becoming hyperglycemic, indicating the destruction of the majority of their pancreatic beta cells and the corresponding inability of the pancreas to produce sufficient insulin. Thus, both the cause and the progression of the disease are similar to human IDDM
patients.
112841 In vivo assays of efficacy of the immunization regimens can be assessed in female NODTLLI mice (commercially available from The Jackson Laboratory, Bar Harbor, Me.). In the literature, it's reported that 80% of female mice develop diabetes by 24 weeks of age and onset of insulitis begins between 6-8 weeks age. NOD mice are inbred and highly responsive to a variety of immunoregulatory strategies. Adult NOD mice (6-8 weeks of age) have an average mass of 20-25 g.
[128.5) These mice can be either untreated (control), treated with the therapeutics of the subject invention (e.g., albumin fusion proteins of the invention and fragments and variants thereof), alone or in combination with other therapeutic compounds stated above.
The effect of these various treatments on the progression of diabetes can be measured as follows:
[12861 At 14 weeks of age, the female NOD mice can be phenotyped according to glucose tolerance. Glucose tolerance can be measured with the intraperitoneal glucose tolerance test (1PGTT). Briefly, blood is drawn from the paraorbital plexus at 0 minutes and 60 minutes after the intraperitoneal injection of glucose (I g/kg body weight). Normal tolerance is defined as plasma glucose at 0 minutes of less than 144 mg ,4, or at 60 minutes of less than 160 mg %. Blood glucose levels are determined with a Glucometer Elite apparatus.
[1.287] Based upon this phenotypic analysis, animals can be allocated to the different experimental groups. In particular, animals with more elevated blood glucose levels can be assigned to the impaired glucose tolerance group. The mice can be fed ad libitum and can be supplied with acidified water (pH 2.3).
[1288] The glucose tolerant and intolerant mice can be further subdivided into control, = albumin fusion proteins of the subject invention, and albumin fusion proteins/therapeutic compounds combination groups. Mice in the control group can receive an interperitoneal injection of vehicle daily, six times per week. Mice in the albumin fusion group can receive an interperitoneal injection of the therapeutics of the subject invention (e.g., albumin fusion proteins of the invention and fragments and variants thereof) in vehicle daily, six times per week. Mice in the albumin fusion proteins/therapeutic compounds combination group can receive both albumin fusion proteins and combinations of therapeutic, compounds as described above.
[12891 The level of urine glucose in the NOD mice can be determined on a bi-weekly, basis using Labstix (Bayer Diagnostics, Hampshire, England). Weight and fluid intake can also be determined on a bi-weekly basis. The onset of diabetes is defined after the appearance of glucosuria on two consecutive determinations. After 10 weeks of treatment, an additional IPGTT can be performed and animals can be sacrificed the following day.
[12901 Over the 10 week course of treatment, control animals in both the glucose tolerant and glucose intolerant groups develop diabetes at a rate of 60% and 86%, respectively (see US patent No. 5,866,546, Gross et at). Thus, high rates of diabetes occur even in NOD mice which are initially glucose tolerant if no intervention is made.
[12911 Results can be confirmed by the measurement of blood glucose levels in NOD
mice, before and after treatment. Blood glucose levels are measured as described above in both glucose tolerant and intolerant mice in all groups described.

(12921 In an alternative embodiment, the therapeutics of the subject invention (e.g., specific fusions disclosed as SEQ ID NO:Y and fragments and variants thereof) can be quantified using spectrometric analysis and appropriate protein quantities can be resuspended prior to injection in 50 µ1 phosphate buffered saline (PBS) per dose. Two injections, one week apart, can be administered subcutaneously under the dorsal skin of each mouse.
Monitoring can be performed on two separate occasions prior to immunization and can be performed weekly throughout the treatment and continued thereafter. Urine can be tested for glucose every week (Keto-Diastix®; Miles Inc., Kankakee, 111.) and glycosuric mice can be checked for serum glucose (ExacTech®, MediSense, Inc., Waltham, Mass.).
Diabetes is diagnosed when fasting glycemia is ga-eater than 2.50¨

Example 45: Histological Examination of NOD Mice.
[1293] Histological examination of tissue samples from NOD mice can demonstrate =
the ability of the compositions of the present invention, and/or a -combination of the compositions of the present invention with other therapeutic agents for diabetes, to increase the relative concentration of beta cells in the pancreas. The experimental method is as follows:
[1294] The mice from Example 44 can be sacrificed at the end of the treatment period =
and tissue samples can be taken from the pancreas. The samples can be fixed in 10% formalin in 0.9% saline and embedded in wax. Two sets of 5 serial 5 µm sections. can be cut for ..
immunolabelling at a cutting interval of 150 µm. Sections can be immunolabelled for insulin (guinea pig anti-insulin antisera dilution 1:1000, ICN Thames U.K.) and glucagon (rabbit anti-pancreatic glucagon antisera dilution 1:2000) and detected with peroxidase conjugated anti-guinea pig (Dako, High Wycombe, U.K.) or peroxidase conjugated anti-rabbit antisera (dilution 1:50, Dako).
[1295] The composition of the present invention may or may not have as strong an effect on the visible mass of beta cells as it does on the clinical Manifestations of diabetes in glucose tolerant and glucose intolerant animals.
paamin ala6j_tcrerrtie BeAkp.titqation IC.Oiribittation Thergot [1296] Transplantation is a common form of treatment of autoirnmtme disease, especially when the target self tissue has been severely damaged. For example, and not by way of limitation, pancreas transplantation and islet cell tranSpla.ntation are common treatment options for LDDM (See, e.g., Stewart et al., Journal of Clinical Endocrinology &
Metabolism 86 (3): 984-988 (2001); 13runicardi, Transplant. Proc. 28: 2138-40 (1996);
Kendall & Robertson, Diabetes Metab. 22: 157-163 (1996); Hamano et al., Kobe J. Med. Sci.
42: 93-104 (1996); Larsen & Stratta, Diabetes Metab. 22: 139-146 (1996); and Kinkhabwala, et al., Am. .1. Surg, 171: 516-520 (1996)). As with any transplantation method, transplantation therapies for autoimmune disease patients include treatments to minimize the risk of host rejection of the transplanted tissue. However, autoimmune disease involves the additional, independent risk that the pre-existing host autoimmune response which damaged the original self -tissue will exert the same damaging effect on the transplanted tissue.
Accordingly, the present invention encompasses methods and compositions for the treatment of autoimmune pancreatic disease using the albumin fusion proteins of the subject invetion in combination with immunomodulators/immunosuppressants in individuals undergoing transplantation therapy of the autoirnmune disease.
[1297] In accordance with the invention, the albumin fusion-based compositions and formulations described above, are administered to prevent and treat damage to the transplanted organ, tissue, or cells resulting from the host individual's autoinunurie response initially directed against the original self tissue. Administration may be carried out both prior and subsequent to transplantation in 2 to 4 doses each one week apart.
[1298]! The following immunomodulatorstimmunosuppressants including, but not . limited to, A1-401, CDP-571 (anti-TNF monoclonal antibody), CG-1088, Diamyd (diabetes vaccine), ICM3 (anti-ICAM-3 monoclonal antibody), linomide (Roquinimex), NBI-(altered peptide ligand), TM-27, VX-740 (11MR-3480), caspase 8 protease inhibitors, thalidomide, hOKT3gammal (Ala-ala) (anti-CD3 monoclonal antibody), Oral Interferon-Alpha, oral lactobacillus, and LymphoStat-BTM can be used together with the albumin fusion therapeutics of the subject invention in islet cell or pancreas transplantation.
Example 47!n vivo Mouse Model of NJDDM
[1299] Male C57BL/6J mice from Jackson Laboratory (Bar Harbor, ME) can be obtained at 3 weeks of age and fed on conventional chow or diets enriched in either fat (35.5% wt)wt; Bioserv.Frenchtown, NJ) or fructose (60% wt/wt; Harlan Teklad, Madison, WO. The regular chow is composed of 4.5% wilwt fat, 23% wt/wt protein, 31.9%
wt/wt starch, 3.7% wt/wt fructose, and 5.3% wt/wt fiber. The high-fat (lard) diet is composed of 35.5% wt/wt fat, 20% wt/wt protein, 36.4% wt/vvt starch, 0.0% wt/wt fructose, and 0.1%

wt/wt fiber. The high-fructose diet is composed of 5% wt/wt fat, 20% wt/wt protein, 0.0%
wt/wt starch, 60% wt/wt fructose, and 9.4% wt/wt fiber. The mice may be housed no more than five per cage at 22 -4/- 3 C temperature- and 50% +1- 20% humidity-controlled room with a 12-hour Edit (6 am to 6 prn)/dark cycle (Lilo et al., 1998, Metabolism 47(6): 663-8, "Nongenetic mouse models of non-insulin-dependent diabetes mellitus"; Larsen et at., Diabetes 50(11): 2530-9 (2001), "Systemic administration of the long-acting derivative NN2211 induces lasting and reversible weight loss in both normal arid obese rats"). After exposure to the respective diets for 3 weeks, mice can be injected intraperitoiieally with either streptozotocin, "STZ" (Sigma, St. Louis, MO), at 100 mg/kg body weight or vehicle (0.05 mol/L. citric acid, pH 4.5) and kept on the same diet for the next, 4 weeks. Under nonfasting conditions, blood is obtained 1, 2, and 4 weeks post-STZ by nipping the distal part of the tail. Samples are used to measure nonfasting plasma glucose and insulin concentrations. Body weight and food intake are recorded weekly.
[13001 To directly determine the effect of the high-fat diet on the ability of insulin to stimulate glucose disposal, the experiments can be initiated on three groups of mice, fat-fed, chow-fed injected with vehicle; and fat-fed injected with STZ at the end of the 7-week period described above. Mice can be fasted for 4 hours before the experiments. In the first series of experiments, mice can be anesthetized with tnethoxyflurane (Pitman-Moor, Mundelein, IL) inhalation. Regular insulin (Sigma) can be injected intravenously ([IV} 0.1 U/kg body weight) through a tail vein, and blood can be collected 3, 6, 9, 12, and 15 minutes after the injection from a different tail vein. Plasma glucose conoctitTations can Oe.
determin$
these samples, and the-fialf-life (tA) of glucose diaappearanee frona plasina Oath* caleulatad using WinNonlin (Scientific Consulting, Apex, NC::), phamticokittetirOp,harmiefodynamios software program.
[1301] In the second series of experiments, mice can be anesthetized with intraperitoneal sodium pentobarbital (Sigma). The abdominal cavity is opened, and the main abdominal vein is exposed and catheterized with a 24-gauge IV catheter (Johnson-Johnson Medical, Arlington, TX). The catheter is secured to muscle tissue adjacent to the abdominal vein, cut on the bottom of the syringe connection, and hooked to a prefilled PE50 plastic tube, which in turn is connected to a syringe with infusion solution. The abdominal cavity is then sutured closed. With this approach, there would be no blockage of backflow of the blood from the lower part of the body. Mice can be infused continuously with glucose (24.1 mg/kg/min) and insulin (10 rtaUlkg/min) at an infusion volume of 10 iIJmin.
Refto-orbital blood samples (70 pi, each) can be taken 90, 105, 120, and 135 minutes after the start of infusion for measurement of plasma glucose and insulin concentrations. The mean of these four samples is used to estimate steady-state plasma glucose (SSPG) and insulin (SSPI) concentrations for each animal.
[13021 Finally, experiments to evaluate, the ability of the albumin fusion proteins, the therapeutic compositions of the instant application, either alone or in combination with any one or more of the therapeutic drugs listed for the treatment of diabetes mellitus, to decrease plasma glucose can be performed in the following two groups of "NIDDM" mice models that are STZ-injected: (I) fat-fed C57BL/6,1, and (2) fructose-fed C57BU6.1. Plasma glucose concentrations of the mice for these studies may range from 255 to 555 mg/dL.
Mice are randomly assigned to treatment with either vehicle, albumin fusion therapeutics of the present invention either alone or in combination with any one or more of the therapeutic drugs listed for the treatment of diabetes mellitus. A total of three doses can be administered. Tail vein blood samples can be taken for measurement of the plasma glucose concentration before the first dose and 3 hours after the final dose.
[13031 Plasma glucose concentrations can be determined using the Glucose Diagnostic Kit from Sigma (Sigma No. 315), an enzyme colorimetric assay.
Plasma insulin levels can be determined using the Rat Insulin RLA Kit from Linco Research (#111-13K; St.
Charles, MO).
Example 48: In vitro 1141k -SEAP Reporter Aitari.Ettabliching tavolivaineatin Action.
The Various 1-1411e Reporters [13041 WIlle/rMEP-SEAP: The malic enzyme promoter isolated from rat (rMEP) contains a PPAR-gamma element which is in the insulin pathway. This reporter construct is stably transfected into the liver 11411e cell-line.
[13051 H411e/SREBP-SEAP: The sterol regulatory element binding protein (SREBP-1c) is a transcription factor which acts on the promoters of a number of insulin-responsive genes, for example, fatty acid synthetase (FAS), and which regulates expression of key genes in fatty acid metabolism in fibroblasts, adipocytes, and hepatocytes. SREBP-lc, also known as the adipocyte determination and differentiation factor I (ADD-1), is considered as the primary mediator of insulin effects on gene expression in adipose cells. It's activity is modulated by the levels of insulin, sterols, and glucose. This reporter construct is stably transfected into the liver fI4IIe cell-line.
113061 114ilefFAS-SEAP: The fatty acid synthetase reporter constructs contain a minimal SREBP-responsive FAS promoter. This reporter construct is stably transfected into the liver 11411e cell-line.

H4fle/PEPCK-SEkP: The phosphoenolpyruvate carboxylcinase (PEPCK) promoter is the primary site of hormonal regulation of PEPCK gene transcription: modulating PEPCK activity. PEPCK catalyzes a committed and rate-limiting step in hepatic gluconeogenesis and must therefore be carefully controlled to maintain blood glucose levels within normal limits. This reporter construct is stably transfected into the.
liver 11411e cell-line.
113681 These reporter constructs can also be stably transfected into 3T3-L1 fibroblasts and L6 rayoblasts. These stable cell-lines are then differentiated into 3T3-L1 adipocytes and L6 myotubes as previously described in Example 41. The differentiated cell-lines can then be used in the SEAT* assay described below.
Growth and Assay Medium 113091 = The growth medium comprises 10% Fetal Bovine Serum (FRS), 10% Calf Serum, 1% NEAA, lx penicillin/streptomycin, and 0.75 mg/mL 0418 (for H411e/rFAS-SEAP
and Ii411e/SREBP-SEAP) or 0.50 mg/mL G418 (for H411e/rMEP-SEAP). For 11411e/PEPCK-SEAP, the growth medium consists of 10% FRS, 1%
penicillin/streptomycin, 15 raM HUES buffered saline, and 0.50 rng/mL G418.
113101 - The =
assay medium consists of low glucose DMEM medium (Life Technologies), . NEAA, lx penicillin/streptomycin for the 1-1411e/rEAS-SEAP,' 114.11e/SREBP-SEAP, 1-1411e/r/v1EP-SEAP reporters. The assay medium for 11411e/PEPCK-SEAP reporter consists of 0.1% F13S, 1% penicillirdstreptornycin, and 15 mivi ITEPES
buffered saline.
Method 113111 The 96-well plates are seeded at 75,000 cells/well in 100 pllwell of growth medium until cells in log growth phase become adherent. Cells are starved far 48 hours by replacing growth medium with assay medium, 200 uL/well. (For H411e/PEPCK-SEAP
cells, assay medium containing 0.5 44 dexamethasone is added at 100 }IL/well and incubated for approximately 20 hours). The assay medium is replaced thereafter with 100 ItLiwell of fresh assay medium, and a 50 1.11:. aliquot of cell supernatant obtained from transfected expressing the therapeutics of the subject invention (e.g., albumin fusion proteins of the invention and fragments and variants thereof) is added to the well.
Supernatants from empty vector transfected cell-lines are used as negative control* Addition of 10 alvl and/or 100 nM
insulin to the wells is used as positive control. After 48 hours of incubation, the conditioned media are harvested and SEAP activity measured (Phospha-Light System protocol, Tropix #BP2500). Briefly, samples are diluted 1:4 in dilution buffer and incubated at 65 C for 30 minutes; to inactivate the endogenous non-placental form of SEAP. An aliquot of 50 pL of the diluted samples is mixed with 50 plõ of SEAP Assay Buffer which contains a mixture of inhibitors active against the non-placental SEAP isoenzymes and is incubated for another 5 minutes. An aliquot of 50 al, of CSPD chemiluminescent substrate which is diluted 1:20 in Emerald luminescence enhancer is added to the mixture and incubated for 15-20 minutes.
Plates are read in a Dynex plate luminometer.
Example 49: Preparation of 1:1A-evrokine. or RA-trowtii factor fusion proteins (sorb os EPO, GMCSF, GCSF).
[13121 The cDNA
for the cytokine or growth factor of interest, such as EPO, can be isolated by a variety of means including from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for all of these proteins are known and available, for instance, in U.S.
Patents 4,703,008, 4,810,643 and 5,908,763. The cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers, can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. EPO (or other cytokine) cDNA is cloned into a vector such as pPPC0005 (Figure 2), pSeCHSA, pScNHSA , or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasrnid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines, a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 1). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 50: Preparation of HA-IFN fusion proteins (such as IFNa).

[1313) The cDNA for the interferon of interest such as IFNa can be isolated by a variety of means including but not exclusively, from cDNA libraries, by 12T-FCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for interferons, such as &Nix are known and available, for instance, in U.S. Patents 5,326,859 and 44588,585, in EP 32134, as well as in public databases such as GenBank. The cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonueleotide linkers can be used to clone the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus of the HA
sequence, with or without the use of a spacer sequence. The IFNct (or other interferon) cDNA
is cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pScl\THSA , or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the eipressioit cassette used employs a mammalian promoter, leader sequence and temainatdr -(See Example 1). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Maximum protein recovery from vials [13141 The albumin fusion proteins of the invention have a high degree of stability even when they are packaged at low concentrations. In addition, in spite of the low protein concentration, good fusion-protein recovery is observed even when the aqueous solution includes no other protein added to minimize binding to the vial walls. The recovery of vial-stored HA-IFN solutions was compared with a stock solution. 6 or 30 [Lem! HA-EFN
solutions were placed in vials and stored at 4 C. After 48 or 72 hrs a volume originally equivalent to 10 ng of sample was removed and measured in an IFN sandwich ELISA. The estimated values were compared to that of a high concentration stock solution.
As shown, there is essentially no loss of the sample in these vials, indicating that addition of exogenous material such as albumin is not necessary to prevent sample loss to the val1 of the vials In vi..bk)___Ltigi_latsfg.:A.,47,1m4_11:th ions.
[13151 To determine the in vivo stability and bioavailability of a HA-ct-IFN fusion molecule, the purified fusion molecule (from yeast) was administered to monkeys.
Pharmaceutical compositions formulated from HA-a-IFN fusions may account for the extended serum half-life and bioavailability. Accordingly, pharmaceutical compositions may be formulated to contain lower dosages of alpha-interferon activity compared to the native alpha-interferon molecule.
[1316] Pharmaceutical compositions containing HA-a-IFN fusions may be used to treat or prevent disease in patients with any disease or disease state that can be modulated by the administration of a-IFN. Such diseases include, but are not limited to, hairy cell leukemia, Kaposi's sarcoma, genital and anal warts, chronic hepatitis 13, chronic non-A, non-B hepatitis, in particular hepatitis C, hepatitis D, chronic myelogenous leukemia, renal cell carcinoma, bladder carcinoma, ovarian and cervical carcinoma, skin cancers, recurrent respirator papillomatosis, non-Hodgkin's and cutaneous T-cell lymphomas, melanoma, multiple myeloma , AIDS, multiple sclerosis, gliobastoma, etc. (see Interferon Alpha, In:
AHFS Drug Information, 1997.
[1317] Accordingly, the invention includes pharmaceutical compositions containing a HA-a-IFN fusion protein, polypeptide or peptide formulated with the proper dosage for human administration. The invention also includes methods of treating patients in need of such treatment comprising at least the step of administering a pharmaceutical composition containing at least one HA-a-IFN fusion protein, polypeptide or peptide.
Arynctional HA-a-IFN fusions [1318] A HA-ct-1FN expression .vector may be modified to include an insertion for the expression of bifunctional HA-a-IFN fusion proteins. For instance, the cDNA
for a second protein of interest may be inserted in frame downstream of the "rHA-IFN"
sequence after the double stop codon has been removed or shifted downstream of the coding sequence.
[1319] In one version of a bifunctional HA-a-MN fusion protein, an antibody or fragment against B-lymphocyte stimulator protein (GenBank Ace 4455139) or polypeptide may be fused to one end of the HA component of the fusion molecule. This biffinctional protein is useful for modulating any immune response generated by the a-IFN
component of the fusion.
:Itta.ittple 51; Pre ants jiasili6Liormont, usion 'ototeitt (such t as :insulio,LIL:FS11) [1320] " The cDNA for the hormone of interest such as insulin can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR
and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for all of these proteins are known and available, for instance, in public databases such as Gen13ank. The cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The hormone eDNA is cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNTISA , or pC4:11SA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 1). This expression cassette is then excised and inserted into a plasmic!. =
suitable for the transfection of mammalian cell lines.
Exatapte 52: Preparation of HA-soluble receptor or HA-bindin 0rotein fusion mtein such as HA-TNF receptor.
11321.1 The cDNA
for the soluble receptor or binding protein of interest such as TNF
receptor can be isolated by a variety of means including but not exclusively, from cDNA
libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for all of these proteins are known and available, for instance, in GenBank. The cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the eDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The receptor cDNA is cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pSeNHSA , or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 1). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
EXample 53: Prepuration 90:1.A..-growth factors such as HA4.QE4 fusion protein.
[13221 The cDNA for the growth factor of interest such as IGF-1 can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR
and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods (see GenBank Ace. No.NP_000609). The cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The growth factor cDNA is cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA , or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 1). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 54: Preparation of HA-single chain antibody fusion proteins.
[1323] Single chain antibodies are produced by several methods including but not limited to: selection from phage libraries, cloning of the variable region of a specific antibody by cloning the cDNA of the antibody and using the flanking constant regions as the primer to clone the variable region, or by synthesizing an oligonucleotide corresponding to the variable region of any specific antibody. The cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The cell cDNA is cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA or pC4:FISA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast.
[13241 In fusion molecules of the invention, the VH and NIL can be linked by one of the following means or a combination thereof: a peptide linker between the C-terminus of the Ve and the N-terminus of the Ve ; a Kex2p protease cleavage site between the VH and V.
such that the two are cleaved apart upon secretion and then self associate;
and eystine residues positioned such that the VH and Ve can form a disulphide bond between them to link them together. An alternative option would be to place the VH at the N-terminus of HA or an HA domain fragment and the Ve at the C-terminus of the HA or HA domain fragment.
113251 The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 1). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines. = The antibody produeed in this manner can be purified from media and tested for its binding to its antigen using standard immunochernical methods.
EXampie 55: Preparation of ILA-cell adhesion 'molecule fusion proteins, [1326] The cDNA for the cell adhesion molecule of interest can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR
and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. . The nucleotide sequences for the known cell adhesiOn 'molecules are known and available, for instance, in GenBank. The cDNA can be tailored at the 5' and 3' ends to generate restriction "sites, such that oligonucleotide *linkers Can be used, for cloning of the cDNA into a vector containing the cDNA for -HA. This can be at th4N or C-terminus with or = en, without the use of a spacer sequence. The cell adhesion molecule cDNA is cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pSeNHSA or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 1). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Ekon' le 56: Preparation of rn4 factprs and pepUdesloaA. fusion proteins (such as HA-antiviral HA-antibiotic II; -err me inhibitor and HA-anti-allergic proteins).

113271 The cDNA for the peptide of interest such as an antibiotic peptide can be isolated by a variety of means including but not exclusively, from cDNA
libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA
into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The peptide cDNA is cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pScNHSA , or pC4:HS A from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. Forexpression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 1). This expression cassette is then excised and inserted into a plasmid suitable for the tra.nsfection of mammalian cell lines.
Example 57: Preparation of targeted HA fusion proteins.
113281 . The cDNA for the protein of interest can be isolated from cDNA
library or can be made synthetically using several overlapping oligonucleotides using standard molecular biology methods. The appropriate nucleotides can be engineered in , the cDNA
to form convenient restriction sites and also allow the attachment of the. protein cDNA to albumin cDNA similar' to the method described for hGH. Also a targeting protein or peptide cDNA
such as single chain antibody or peptides, such as nuclear localization signals, that can direct proteins inside the cells can be fused to the other end of albumin. The protein of interest and the targeting peptide is cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pSeNHSA , or pC4:HSA which allows the fusion with albumin cDNA. In this manner both N- and C-terminal end of albumin are fused to other proteins. The fused cDNA
is then excised from pPPC0005 and is inserted into a plasmid such as pSAC35 to allow the expression of the albumin fusion protein in yeast. All the above procedures can be performed using standard methods in molecular biology. The albumin fusion protein secreted from yeast can be collected and purified from the media and tested for its biological activity and its targeting activity using appropriate biochemical and biological tests.

Example 5$: Preparation of flA7entyllies tus4m&
113291 The cDNA for the enzyme of interest can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The cDNA can be tailored at the 5' and 3' ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the eDNA into a vector containing the cDNA for HA.
This can be at the N or C-terminus with or without the use of a spacer sequence. The enzyme cDNA is cloned into a vector such as pPPC0005 (Figure 2), pScCHSA, pSeNHSA, or pC4:HSA
from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 1). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
.'Example 59: Bacterial Expression of an AlbOrnitt Fuswn Prote40.
[1.330l A Polynucleotide encoding an albumin fusion Protein of the present invention comprising a bacterial signal sequence is amplified using. PCR oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence, to synthesize insertion fragments.
The primers used to amplify the polynucleotide encoding insert. should preferably contain restriction sites, such as Hamill and Xbal, at the 5' end of the primers in order to clone the amplified product into the expression vector. For example, RanillI and Xbar correspond to the restriction enzyme sites on the bacterial expression vector pQE-9.
(Qiagen, Inc., Chatsworth, CA), This plasmid vector encodes antibiotic resistance (Amp!), a bacterial origin of replication (on), an 17PTG-regulatable promoter/operator (P/0), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
[13311 The pQE-9 vector is digested with Baniffl and Xbal and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid PREP4, which expresses the lad iepressor and also confers kanamycin resistance (Kan'). Transformants are identified by their ability to grow on LB plates and anwicillinikanarnycin resistant colonies are selected.
Plasmid DNA is isolated and confirmed by restriction analysis.
113321 Clones containing the desired constructs are grown overnight (0(N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml).
The 0/N
culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (0.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lad repressor, clearing the P/0 leading to increased gene expression.
[13331 Cells are grown for an. extra 3 to 4 hours. Cells are then harvested by cen.tri fugation (20 mins at 6000Xg). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine Ha or preferably in, 8 M urea and concentrations greater than 0.14 M 2-mercaptoethanol by stirring for 3-4 hours at 4 C (see, e.g., Burton et al., Eur. J. Bioehem.
/79:379-387 (1989)). The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the .
Ni-NTA resin with high affinity and can be purified in a simple one-,step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra). -[13341 Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCI, pH
8. The column is first washed with 10 volumes of 6 M guanidine-HC1, pH 8, then washed with 10 volumes of 6 M guanidine-HCI pH 6, awl finally the polypeptide is eluted with 6 M
guanidine-HC1, pH 5.
[13351 The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pli 6 buffer plus 200 ruhl NaCl.
Alternatively, the protein can be successfully refolded while immobilized on, the Ni-NTA
column.
Exemplary conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM
NaC1, 20% glycerol, 20 mM Tris/HC1 pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM inunidazole. Immidazole is removed by a final dialyzing step against PBS or 50 inlvi sodium acetate pH 6 buffer plus 200 mM
NaCl. The purified protein is stored at 4 C or frozen at -80 C.
113361 In addition to the above expression vector, the present invention further includes an expression vector, called pliE4a (ATCC Accession Number 209645, deposited on February 25, 1998) which contains phage operator and promoter elements operatively linked to a polynucleotide encoding an albumin fusion protein of the present invention, called pHE4a. (ATCC Accession Number 209645, deposited on February 25, 1998.) This vector contains: 1) a neonaycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-DeIgarno sequence, and 6) the lactose operon repressor gene (tacit . The origin of replication (oriC) is derived from pUC19 (LT1, Gaithersburg, MD). The promoter and operator sequences are made synthetically.
113371 DNA can be inserted into the pHE4a by restricting the vector with Ndei and Xbal, BallaHI, XhoI, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA
insert is generated according to PCR protocols described herein or otherwise known in the art, using PCR primers having restriction sites for NdeI (5' primer) and XbaI, BarnHI, Xhol, or Asp718 (3' primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.
(13381 The engineered vector may be substituted in the above protheol to express protein in a bacterial system.
Example 60i Expression of an Albumin Fusion Protein in Mammalian Cells.
[13391 The albumin fusion proteins of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of triRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLV1, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
113401 Suitable expression vectors for use in practicing the present invention include, for example, vectors such as, pSVL and pMSG (Phamiacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3Ø Mammalian host cells that could be used include, but are not limited to, human Hela, 293, H9 and Juricat cells, mouse NIFI3T3 and C127 cells, Cos 1, Cos 7 and CV I, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[13411 Alternatively, the albumin fusion protein can be expressed in stable cell lines containing the polynucleotide encoding the albumin fusion protein integrated into a chromosome. The co-transfection with a selectable marker such as DHFR, gpt, neomycin, or hypomycin allows the identification and isolation of the transfected cells.
113421 The transfected polynucleotide encoding the fusion protein can also be amplified to express large amounts of the encoded fusion protein. The DHFR
(dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt et al., J. Biol.
Chem. 253:1357-1370 (1978); Hamlin et al., Biochern. et Biophys. Acta, 1097:107-143 (1990); Page et al., Biotechnology 9:64-68 (1991)). Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Rio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
[13431 Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fi=agment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.
[13441 Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.
[13451 A polynucleotide encoding an albumin fusion protein of the present invention is generated using techniques known in the art and this polynucleotide is amplified using PCR technology known in the art. If a naturally occurring signal sequence is used to produce the fusion protein of the present invention, the vector does not need a second signal peptide.
Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No. WO

96/34891.) [13461 The ampli Fied fragment encoding the fusion protein of the invention is isolated from a I% agarose gel using a commercially available kit ("Geneclean," BIC) 101 Inc., La Jolla, Ca.). The fragment then is digested with appropriate restriction enzymes and again purified on a I% agarose gel.
[13471 The amplified fragment encoding the albumin fusion protein of the invention is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA
ligase. E.
calf 1113101 or XL-! Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
11348j Chinese hamster ovary cells lacking an active DHFR gene is used for transfection. Five lig of the expression plasmid pC6 or pC4 is cotransfected with 0.5 ag of the plasmid pSVneo using lipofectin (Feigner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha. Minus MEM supplemented with 1 Mg/ml 0418. After 2 days, the cells are tryPsinized and seeded in hYbri.cloma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of rnethotrexate plus I mg/nil 0418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM,. 800 nM).
Clones growing at the highest concentrations of methetrexate are then transferred to new 6-well plates containing even higher concentrations of methotrex ate (1 !AM, 2 tiM, 5 aM, 10 inM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 FLM. Expression of the desired fusion protein is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 61: Multifusion Fusions.
113401 The albumin fusion proteins (e.g,. containing a Therapeutic protein (or fragment or variant thereof) fused to albumin (or a fragment or variant thereof)) may additionally be fused to other proteins to generate "multifusion proteins".
These multifusion proteins can be used for a variety of applications. For example, fusion of the albumin fusion proteins of the invention to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See e.g,. EP A 394,827; Traunecker et al., Nature 331;84-86 (1988)). Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of an albumin fusion protein.
Furthermore, the fusion of additional protein sequences to the albumin fusion proteins of the invention may further increase the solubility and/or stability of the fusion protein. The fusion proteins described above can be made using or routinely modifting techniques blown in the art and/or by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG
molecule.
[13501 Briefly, the human Fe portion of the IgG molecule can be PCR
amplified, using primers that span the 5' and 3' ends of the sequence described below.
These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian or yeast expression vector.
[13511 For example, if pC4 (ATCC Accession No. 209646) is used, the human Fe portion can be ligated into the BarnHI cloning site. Notes that the 3' BantHI
site should be destroyed. Next, the vector containing the human Fe portion is re-restricted with BrunHI, linearizing the vector, and a polynucleotide encoding an albumin fusion protein of the present invention (generateed and isolated using techniques known in the art), is ligated into this Bamill site. Note that the polynucleotide encoding the fusion protein of the invention is cloned without a stop eodon, otherwise a Fe containing fusion protein will not be produced.
[1352] If the naturally occurring signal sequence is used to produce the.
albumin fusion protein of the present invention, pC4 does not need a second signal peptide.
Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No.
WO 96/34891.) Human IgG Fe region:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAG
CACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA
CACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGC
CACGAAGACCCTGAGGTCAAGTTC AACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC
AGCGTCCTCACCGTC CTG C A CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAMWCCcTCCQAACCCCATCGAQAAAACCacTPCWQCC

AAA.GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG
CTGACCA.AGAACCAGGTCAGCCTGACCTGCCTG GTCAAAG GCTTCTATCCAAGC
GACATCGCCGTGGAGTGGGA.GAGCAATGGGCAGCCGGAGAACAACTACAAGAC
CACGCCTCCCGTGCTGGACTCCGACGGCTCCTICITCCTCTACAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT
GAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID NO: 1112) Example 62: Productinti of an -Antibody from anAjbuniin Fusion Protein,.
Hybridoma Technology [1353] Antibodies that bind the albumin fusion proteins of the present invention and portions of the albumin fusion proteins of the present invention (e.g., the Therapeutic protein portion or albumin portion of the fusion protein) can be prepared by a variety of methods.
(See, Current Protocols; Chapter 2.) As one example of such methods, a preparation of an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention is prepared and purified to render it substantially free of natural contaminants.
Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
113541 Monoclonal antibodies specific for an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention, are prepared using hybridorna technology (Kohler et al., Nature 256:495 (1975); Kohler et al., (Elm J.
Immunol. 6:511 (1976); Kohler et al., Eur. J. Irrununol. 6:292 (1976); Hanunerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention. The splenocytes of such mine are extracted and fused with a suitable myelorna cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in FIAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)).
The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention.

1355] Alternatively, additional antibodies capable of binding to an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the an albumin fusion protein of the invention (or portion of an albumin fusion protein of the invention) -specific antibody can be blocked by the fusion protein of the invention, or a portion of an albumin fusion protein of the invention. Such antibodies comprise anti-idiotypic antibodies to the fusion protein of the invention (or portion of an albumin fusion protein of the invention) -specific antibody and are used to immunize an animal to induce formation of further fusion protein of the invention (or portion of an albumin fusion protein of the invention) -specific antibodies.
113561 For in viva use of antibodies in humans, an antibody is "humanized".
Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S.
Patent No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO
8601533; Robinson et al., International Publication No. WO 8702671; Bonhomie et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985)).
(13571 Isolation Of Antibody Fragments Directed Against an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention From A Library Of scFvs. Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reaetivities against an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention, to which the donor may or may not have been exposed (see e.g., U.S. Patent 5,885,793), (13581 Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in International Publication No. WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coil harboring the phagemid are used to oculate 50 ml of 2xTY containing 1% glucose and 100 p.g/m1 of ampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to inoculate 50 ml of 2xTY-AMP-GLU, 2 x 108 TU of delta gene 3 helper (MI3 delta gene III, see International Publication No. WO 92/01047) are added and the culture incubated at 37 C for 45 minutes without shaking and then at 37 C for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2xTY
containing 100 in/m1 ampicillin and 50 ug/ml kanamycin and grown overnight.
Phage are prepared as described in International Publication No. WO 92/01047.
(1359] M13 delta gene ER is prepared as follows; M13 delta gene III helper phage does not encode gene In protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene 111 particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene ifi protein during phage morphogenesis. The culture is incubated for 1 hour at 37 C without shaking and then for a further hour at 37 C with shaking. Cells are spun down (rEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2xTY broth containing 100 fig ampicillin/ml and 25 lag kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37 C.
Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 pm filter (Minisart NML; Sartotius) to give a final concentration of approximately 1013 transducing units/m1 (ampicillin-resistant clones).
[1360] Panning of the Library. Imnumotubes (Nunc) are coated overnight in PBS
with 4 ml of either 100 1.1g/m1 or 10 Wmi of an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37 C and then washed 3 times in PBS. Approximately 1013 TU
of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tweelr-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCI, pH 7.4.
Phage are then used to infect 10 ml of mid-log E. coil TG1 by incubating eluted phage with bacteria for 30 minutes at 37 C, The E. call are then plated on TYE plates containing 1%
glucose and 100 jig/m1 ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection.
This process then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tweeitkl20 and 20 times with PBS for rounds 3 and 4.
[13611 Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coil HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/m1 of an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention, in 50 rriM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., International Publication No. WO
92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.
Exartmle 63: Method of Treatment Using Gene Therapy-Ex Vivo.
[1362] One method of gene therapy transplants fibroblasts, which are capable of expressing an albumin fusion protein of the present invention, onto a patient.
Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask.
The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12, media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C for approximately one week.
[1363] At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
[1364) pMV-7 (Kirschmeier,P.T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI
and HindIII
and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
[13651 Polynucleotides encoding an albumin fusion protein of the invention can be generated using techniques known in the art amplified using PCR primers which correspond to the 5' and 3' end sequences and optionally having appropriate restriction sites and initiationistop codons, if necessary. Preferably, the 5' primer contains an EcoRI site and the 3' primer includes a HindIff site. Equal quantities of the Moloney 111116118 sarcoma virus linear backbone and the amplified EcoRI and IfindIE fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanarnycin for the purpose of confirming that the vector has the gene of interest properly inserted.
113661 The amphotropic pA317 or GP+am12 packaging cells are gown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
[1367] Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer tells and this media is then used to. infect fibroblast cells. Media is removed from A sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is. high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether the albumin fusion protein is produced.
113681 The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 rnicrocarrier beads.
Example 64: Method of Treatment Using Gene Theratnt Vieo,., 113691 Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences encoding an albumin fusion protein of the invention into an animal;
Polynucleotides encoding albumin fusion proteins of the present invention may be operatively linked to (i.e., ssociated with) a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, W090/11092, W098/11779; U.S. Patent NO.
5693622, 5705151, 5580859; Tabata et al., Cardiovase. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997);
Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996).
[1370] The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
[1371] The term "naked" polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, polyrmcleotides encoding albumin fusion proteins of the present invention may also be delivered in liposome formulations (such as those taught in Feigner P.L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.
[1372] The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
[1373] The polynucleotide construct can be delivered to the interstitial space of tissues within an animal, including muscle, skin, brain, lung, liver, spleen, bone tilaITONV, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue.
Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. in vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
(13741 For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space Of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues,.
throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
[1375) The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. , Suitable template DNA for production of m.RNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.
[13761 Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1,5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is -iosed with stainless steel clips.
[13771 After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 urn cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for fusion protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different tunes. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be used to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.
Example 65: Transgenie Animals.
(1.3781 The albumin fusion proteins of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
In a specific embodiment, techniques described herein or otherwise known in the art, are used to express fusion proteins of the invention in humans, as part of a gene therapy protocol.
[13791 Any technique known in the art may be used to introduce the polynucleotides encoding the. albumin fusion proteins of the invention into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol, Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell.
Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989).

[13801 Any technique known in the art may be used to produce transgenic clones containing polynucleotides encoding albumin fusion proteins of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wi!mut et al., Nature 385:810-813 (1997)).
[13811 The present invention provides for transgenic animals that carry the polynucleotides encoding the albumin fusion proteins of the invention in all their cells, as well as animals which carry these polynucleotides in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et at. (Lasko et al., Proc.
Natl. Acad. Sci. USA
89:6232-6236 (1992)): The regulatory sequences required for such a cell-type specific activation will depend upon 'the particular cell type of interest, and will be apparent to 'those of skill in the art When it is desired that the polynucleotide encoding the fusion proteinof-the invention be integrated- into the chromosomal site of the endogenous gene corresponding to the Therapeutic protein portion or ablurnin portion of the fusion protein of the invention, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for -the purpose of integrating, via hOmologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene.
The transgene may als.Cilie selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, foi= example, the teaching of Gu et al.
(Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
113821 Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the polynucleotide encoding the fsuion protien of the invention has taken place. The level of niRNA expression of the polynucleotide encoding the fusion protein of the invention in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of fusion protein-expressing tissue may also be evaluated immunocytochemically or inununohistochemically using antibodies specific for the fusion protein.
113831 Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis;
crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene (i.e., polynucleotide encoding an albumin fusion protein of the invention) .on a distinct background that is appropriate for an experimental model of .
interest.
(13841 Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of fusion proteins of the invention and the Therapeutic protein and/or albumin component of the fusion protein of the invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions andior, disorders.
gltantole 66 Assavs. Detecting Stimulation Or Inhibition of B cell Proliferation and Differentiation.
113851 Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their microenvironment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway. To date, numerous stimulatory and inhibitory signals have been found to influence 13 cell responsiveness including IL-2,. IL-4, IL-5, IL-6, IL-7, 11,10, M-13, IL-14 and M-15.
Interestingly, these signals are by themselves weak effectors but can, in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, horning, tolerance and death among B cell populations.

[1386) One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27, and CD30 along with their respective ligands CD154, CD70, and CD153 have been found to regulate a variety of immune responses.
Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.
113871 In Vitro Assay- Albumin fusion proteins of the invention (including fusion proteins containing fragments or variants of Therapeutic proteins and/or albumin or fragments or variants of albumin) can be assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of an albumin fusion protein of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgIvr antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incorporation. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220) .
[13811 Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 105 B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5 X 10-5M 2ME, 10013/m1 penicillin, 1 Oug/ml streptomycin, and 10-5 dilution of SAC) in a total volume of 150u1. Proliferation or inhibition is quantitated by a 20h pulse (luCi/well) with 311-thymidine (6.7 CilmM) beginning 7211 post factor addition. The positive and negative controls are IL2 and medium respectively.
[1389] In vivo Assay- BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of an albumin fusion protein of the invention (including fusion proteins containing fragments or variants of Therapeutic proteins and/or albumin or fragments or variants of albumin). Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues arid serum collected for analyses.
Comparison of H&E
sections from normal spleens and spleens treated with the albumin fusion protein of the invention identify the results of the activity of the fusion protein on spleen cells, such as the diffusion of pen-arterial lymphatic sheaths, and/or significant increases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochemical studies using a B
cell marker, anti-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.
[1390] Flow cytornetric analyses of the spleens from mice treated with the albumin fusion protein is used to indicate whether the albumin fusion protein specifically increases the proportion of ThB+, CD45R(B220)dull B cells over that which is observed in control mice.
[1391] Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and fusion protein treated mice.
Example 67: T Cell Proliferation Assay.
[1392] A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of 3H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 ill/well of mAb to CD3 (HI13a, Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4 degrees C (1 Wail in .05M bicarbonate buffer, pH
9.5), then washed three times with PBS. PBMC are isolated by F/1.1 gradient centrifugation from human peripheral blood and added to quadruplicate wells (5 x 104/well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of an albumin fusion protein of the invention (including fusion proteins containing fragments or variants of Therapeutic proteins and/or albumin or fragments or variants of albumin) (total volume 200 up. Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37 degrees C, plates are spun for 2 min. at 1000 rpm and 100 ill of supernatant is removed and stored ¨20 degrees C for measurement of 11-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 ul of medium containing 0.5 tiCi of 3H-thymidine and cultured at 37 degrees C for 18-24 hr. Wells are harvested and incorporation of 3H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. I1-2 (100 Uhril) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative control for the effects of fusion proteins of the invention.
Exam le C: Effect of Fits 'nn roteinS of the 'nvention O. the Et resSion Classfl rostimulatory and Adhesion Molecules and ...00 Differentiation of -Mputicytes and Monocyte-Derived Human Dendritic Cells.
113931 Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or eluttiated moriocytie fractions are cultured for 7-10 days with GM-CSF (50 ngirril) and 1L-4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and IVII-IC class H antigens). Treatnient with activating factors, such as TNF-a, causes a rapid change in surface phenotype (increased expression of MIIC class I and fl, costimulatory and adhesion molecules, downregulation of FC7RII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.
[1394] FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of an albumin fusion protein of the invention or LPS
(positive control), washed with PBS containing 1% BSA and 0.02 rriM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies . .
for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytornetry on a FACScan (Becton Dickinson), [1395] Effect on the production of cytokin.es. Cytokines generated by dendritic cells, in particular 1L-12, are important in the initiation of 1-cell dependent immune responses. IL-12 strongly influences the development of Thl helper 1-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the 1L-12 release as follows.
Dendritic cells (106/m1) are treated with increasing Concentrations of an albumin fusion protein of the invention for 24 hours. LPS (100 rig,/m1) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-I 2 content using commercial ELISA kit (e.g., R & D Systems (Minneapolis, MN)). The standard protocols provided with the kits are used.
[1396] .Fffeet 01A the ootoso4o of WIC Cas 11, costionditoty and.
arthdsieni.

molecules. Three major families of cell surface antigens can be identified on monocytes:
adhesion molecules, molecules involved in antigen presentation, and Fe receptor.
Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICA1v1-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increased expression of Fe receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.
[13971 FACS analysis is used to examine the surface antigens as follows.
Monocytes are treated 1-5 days with increasing concentrations of an albumin fusion protein of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0_02 niM
sodium nide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
[13981 Monocyte activation and/or increased survival. Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in.the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Albumin fusion proteins of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, MD) by centrifugation through a Histopaque gradient (Sigma). Monocytes are isolated from PBMC
by counterflow centrifugal elutriation.
[13991 Monocyte Survival Assay. Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from -internally regulated processes (apoptosis). Addition to the culture of activating factors, such as TINIF-alpha dramatically improves cell survival and prevents DNA
fragmentation.
Propidium iodide (PI) staining is used to measure apoptosis as follows.
Monocytes are cultured for 48 hours in polypropylene tubes in serum-free medium (positive control), in the presence of 100 ng/m1 TNF-alpha (negative control), and in the presence of varying concentrations of the fusion protein to be tested. Cells are suspended at a concentration of 2 x 106/m1 in PBS containing PI at a final concentration of 5 p.girril, and then incubated at room temperature for 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.
114001 Effect on cytokine release. An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation. An EL1SA to measure cytolcine release is performed as follows. Human monocytes are incubated at a density of 5x105 cells/m.1 with increasing concentrations of an albumin fusion protein of the invention and under the same conditions, but in the absence of the fusion protein. For 1L-12 production, the cells are primed overnight with IFN (100 Ultril) in the presence of the fusion protein. LPS (10 neat) is then added.
Conditioned media are collected after 24h and kept frozen until. use.
Measurement of TNF-alpha, 1L-10, MCP -1 and 1L-8 is then performed using a commercially available EL1SA kit (e.g., R & D Systems (Minneapolis, MN)) and applying the standard protocols provided with the kit.
114011 Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1x105 cell/well. Increasing concentrations of an albumin fusion protein of the invention are added to = the wells in a total volume of 0.2 ml culture medium (RPMI 1640 + 10%
FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaC1, 10 niM potassium phosphate buffer pH 7.0, 5.5 m.M dextrose, 0.56 rnM
phenol red and 19 U/m1 of FIRPO) is added, together with the stimulant (200 nM PMA).
The plates are incubated at 37 C for 2 hours and the reaction is stopped by adding 20 id 1N NaOH per welt The absorbance is read at 610 um. To calculate the amount of 11202 produced by the macrophages, a standard curve of a 11202 solution of known molarity is performed for each experiment.
Example 6% 'ffloletical Effects of ftsititt Proteins of the Invention.
Astrocyte and Neuronal Assays.
11402] Albumin fusion proteins of the invention can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent Tression of FGF- I and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A
thymidine incorporation assay, for example, can be used to elucidate an albumin fusion protein of the invention's activity on these cells.

Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., "Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension." Proc. Natl.
Acad. Sci.
USA 83;3012-3016. (1986)).
However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of an albumin fusion protein of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.
Fibroblast and endothelial cell assays.
[1404] Human lung fibroblasts are obtained from Clonetics (San Diego, CA) and maintained in growth media from Clonetics. Dermal rnicrovascular endothelial cells are obtained from Cell Applications (San Diego, CA). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test fusion protein of the invention proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, CA) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor fluorescence reader. For the PGE2 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA
basal medium, the cells are incubated with FGF-2 or fusion protein of the invention with or without IL-la for 24 hours. The supernatants are collected and assayed for PGE2 by ETA kit (Cayman, Ann Arbor, MI). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to OA% BSA basal medium, the cells are incubated with FGF-2 or with or without an albumin fusion protein of the invention andlor IL-la for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, MA).
[14051 Human lung fibroblasts are cultured with FGF-2 or an albumin fusion protein of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10 -2500 ng/m1 which can be used to compare stimulation with the fusion protein of the invention.
Cefl Prolifiar4tiOn based on InIlthymidine incorpOration.
[14061 The following [31-1]Thyrnidine incorporation assay can be used to measure the effect of a Therapeutic proteins, e.g., growth factor proteins, on the proliferation of cells such as fibroblast cells, epithelial cells or immature muscle cells.
(14071 Sub-confluent cultures are arrested in GI phase by an 18 h incubation in serum-free medium. Therapeutic proteins are then added for 24 Ii and during the last 4 le the cultures are labeled with [3H]thymidine, at a final concentration of 0.33 iM
(25 Ciimmol, Arnersharn, Arlington Heights, IL). The incorporated [31-1]th3anidine is precipitated with ice-cold 10% trichloroacetic acid for 24 h. Subsequently, the cells are rinsed sequentially with ice-cold 10% trichloroacetic acid and then with ice-cold water. Following lysis in 0.5 M
NaOH, the lysates and PBS rinses (500 ml) are pooled, and the amount of radioactivity is measured.
Parkinson Models, 114081 The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized.
involves the systemic administration of 1-methy1-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, l>.4PTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methy1-4-phenyl pyridine (IVIPP) and released. Subsequently, MPP+ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP+ is then concentrated in mitochondria by the electrochemical gadient and selectively inhibits nicotida.mide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.
[14091 It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev.
Biol. 1989).
Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with lvIPTP exposure (Otto and Unsicker, J.
Neuroscience, 1990).
[14101 Based on the data with FG-F-2, an albumin fusion protein of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP
treatment. The potential effect of an albumin fusion protein of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm2 on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and medium containing hormonal supplements (N1). The cultures arc fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopaminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.
[1411] Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase irnmunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro.
Therefore, if a therapeutic protein of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the fusion protein may be involved in Parkinson's Disease.
Example 7O TheEffeet of Allmmat.Pusion Proteins of the Invention on the Growth of Vascular Endothelial Cells.
[14121 On day 1, human umbilical vein endothelial cells (1-11.NEC) are seeded at 2-5x104 cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (PBS), 16 units/m1 heparin, and SO units/nil endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10%
FBS, 8 units/m1 heparin. An albumin fusion protein of the invention, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.
[14131 An increase in the number of HUVEC cells indicates that the fusion protein may proliferate vascular endothelial cells, while a decrease in the number of IILTVEC cells indicates that the fusion protein inhibits vascular endothelial cells.
Example 71: Rat Conical Wound Healing Model [14141 = This animal model shows the effect of an albumin fusion protein of the invention on neovascularization. The experimental protocol includes:
Making a 1-1.5 ram long incision from the center of cornea into the stromal layer.
Inserting a spatula below the lip of the incision facing the outer corner of the eye..
Making a pocket (its base is 1-1.5 mm form the edge of the eye).
Positioning a pellet, containing 5Ong- 5ug of an albumin fusion protein of the invention, within the pocket.

Treatment with an an albumin fusion protein of the invention can also be applied' topically to the corneal Wounds in a dosage range of 20mg 500mg (daily treatment for five days). =
Ejtarapie 72: Diabetic Mott.Se.atid aliaceetwiticoid-impaired Wound Iteal1it2 Models, Diabetic db+Idb+ Mouse Model.
[14161 To demonstrate that an albumin. fusion protein of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M.H. et al.õ 1. Sztrg. Res. 52:389 (1992); Greenhalgh, D.G. et al., Am. .1 Pathol. 1.36:1235 (1990)).
[14171 The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (dbaldb+) mice are obese in comparison to their normal heterozygous (db+/-1-m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad.
Sci. USA
77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., .1 Immunol. 120:1375 (1978);
Debray-Sachs, M. et al., C'lin. Exp. hnmunol. 51(1):1-7 (1983); Leiter et al., Am. I
of PathoL 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984);
Robertson et al., Diabetes 29(0:60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979); Coleman, Di., Diabetes 31 (Suppl):1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type 11 diabetes (Mandel et al., Immunol. 120:1375-1377 (1978)).
[1418] The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et aL, Am. J. of PathoL 136:1235-1246 (1990)). =
0.4191 Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littemiates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and tJse of Laboratory Animals.
[1420] Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rificin, D.B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methy1-2-butanol dissolved in &ionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given =
topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
114211 Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
114221 An albumin fusion protein of the invention is administered using at a range different doses, from 4mg to 500mg per wound per day for 8 days in vehicle.
Vehicle control groups received 5OrnL of vehicle solution.
[14231 Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered forrnalin in tissue cassettes between biopsy sponges for further processing.
[14241 Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.
[1425] Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day I is 64mm7, the corresponding size of the dermal punch.
Calculations are made using the following fommla:
a, [Open area on day 8] - [Open area on day 1]! [Open area on day 1]
114261 Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5nun) and cut using a Reichert-Jung rnicrotome. Routine hernatoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with an albumin fusion protein of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)). A
calibrated lens micrometer is used by a blinded observer.
[14271 Tissue sections are also stained immunohistochemieally with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control, Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a =
calibrated lens micrometer.
114281 Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is -demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system.
Human colon cancer served as a positive tissue control and human brain tissue is used as a negative tissue control. Each specimen included a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.
[1429] Experimental data are analyzed using an unpaired t test. A p value of < 0.05 is considered significant.
Steroid Impaired Rat Model [1430] The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing.
In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989);
Wahlet al., 11 Immunol, 115: 476-481 (1975); Werb et al., J. Exp. Med. /47:16844694 (1978)).
Glucocorticoids . retard wound healing by inhibiting angiogenesis, .decreasing vascular permeability (Ebert et aL, An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et aL, Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin.
Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, "Glucocorticoids and wound healing", In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impaired wound =
healing is a well establish phenomenon in rats (Beck et al., Growth Factors.
5: 295-304 (1991); Haynes et aL, .1. OM. Invest. 61: 703-797 (1978); Wahl, "Glucocorticoids and wound healing", In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA
86: 2229-2233 (1989)).
[1431] To demonstrate that an albumin fusion protein of the invention can accelerate the healing process, the effects of multiple topical applications of the fusion protein on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of rnethylprednisolone is assessed.
[1432] Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad-libitum. All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
[1433] The wounding protocol is followed according to that described above.
On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch.
The wounds are left open for the duration of the experiment: Applications of the testing materials are given topically once iµday for 7 consecutive days commencing on the day of wounding and subsequent to rnethylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
(1434j Wounds are visually examined and photogaphed it a fixed distance at the day of wounding and at the end of treatment. Wound Closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
[1435] The fusion protein of the invention is administered using at a range different doses, from 4mg to 500mg per Wound per day for 8 days in vehicle. Vehicle control groups received 50nil, of vehicle solution.
[1436] Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300mgfkg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered forinalin in tissue cassettes between biopsy sponges for further processing.
[1437] Three groups of 10 animals each (5 with methylprednisolone and 5 without =
glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups, [14381 Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64nun2, the corresponding size of the dermal punch.
Calculations are made using the following formula:
a. [Open area on day 8] - [Open area on day 1] / [Open area on day 1]
[1439] Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5nun) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with an albumin fusion protein of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.
[1440] Experimental data are analyzed using an unpaired t test. A p value of < 0.05 is considered significant.
Example 73: Lvmphedema Animal Model.
11441] The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of an albumin fusion protein of the invention in lyrnphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 3-4 weeks.
114421 . Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately ¨350g are dosed with Pentobarbital.
Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% Et0H. Blood is drawn for serum total protein testing.
Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1% Evan's Blue.
Circumference and volumetric measurements are then made following injection of dye into paws.
[1443] Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and hemostats are used to dissect and separate the skin flaps. After locating the feinoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated or suture ligated.
[14441 Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected. The popliteal lymph node is then located.
The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node are then ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues.
[1445] Care is taken to control any mild bleeding resulting from this procedure. After lymphatics are occluded, the skin flaps are sealed by using liquid skin (Vetbond) (AJ Buck).
The separated skin edges are sealed to the underlying muscle tissue while leaving a gap of ¨0.5 cm around the leg. Skin also may be anchored by suturing to underlying muscle when necessary.
[14461 To avoid infection, animals are housed individually with mesh (no bedding).
Recovering animals are checked daily through the optimal edematous peak, which typically occurred by day .5-7. The plateau edematous peak are then observed. To evaluate the intensity of the lymphedema, the circumference and volumes of 2 designated places on each paw before oper.atton and daily for 7 days are measured. The effect of plasma proteins on lymphedema is determined and whether protein analysis is a useful testing perimeter is also investigated. The weights of both control and edematous limbs are evaluated at 2 places.
Analysis is performed in a blind manner.
[1447j Circumference Measurements: Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb circumference. Measurements are done at the ankle bone and dorsal paw by 2 different people and those 2 readings are averaged.
Readings are taken from both control and edematous limbs.
[1448] Volumetric Measurements: On the day of surgery, animals are anesthetized with Pentobarbital and are tested prior to surgery. For daily volumetrics animals are under brief halothane anesthetic (rapid immobilization and quick recovery), and both legs are shaved and equally marked using waterproof marker on legs. Legs are first dipped in water, then dipped into instrument to each marked .level then measured by Buxco edema soft-ware(ChenNictor). Data is recorded by one person, while the other is dipping the limb to marked area.
114491 Blood-plasma protein measurements; Blood is drawn, spun, and serum separated prior to surgery and then at conclusion for total protein and Ca2+
comparison.
[14591 Limb Weight Comparison: After drawing blood, the animal is prepared for tissue collection. The limbs are amputated using a quillitine, then both experimental and control legs are cut at the ligature and weighed. A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed.
[1451] Histological Preparations: The transverse muscle located behind the knee (popliteal) area is dissected and arranged in a metal mold, filled with freezeGel, dipped into cold methylbutane, placed into labeled sample bags at - NEC until sectioning.
Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics..
Example 14 Suppression of TNF alpha-Induced Adhesion Molecule Expression by an Albumin Fusion Protein of the Invention.
[1452] The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-I (ICAM-1), vascular cell adhesion molecule-1 (VCAIVI-1), and endothelial leukocyte adhesion molecule-I (E-selectin) expression on endothelial cells (EC).
The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytoldnes and growth factor participate in the modulation of the expression of these CAMs.
[1453] Tumor necrosis factor alpha (TNF-a), a potent proinflarnmatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome.
114541 The potential of an albumin fusion protein of the invention to mediate a suppression of TNF-a induced CAM expression can be examined. A modified ELISA
assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM
expression on TNF-a treated ECs when co-stimulated with a member of the FGF
family of proteins.
114551 To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM-2;
Clonetics, San Diego, CA) supplemented with 10% FCS and 1%
penicillin/streptomycin in a 37 degree C humidified incubator containing 5% CO2. HUVECs are seeded in 96-well plates at concentrations of 1 x 104 cells/well in EGM medium at 37 degree C for 18-24 bra or until confluent. The monolayers are subsequently washed 3 times with a serum-free solution of RPM1-1640 supplemented with 100 Um' penicillin and 100 mg/ml streptomycin, and treated with a given cytokine and/or growth factor(s) for 24 h at 37 degree C.
Following incubation, the cells are then evaluated for CAM expression.
114561 Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence. Growth medium is removed from the cells and replaced with 90 ul of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 ul volumes). Plates are incubated at 37 degree C for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 1000 of 0.1% paraformaldehyde-PBS(with Ca++ and Mg) is added to each well. Plates are held at 4 C for 30 min.
114571 Fixative is then removed from the wells and wells are washed IX
with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10 ul of diluted primary antibody to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 p.g/m1 (1:10 dilution of 0.1 inWnal stock antibody). Cells are incubated at 37 C for 30 min. in a humidified environment.
Tells are washed X3 with PBS(+Ca,Mg)+0.5% BSA.
114581 Then add 20 gl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000 dilution) to each well and incubated at 37 C for 30 min. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 il of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer 1:5,000 (10 ) > 1(Y 5> 10> 10-1'5. 5 RI
of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 ill of pNNP reagent must then be added to each of the standard wells. The plate must be incubated at 37 C for 4h. A volume of 50 p.1 of 3M
NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to indicate the concentration of AP-conjugate in each standard well [ 5.50 ng; 1.74 ng;
0.55 ng; 0.18 ng}. Results are indicated as amount of bound AP-conjugate in each sample.
Example 75: Construction of GAS Reporter Construct, 114591 One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the hies-STATs pathway bind to gamma activation site "GAS" elements or interferon-sensitive responsive element ("ISRE"), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.
114601 GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or "STATs." There are six members of the STATs family. Stat 1 and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class 1, cells after treatment with 11-12. ..Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.
[14611 The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase ("Jaks") family.
Jaks represent a distinct family of soluble tyrosine Idnasei and include Tyk2, Jakl, Jak2, and Jak3.. These kina.ses display significant sequence similarity and are generally catalytically inactive in resting cells.
[14621 The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochent 64:621-(1995)). A cytoldne receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, 11-3. IL-4, IL-6, IL-7, M-9, 11-11, Epo, PRL, GH, G-CSF, GM-CSF, LE, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and M-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO: 1113)).
114631 Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway. Therefore, activation of the Inks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jalcs-STATs pathway (See Table 5, below). Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified.

Table 5 JARS STATS GAS(elements) or IS_RE
Ljg tyk2 Jakl ak2 Jak3 TIN family + + - 1,2,3 ISRE
IFN-g + + 1 GAS (TRF1>Lys6>IFP) 11-10 + ? ? 1,3 fro130 family 1L-6 (Pleiotropic) + + + ? 1,3 GAS(TRF1>Lys6>IFP) 11-1 1(Pleiotropic) ? + ? ? 1,3 OnM(Pleiotropic) ? + + ? 1,3 L1F(Pleiotropic) ? + + ? 1,3 CNTF(Pleiotropic) 4+ + ? 1,3 G-CSF(Pleiotropic) ? + ? ? 1,3 IL-12(Pleiotropic) + + + 1,3 g-C family IL-2 (lymphocytes) + - + 1,3,5 GAS
11,4 (lymph/myeloid) + + 6GAS(IRF1=1FP Ly6)(IgH) IL-7 (lymphocytes) + - + 5 GAS
IL-9 (lymphocytes) + - + 5 GAS
1L-13 (lymphocyte) + ? ? 6 GAS
LL-15 ? + ? + 5 GAS
go140 family IL-3 (myeloid) + - 5 GAS(TEF1>IFP Ly6) = II-5 (myeloid) + - 5 GAS
GM-CSE (myeloid) - + - 5 GAS
Growth hormone family GH ? + - 5 PRL ? + - 1,3,5 EPO ? + 5 GAS
(B-CAS>IRF1=IFP Ly6) Receptor Tyrosine Kinases EGF ? + + - 1,3 GAS (IRM) PDGF ? + + - 1,3 CSF4 ? + + 1,3 GAS(not 1RF1) 114641 To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 78-80, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5' primer contains four tandem copies of the GAS binding site found in the IRF 1 promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al, Immunity 1:457-468 (1994).), although other GAS or ISRE elements can be used instead. The 5' primer also contains 18bp of sequence complementary to the SV40 early promoter sequence and is flanked with an Xhol site. The sequence of the 5' primer is:
5' :GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAA
TGATTTCCCCGAAATATCTGCCATCTCAATTAG:3' (SEQ ID NO: 1114) [14651 The downstream primer is complementary to the SV40 promoter and is flanked with a Hind Ill site: 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID
NO: 1115) - [1466) PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clonteeh. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence:
5':CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATT
TCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACT
CCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCr GACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTC
CAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGGIT:3' (SEQ ID NO:1116) (1467) With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or "SEAP." Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well !mown reporter molecules that can be used instead of SEAP include chloramphenicol aeetyltransferase (CAT), lueiferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.
[1468] The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIll and Xhol, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
[14691 Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using Sall and Notl, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 78-80.
[14701 Other constructs can be made using the above description and replacing GAS
with a different promoter sequence. For example, construction of reporter molecules containing EGR and NF-KB promoter sequences are described in Examples 78-82.
However, many other promoters can be substituted using the protocols described in these ;
Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, II-2/NFAT, or NF-KB/GAS).
Similarly, other cell lines can be used to test reporter construct activity, such as HELA
(epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC
(aortic), or Cardiornyocyte.
Example 76: Assay for SEA.P Activity.
[14711 As a reporter molecule for the assays described in examples disclosed herein, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.
[1472] Prime a dispenser with the 2.5x Dilution Buffer and dispense 15 ul of 2.5x dilution buffer into Optiplates containing 35 ul of a solution containing an albumin fusion protein of the invention. Seal the plates with a plastic sealer and incubate at 65 degree C for 30 min. Separate the Optiplates to avoid uneven heating.
[1473] Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature min. Empty the dispenser and prime with the Reaction Buffer (see the Table below). Add 50 ul Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminesecnt signal is time dependent, and it takes about 10 minutes to read 5 plates on a luminometer, thus one should treat 5 plates at each time and start the second set minutes later.

114741 Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.
Table 6 # of plates itxn buffer CSPD (ml) - # of plates Rxn buffer CSPD (m1) diluent (m1) diluent (m1) 60 3 31 165 8.2$
11 65 3.25 32 170 8.5 ...... ___________ _ 12 70 3.5 33 175 835 13 75 3.75 34 180 9

14 80 4 185 9.25 UM " 4.25 36 190 9.5 _______________________ . NM 9'75 17 95 4.75 38 200 10 =
18 100 5 39 205 10.25 19 105 5.25 40 210 10.5 110 5.5 41 215 10.75 21 115 _______ EOM 42 220 11 22 120 6 43 IMINII 11.25 23 125 6.25 44 230 11.5 , c., _______________________________________________________________ 24 ,130 6.5 45 235 11.75 135 6.75 46 240 12 =
26 140 7 47 245 12.25 27 145 7.25 48 250 12.5 28 150 7.5- 49 255 12.75 29 155 7.75 50 260 13 ¨ -, ______________________________ Example 77: Assay Identifying Neuronal Activity. .
[14751 When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation.

The promoter of EGR1 is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, the ability of fusion proteins of the invention to activate cells can be assessed.
114761 Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGRI gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells by an albumin fusion protein of the present invention can be assessed.
[14771 The EGR/SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (-633 to 1)(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers:
First primer: 5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3' (SEQ
NO: 1117) Second primer: 5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3' (SEQ ID
NO: 1118) 114781 Using the GAS:SEAP/Neo vector produced in Example 75, EGRI
amplified product can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes Xliol/Hind111, removing the GAS/SV40 stuffer. Restrict the amplified product with these same enzymes. Ligate the vector and the EGRI
promoter.
114791 To prepare 96 well-plates for cell culture, two rills of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr.
[14801 PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (11W BIOSC1ENCES, Cat. # 12449-7811), 5% heat-inactivated fetal bovine serum (PBS) supplemented with 100 units/ml penicillin and 100 ug/m1 streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.
11481] Transfect the EGR/SEAP/Neo construct into PCI2 using techniques known in the art. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 ug/m1 G418.

The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 ug/ml 6418 for couple of passages.
[14821 To assay for neuronal activity, a 10 cm plate with cells around 70 to 80%
confluent is screened by removing the old medium. Wash the cells once with PBS

(Phosphate buffered saline). Then starve the cells in low serum medium (RPM1-containing 1% horse serum and 0.5% PBS with antibiotics) overnight.
114831 The next morning, remove the medium and wash the cells with PBS.
Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium.
Count the cell number and add more low serum medium to reach final cell density as 5x105 cells/ml.
[1484] Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to 1 x105 cells/well). Add a series of different concentrations of an albumin fusion protein of the inventon, 37 degree C for 48 to 72 hr. As a positive control, a growth factor known to activate PC12 cells through EGR can be used, such as 50 ng/u1 of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells.
SEAP assay may be routinely performed using techniques blown in the art and/or as described in Example 76.
Example 78: Assay for T-eell Activity.
[14851 The following protocol is used to assess T-cell activity by identifying factors, and determining whether an albumin fusion protein of the invention proliferates and/or differentiates T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 75. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. 1.18-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.
[1486] Jurkat T-cells are lymphoblastic CD4+ Thl helper cells. In order to generate stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure described below). The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml genticin selected. Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma.
The dose response of a selected clone is demonstrated.

[14871 Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 ul of cells. Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI + 10% serum with 1%Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C
and incubate at room temperature for 15-45 mins.
114881 During the incubation period, count cell concentration, spin down the required number of cells (107 per transfection), and resuspend in OPTI-MEM to a final concentration of 107 cells/ml. Then add lml of I x 107 cells in OPTI-MEM to T25 flask and incubate at 37 degree C for 6 hrs. After the incubation, add 10 ml of RPMI + 15% serum.
[14891 The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI +
10%
serum, I mg/ml Genticin, and 1% Pen-Strep. These cells are treated with varying concentrations of one or more fusion proteins of the present invention.
[14901 On the day of treatment with the fusion protein, the cells should be washed and resuspended in fresh RPM' + 10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the number of fusion proteins and the number of different concentrations of fusion proteins being screened. For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required.
[14911 The well dishes containing Jurkat cells treated with the fusion protein are placed in an incubator for 48 Firs (note: this time is variable between 48-72 hrs). 35 ul samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at -20 degree C until SEAP assays are performed according to Example 76. The plates containing the remaining treated cells are placed at 4 degree C and serve as a source of material for repeating the assay on a specific well if desired.
[1492] As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.
[14931 The above protocol may be used in the generation of both transient, as well as, stable transfeeted cells, which would be apparent to those of skill in the art.
Example 79: Assay for T-cell Activity.
[1494] NY-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-I and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB
regulates the expression of genes involved in immune cell activation, control of apoptosis (NF- KB
appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses.
[14951 In non-stimulated conditions, NF- KB is retained in the cytoplasm with [-KB
(Inhibitor KB). However, upon stimulation, I- KB is phosphorylated and degraded, causing NF- KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF- KB include IL-2, IL-6, GM-CSF, ICAM-1 and class l MEC.
[14961 Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the fusion protein.
Activators or inhibitors of NF-KB would be useful in treating, preventing, and/or diagnosing diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis.
114971 To construct a vector containing the NF-KB promoter element, a PCR
based strategy is employed. The upstream primer contains four tandem copies of the NF-KB
binding site (GGGGAC rn. ceC) (SEQ TO NO: 1119), 18 bp of sequence complementary to the 5' end of the SV40 early promoter sequence, and is flanked with an XhoI
site:
5':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGAC1TTCCGGGACTTTC
CATCCTGCCATCTCAATTAG:3' (SEQ ID NO: 1120) 114981 The downstream primer is complementary to the 3' end of the SV40 promoter and is flanked with a Hind III site:
5' :GCGGCAAGCTT __ ITTGCAAAGCCTAGGC:3' (SEQ ID NO: I I I 5) 114991 PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with Xhol and Hind III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence:
5' :CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTG
CCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCC
CTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTAT
TTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGG
AGGCTTITTIGGAGGCCTAGGCTTTTGCAAAAAGCTT:3' (SEQ ID NO: 1121) 115001 Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-I<B/SV40 fragment using XhoI and Hind111.
However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
[1501] In order to generate stable mammalian cell lines, the NF-cassette is removed from the above NF-KB/SEAP vector using restriction enzymes Sall and Notl, and inserted into a vector containing neomycin resistance. Particularly, the NF-ICBISV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP
gene, after restricting pGFP-1 with Sall and NotI.
[1502] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 76.
Similarly, the method for assaying fusion proteins with these stable Jurkat T-cells is also described in Example 76. As a positive control, exogenous TNF alpha (0.1,1, 10 ng) is added to wells H9, 1110, and Hi I., with a 5-10 fold activation typically observed.
Example 80: Assay Identifying Myeloid Activity.
115031 = The following protocol is used to assess myeloid activity of an albumin fusion protein of the present invention by determining whether the fusion protein proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 75. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, 11L60, or KG1 can be used.
[1504] To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in Example 75, a DEAE-Dextran method (Itharbanda et. al., 1994, Cell Growth &
Differentiation, 5:259-265) is used. First, harvest 2x107 U937 cells and wash with PBS. The U937 cells are usually grown in RPM' 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/m1 penicillin and 100 mg/m1 streptomycin.
[1505] Next, suspend the cells in 1 ml of 20 mM Tris-HC1 (pH TA) buffer containing 0.5 mg/m1 DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCI, 5 m/v1 KC1, uM Na211PO4.7H20, 1 mM MgC12, and 675 uM CaCl2. Incubate at 37 degrees C for min.
[1506] Wash the cells with RPM! 1640 medium containing 10% FRS and then resuspend in 10 ml complete medium and incubate at 37 degree C for 36 hr.
[15071 The GAS-SEAP/U937 stable cells are obtained by growing the cells in ug/ml G418. The 0418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 ug/ml 0418 for couple of passages.
[15081 These cells are tested by harvesting lx10 cells (this is enough for ten 96-well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5x105 cells/ml. Plate 200 ul cells per well in the 96-well plate (or lx105 cells/well).
[15091 Add different concentrations of the fusion protein. incubate at 37 degee C for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells. Over 30 fold induction is typically observed in the positive control wells. SEA? assay the supernatant according to methods known in the art and/or the protocol described in Example 76.
txample 81: Assay Identifying ChartLes in Small ytolecule Concentration and Membrane Permeability.
[15101 Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential.
These alterations can be measured in an assay to identify fusion proteins which bind to receptors of a particular cell Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.
[1511) The following assay uses FluorornetTie imaging Plate Reader ("PUPA") to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules.
Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.
[15121 For adherent cells, seed the cells at 10,000 -20,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO2 incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 ul of HBSS
(Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.
[15131 A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/m1 lino-4 is added to each well.
The plate is incubated at 37 degrees C in a CO2 incubator for 60 min. The plate is washed four times in the Biotek washer with HESS leaving 100 ul of buffer.
[15141 For non-adherent cells, the cells are spun down from culture media.
Cells are re-suspended to 2-5x106 cells/m1 with HBSS in a 50-ml conical tube. 4 ul of 1 mg/m1 fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degrees C water bath for 30-60 min. The cells are washed twice with HESS, resuspended to 1 x106 cells/ml, and dispensed into a microplate, 100 ul/well.
The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley Cell Wash with 200 ul, followed by an aspiration step to 100 ul final volume.
[15151 For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4 . The fusion protein of the invention is added to the well, and a change in fluorescence is detected 115161 To measure the fluorescence of intracellular calcium, the FL1PR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 second; (3) Camera Fistop is F/2; (4) Excitation is 488 mu; (5) Emission is 530 nm; and (6) Sample addition is 50 ul. Increased emission at 530 nrn indicates an extracellular signaling event caused by an albumin fusion protein of the present invention or a molecule ,induced by an albumin fusion protein of the present invention, which has resulted in an increase in the intracellular Ca ++ concentration.
Example 82: Assay Identifyin2 Tyrosine Kinase Activity.
[1517] The Protein Tyrosine Kinases (PTIO represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase (RPTK) group are receptors for a range of mitagenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.
[15181 Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated .frosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
[15191 Because of the wide range of known factors capable of stimulating tyrosine kinase activity, identifying whether an albumin fusion protein of the present invention or a molecule induced by a fusion proetin of the present invention is capable of activating tyrosine kinase signal transduction pathways is of interest. Therefore, the following protocol is designed to identify such molecules capable of activating the tyrosine kinase signal transduction pathways.
115201 Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, IL), The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/m1), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, MO) or 10% Matrigel .purchased from Becton Dickinson (Bedford,MA), or calf serum, rinsed with PBS and stored at 4 degree C.
Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, CA) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford,MA) are used to cover tile Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.
[15211 To prepare extracts, A431 cells are seeded onto the nylon membranes of Loprodyne plates (20,000/200ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60ng/m1) or a different concentrations of an albumin fusion protein of the invention, the medium was removed and 100 ml of extraction buffer ((20 mM
HEPES pH
7.5, 0.15 M NaC1, 1% Tritoirr X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P207 and a cocktail of protease inhibitors (# 1836170) obtained from Boeheringer Mannheim (Indianapolis, IN)) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 40C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum.
Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4 degree C at 16,000 x g.
115221 Test the filtered extracts for levels of tyrosine kinase activity.
Although many methods of detecting tyrosine kinase activity are known, one method is described here.
[15231 Generally, the tyrosine kinase activity of an albumin fusion protein of the invention is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include FSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and FSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim.
[15241 The tyrosine kinase reaction is set up by adding the following components in order. First, add lOul of 5uM Biotinylated Peptide, then 10u! ATP/Mg2+ (5mM
ATF/50mM
MgC12), then lOul of 5x Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-.
'glyeerophosphate, ImM EGTA, 100mM MgC12, 5 mM MnC12, 0.5 mg/m1 BSA), then Sul of Sodium Vanadate(lmM), and then 5u1 of water. Mix the components gently and preincubate the reaction mix at 30 degree C for 2 min. Initial the reactiOn by adding lOul of the control enzyme or the filtered supernatant. =
[15251 The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120rnin EDTA and place the reactions on ice.
115261 Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degree C for 20 min. This allows the strepta.vidin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300u1/well of PBS four times. Next add 75 ul of anti-phospotyrosine antibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5u/m1)) to each well and incubate at 37 degree C for one hour. Wash the well as above.
[15271 Next add 100u1 of peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 rim by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.

Example 83: Assay Identifying Phosphorylation Activity.
[1528] As a potential alternative and/or complement to the assay of protein tyrosine kinase activity described in Example 82, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, INK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tee, and Janus, as well as any other phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assay.
[15291 Specifically, assay plates are made by coating the wells of a 96-well ELISA
plate with 0.1m1 of protein G (lug/ml) for 2 hr at room temp, (RD. The plates are then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein Opiates are then treated with 2 commercial monoclonal antibodies (100w/well) against Eric-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules.) After 3-5 rinses with PBS, the plates are stored at 4 degree C
until use.
[1530] A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6w/well) or varying concentrations of the fusion protein of the invention for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.
(1531) After incubation with the extract for 1 hr at RT, the wells are again rinsed. As a positive control, a commercial preparation of MAP kinase (lOng/well) is used in place of A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody (lug/m1) which specifically recognizes the phosphorylated epitope of the Eric-I and Erk-2 kinases (1 hr at WI). This antibody is biotinylaied by standard procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Wall= DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation by the fusion protein of the present invention or a molecule induced by an albumin fusion protein of the present invention.

Example 84: Assay for the Stimulation of Bone Marrow CD34+ Cell Proliferation.

[15321 This assay is based on the ability of human CD34+ to proliferate in the presence of hematopoietic growth factors and evaluates the ability of fusion proteins of the inventon to stimulate proliferation of CD34+ cells.
115331 It has been previously shown that most mature precursors will respond to only a single signal. More immature precursors require at least two signals to respond. Therefore, to test the effect of fusion proteins of the invention on hematopoietic activity of a wide range of progenitor cells, the assay contains a given fusion protein of the invention in the presence or absence of hematopoietic growth factors. Isolated cells are cultured for 5 days in the presence of Stem Cell Factor (SCF) in combination with tested sample. SCF
alone has a very limited effect on the proliferation of bone marrow (BM) cells, acting in such conditions only as a "survival" factor. However, combined with any factor exhibiting stimulatory effect on these cells (e.g., IL-3), SCF will cause a synergistic effect. Therefore, if the tested fusion protein has a stimulatory effect on hematopoietic progenitors, such activity can be easily detected. Since normal BM cells have a low level of cycling cells, it is likely that any inhibitory effect .of a given fusion protein might not be detected.
Accordingly, assays for an inhibitory effect on progenitors is preferably tested in cells that are first subjected to in vitro stimulation with SCF+M+3, and then contacted with the compound that is being evaluated for inhibition of such induced proliferation.
[1534] Briefly, CD34+ cells are isolated using methods known in the art.
The cells are thawed and resuspended in medium (QBSF 60 serum-free medium with 1% L-glutarnine (500m1) Quality Biological, Inc., Gaithersburg, MD Catu 160-204-101). After several gentle centrifugation steps at 200 x g, cells are allowed to rest for one hour. The cell count is adjusted to 2.5 x 105 cells/ml. During this time, 100 ul of sterile water is added to the peripheral wells of a 96-well plate. The cytokines that can be tested with an albumin fusion protein of the invention in this assay is rhSCF (R&D Systems, Minneapolis, MN, Cat# 255-SC) at 50 ng/ml alone and in combination with rhSCF and rhIL-3 (R&D Systems, Minneapolis, MN, Cat# 203-ML) at 30 ng/ml. After one hour, 10 ).1.1 of prepared cytokines, varying concentrations of an albumin fusion protein of the invention, and 20 p.1 of diluted cells are added to the media which is already present in the wells to allow for a final total volume of 100 pl. The plates are then placed in a 37 C/5% CO2 incubator for five days.
115351 Eighteen hours before the assay is harvested, 0.5 pCi/well of [311]
Thymidine is added in a 10 tl volume to each well to determine the proliferation rate.
The experiment is terminated by harvesting the cells from each 96-well plate to a filtermat using the Tomtec Harvester 96. After harvesting, the filtermats are dried, trimmed and placed into OmniFilter assemblies consisting of one OmniFilter plate and one OmniFilter Tray. 60 ill Microscint is added to each well and the plate sealed with TopSeal-A press-on sealing film A
bar code 15 sticker is affixed to the first plate for counting. The sealed plates are then loaded and the level of radioactivity determined via the Packard Top Count and the printed data collected for analysis. The level of radioactivity reflects the amount of cell proliferation.
11536] The studies described in this example test the activity of a given fusion protein to stimulate bone marrow CD34+ cell proliferation, One skilled in the art could easily modify the exemplified studies to test the activity of fusion porteins and polynucleotides of the invention (e.g., gene therapy) as well as agonists and antagonists thereof. The ability of an albumin fusion protein of the invention to stimulate the proliferation of bone marrow CD34+ cells indicates that the albumin fusion protein and/or polynucleotides corresponding to the fusion protein are useful for the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the "Immune Activity" and "Infectious Disease" sections above, and elsewhere herein.
Example 85: 4ssav for Extracellular Matrix Enhanced Cell Response (MEM).
[15371 The objective of the Extracellular Matrix Enhanced Cell Response (EMECR) assay is to evaluate the ability of fusion proteins of the invention to act on hematopoietic stem cells in the context of the extracellular matrix (ECM) induced signal.
[15381 Cells respond to the regulatory factors in the context of signal(s) received from the surrounding microenvironment. For example, fibroblasts, and endothelial and epithelial stem cells fail to replicate in the absence of signals from the ECM.
Hematopoietic stem cells can undergo self-renewal in the bone marrow, but not in in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro is dependent upon their interaction with the stromal cells and the ECM protein fibronectin (fn). Adhesion of cells to fa is mediated by the cc5.Po and a4.131 integrin receptors, which are expressed by human and mouse hematopoietic stem cells. The factor(s) which integrate with the ECM
environment and are responsible for stimulating stem cell self-renewal havea not yet been identified. Discovery of such factors should be of great interest in gene therapy and bone marrow transplant applications [15391 Briefly, polystyrene, non tissue culture treated, 96-well plates are coated with fri fragnkcnt at a coating concentration of 02 ug,/ cm2. Mouse bone marrow cells are plated (1,000 cells/well ) in 0.2 ml of serum-free medium. Cells cultured in the presence of IL-3 ( 5 rig/m1 ) + SCF ( 50 ng/ml ) would serve as the positive control, conditions under which little self-renewal but pronounced differentiation of the stem cells is to be expected. Albumin fusion proteins of the invention are tested with appropriate negative controls in the presence and absence of SCF(5.0 ng/ml), where volume of the adrninisted composition containing the albumin fusion protein of the invention represents 10% of the total assay volume. The plated cells are then allowed to grow by incubating in a low oxygen environment ( 5%
CO2, 7% 02, and 88% N2 ) tissue culture incubator for 7 days. The number of proliferating cells within the wells is then quantitated by measuring thyrnidine incorporation into cellular DNA.
Verification of the positive hits in the assay will require phenotypic characterization of the cells, which can be accomplished by scaling up of the culture system and using appropriate antibody reagents against cell surface antigens and FACSean.
[1540] If a particular fusion protein of the present invention is found to be a stimulator of hematopoietic progenitors, the fusion protein and polynucleotides corresponding to the fusion protein may be useful for example, in the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the "Immune Activity" and "Infectious Disease" sections above, and elsewhere herein. The fusion protein may also be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
[15411 Additionally, the albumin fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention, may also be employed to inhibit the proliferation and differentiation of hematopoietic cells and therefore may be employed to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy. This antiproliferative effect may allow administration of higher doses of chemotherapeutic agents and, therefore, more effective chemotherapeutic treatment.
[15421 Moreover, fusion proteins of the invention and polynucleatides encoding albumin fusion proteins of the invention may also be useful for the treatment and diagnosis of hematopoietic related disorders such as, anemia, pancytopenia, leukopenia, thrombocytopenia or ieukemia, since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
Example 86: Human Dermal Fibroblast and Aortic Smooth Muscle Cell Proliferation.
115431 An albumin fusion protein of the invention is added to cultures of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC) and two co-assays are performed with each sample. The first assay examines the effect of the fusion protein on the proliferation of normal human dermal fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts or smooth muscle cells is a part of several pathological processes, including fibrosis, and restenosis. The second assay examines 1L6 production by both NHDF and SMC. 1L6 production is an indication of functional activation. Activated cells will have increased production of a number of cytokines and other factors, which can result in a proinflanunatory or immunomodulatory outcome.
Assays are run with and without co-'rNFa stimulation, in order to check for costimulatory or inhibitory activity. .
115441 Briefly, on day 1, 96-well black plates are set up with 1000 cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 ill culture media. NHDF culture media contains:
Clonetics FB basal media, lmgirril hFGF, 5mg/m1 insulin, 50mg/m1 gentamycin, 2%FBS, while AoSMC culture. media contains Clonetics SM basal media, 0.5 pg/mIlEGF, 5mg/m1 insulin, 1 tig/m1 hFGF, 50ing/m1 gentamycin, 50 oeml Amphotericin B, 5%FBS.
After incubation at 37 C for at least 4-5 hours culture media is aspirated and replaced with growth arrest media. Growth arrest media for NHDF contains fibroblast basal media, 50Ing/m1 gentamycin, 2% FBS, while growth arrest media for AoSMC contains SM basal media, 50mghnl gentarnycin, 50ng/m1 Amphotericin B, 0.4% FBS. Incubate at 37 C until day 2.
[15451 On day 2, serial dilutions and templates of an albumin fusion protein of the invention are designed such that they always include media controls and known-protein controls. For both stimulation and inhibition experiments, proteins are diluted in growth arrest media. For inhibition experiments, TNFa is added to a final concentration of 2ng/m1 (NHDF) or 5ngitn1 (AoSMC). Add 1/3 vol media containing controls or an albumin fusion protein of the invention and incubate at 37 degrees C/5% CO2 until day 5.
[15461 Transfer 600 from each well to another labeled 96-well plate,, cover with a plate-sealer, and store at 4 degrees C until Day 6 (for IL6 EL1SA). To the remaining 100 1.t1 in the cell culture plate, aseptically add Alamar Blue in an amount equal to 10% of the culture olume (10 1). Return plates to incubator for 3 to 4 hours. Then measure fluorescence with excitation at 530nm and emission at 590nm using the CytoPluor. This yields the growth stimulation/inhibition data.
[15471 On day 5, the IL6 ELISA is performed by coating a 96 well plate with 50-100 W./well of Anti-Human 1L6 Monoclonal antibody diluted in PBS, pH 7.4, incubate ON at room temperature.
(1548) On day 6, empty the plates into the sink and blot on paper towels.
Prepare Assay Buffer containing PBS with 4% BSA. Block the plates with 200 ill/well of Pierce Super Block blocking buffer in PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05% Tweeir-20). Blot plates on paper towels. Then add 50 ul/well of diluted Anti-Human 1L-6 Monoclonal, Biotin-labeled antibody at 0.50 mg/ml. Make dilutions of 1L-6 stock in media (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samples to top row of plate.
Cover the plates and incubate for 2 hours at RT on shaker.
(1549) Plates are washed with wash buffer and blotted on paper towels.
Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100 p1/well. Cover the plate and incubate 1 h at RT. Plates are again washed with wash buffer and blotted on paper towels.
[1550] Add 100 Id/well of Enhancement Solution. Shake for 5 minutes. Read the plate on the Walla.c DELF1A Fluorometer. Readings from triplicate samples in each assay were tabulated and averaged.
[15511 A positive result in this assay suggests AoSMC cell proliferation and that the albumin fusion protein may be involved in dermal fibroblast proliferation and/or smooth muscle cell proliferation. A positive result also suggests many potential uses of the fusion protein and polynucleotides encoding the albumin fusion protein. For example, inflammation and immune responses, wound healing, and angiogenesis, as detailed throughout this specification. Particularly, fusion proteins may be used in wound healing and dermal regeneration, as well as the promotion of vasculogenesis, both of the blood vessels and lymphatics. The growth of vessels can be used in the treatment of, for example, cardiovascular diseases. Additionally, fusion proteins showing antagonistic activity in this assay may be useful in treating diseases, disorders, and/or conditions which involve angiogenesis by acting as an anti-vascular agent (e.g., anti-angiogenesis).
These diseases, disorders, and/or conditions are known in the art and/or are described herein, such as, for example, malignancies, solid tumors, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques;
ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye;
rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis;
vasculogenesis;
granulations; hypertrophic scars (keloids); nonunion fractures; scleroderrna;
trachoma;
vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals;
axteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma;
fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis. Moreover, albumin fusion proteins that act as antagonists in this assay may be useful in treating Anti-hyperproliferative diseases and/or anti-inflammatory known in the art and/or described herein.
Example 87: Cellular Adhesion Molecule (CAM Exu ion on Endothelial Cells, .
115521 The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion .molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expressioh on endothelial cells (EC).
The expressioria-these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.
[15531 Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells (HUVECs)) are grown in a standard 96 well plate to confluence, growth medium is removed from the cells and replaced with 100 al of 199 Medium (10% fetal bovine serum (PBS)).
Samples for testing (containing an albumin fusion protein of the invention) and positive or negative controls are added to the plate in triplicate (in 10 11.1 volumes).
Plates are then incubated at 37 C for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 ul of 0.1%
parafomialdehyde-PBS(with Ca++ and Mg-H-) is added to each well. Plates are held at 4 C for 30 min. Fixative is removed from the wells and wells are washed IX with PBS(+Ca,Mg) + 0.5% BSA
and drained. 10 pi of diluted primary antibody is added to the test and control wells. Anti-1CAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 p.g./nal (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37 C for 30 mm. in a humidified environment. Wells are washed three times with PBS(+Ca,Mg) + 0.5%
BSA.
20 ill of diluted ExtrAvidin-Alkaline Phosphatase (1:5,000 dilution, referred to herein as the working dilution) are added to each well and incubated at 37 C for 30 mm.
Wells are washed three times with PBS(4-Ca,Mg)+0.5% BSA. Dissolve I tablet of p-Nitrophenol Phosphate pNPP per 5 ml of glycine buffer (pH 10.4). 100 p.I of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (10 ) > 1040>10-1>10-1-s. 5 itl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 rig, 1.74 ng, 0.55 rig, 0.18 ng. 100 ul of pNNP reagent is then added to each of the standard wells. The plate is incubated at 37 C for 4h. A volume of 50 ul of 3M
NaOH is added to all wells. The plate is read on a plate reader at 405 nm using the background subtraction option on blank wells filled with glycine buffer only.
Additionally, the template is set up to indicate the concentration of AP-conjugate in each standard well [ 5.50 rig; 1.74 ng; 0.55 rig; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.
Example Alanrar Rine Endothelial Cells Proliferation AMY.
[1554] This assay may be used to quantitatively determine protein mediated inhibition of bFGF-induced proliferation of Bovine Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells (BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs).
This assay incorporates a fluorometric growth indicator based on detection of metabolic activity. A standard Alamar Blue Proliferation Assay is prepared in EGM-2MV
with 10 rig /ml of bFGF added as a source of endothelial cell stimulation. This assay may be used with a variety of endothelial cells with slight changes in growth medium and cell concentration.
Dilutions of protein batches to be tested are diluted as appropriate. Serum-free medium (GIBCO SFM) without bFGF is used as a non-stimulated control and Angiostatin or TSP-1 are included as a known inhibitory controls.
[1555] Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of 5000 to 2000 cells/well in a 96 well plate and placed at 37 degeesC overnight.
After the overnight incubation of the cells, the growth media is removed and replaced with GIBCO
EC-SFM. The cells are treated with the appropriate dilutions of an albumin fusion protein of the invention or control protein sample(s) (prepared in SFM ) in triplicate wells with additional bFGF to a concentration of 10 ngi ml. Once the cells have been treated with the samples, the plate(s) is/are placed back in the 37 C incubator for three days. After three days 10 ml of stock alarnar blue (Biosource Cat# DAL1100) is added to each well and the plate(s) is/are placed back in the 37 C incubator for four hours. The plate(s) are then read at 530run excitation and 590nm emission using the CytoFluor fluorescence reader.
Direct output is recorded in relative fluorescence units.
115561 Alamar blue is an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. As cells grow in culture, innate metabolic activity results in a chemical reduction of the immediate surrounding environment. Reduction related to growth causes the indicator to change from oxidized (non-fluorescent blue) fonn to reduced (fluorescent red) form (i.e., stimulated proliferation will produce a stronger signal and inhibited proliferation will produce a weaker signal and the total signal is proportional to the total number of cells as well as their metabolic activity). The background level of activity is observed with the starvation medium alone. This is compared to the output observed from the positive control samples (13FGF in growth medium) and protein dilutions.
Example 89: Detection of Inhibition of a Mixed tiyeaphoevte Reaction.
11557) This assay can be used to detect and evaluate inhibition of a Mixed Lymphocyte Reaction (MLR ) by fusion proteins of the invention. Inhibition of a MLR may be due to a direct effect on cell proliferation and viability, modulation of costimulatory molecules on interacting cells, modulation of adhesiveness between lymphocytes and accessory cells, or modulation of cytolcine production by accessory cells.
Multiple cells may be targeted by the albumin fusion proteins that inhibit MLR since the peripheral blood mononuclear fraction used in this assay includes T, B and natural killer lymphocytes, as well as rnonocytes and dendritic cells.
115581 Albumin fusion proteins of the invention found to inhibit the MLR
may find application in diseases associated with lymphocyte and monocyte activation or proliferation.
These include, but are not limited to, diseases such as asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus, multiple clerosis, glomerulonephritis, inflammatory bowel disease, crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease, host vs. graft disease, hepatitis, leukemia and lymphoma.
[15591 Briefly, PBMCs from human donors are purified by density gradient centrifugation using Lymphocyte Separation Medium (LSM , density 1.0770 g/ml, Organon Teknika Corporation, West Chester, PA). PBMCs from two donors are adjusted to 2 x 106 cells/m1 in RPMI-1640 (Life Technologies, Grand Island, NY) supplemented with 10% FCS
and 2 niM glutamine. PBMCs from a third donor is adjusted to 2 x 105 cells/ml.
Fifty microliters of PBMCs from each donor is added to wells of a 96-well round bottom microtiter plate. Dilutions of the fusion protein test material (50 pi) is added in triplicate to microtiter wells. Test samples (of the protein of interest) are added for final dilution of 1:4; rhulL-2 (R&D Systems, Minneapolis, MN, catalog number 202-EL) is added to a final concentration of 1 Wail; anti-CD4 rnAb (R&D Systems, clone 34930.11, catalog number MAB379) is added to a final concentration of 10 ug/ml. Cells are cultured for 7-8 days at 37 C in 5%
CO2, and 1 pC of [311] thymidine is added to wells for the last 16 hrs of culture. Cells are harvested and thymidine incorporation determined using a Packard TopCount:
Data is expressed as the mean and standard deviation of triplicate determinations.
[15601 Samples of the fusion protein of interest are screened in separate experiments and compared to the negative control treatment, anti-CD4 mAb, which inhibits proliferation of lymphocytes and the positive control treatment, ILA (either as recombinant material or supernatant), which enhances proliferation of lymphocytes.
Example 90: Assays for Protease Activity.
(1561] The following assay may be used to assess protease activity of an albumin fusion protein of the invention.
[15621 Gelatin and casein zymography are performed essentially as described (Heusen et at., Anal. Biochem., 102:196-202 (1980); Wilson et al, Journal of Urology, 149:653-658 (1993)). Samples are run on 10% polyacryamide/0.1% SDS gels containing 1%
gelain orcasein, soaked in 2.5% tritoirat room temperature for 1 hour, and in 0.1M glycine, pH 8.3 at 37 C 5 to 16 hours. After staining in amido black areas of proteolysis apear as clear areas agains the blue-black background. Trypsin (Sigma T8642) is used as a positive control.

,15631 Protease activity is also determined by monitoring the cleavage of n-a-benzoyl-L-arginine ethyl ester (BABE) (Sigma B-4500. Reactions are set up in (25mMNaPO4,1mM EDTA, and 1mM BABE), pH 7.5. Samples are added and the change in adsorbance at 260run is monitored on the Beckman DU-6 spectrophotometer in the time-drive mode. Trypsin is used as a positive control.
[1564] Additional assays based upon the release of acid-soluble peptides from casein or hemoglobin measured as adsorbance at 280 urn or colorimenically using the Folin method are performed as described in Bergmeyer, et al., Methods of Enzymatic Analysis, 5 (1984).
Other assays involve the solubilization of chromogenic substrates (Ward, Applied Science, 251-317 (1983)).
Example 91: Identifyine Serine Protease Substrate Specificity.
[1565] Methods known in the art or described herein may be used to determine the substrate specificity of the albumin fusion proteins of the present invention haying serine protease activity. A preferred method of determining substrate specificity is by the use of positional scanning synthetic combinatorial libraries as described in GB 2 324 529.
Example 92: Ligand BindinE Assays.
115661 The following assay may be used to assess ligand binding activity of an albumin fusion protein of the invention.
[1567] Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format The purified ligand for an albumin fusion protein of the invention is radiolabeled to high specific activity (50-2000 Cihrunol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards the fusion protein. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell polypeptide sources. For these assays, specific polypeptide binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.
Example 93: Functional Assay in Xenopus Oocytes.

[15681 Capped RNA transcripts from linearized plasmid templates encoding an albumin fusion protein of the invention is synthesized in vitro with RNA
polymerases in accordance with standard procedures. In vitro transcripts are suspended in water at a final concentration of 0.2 mg/mi. Ovarian lobes are removed from adult female toads, Stage V
defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocytc) are injected in a 50 ni bolus using a microinjeetion apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus ooeytes in response fusion protein and polypeptide agonist exposure. Recordings are made in Calf- free Barth's medium at room temperature. The Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands.
Example 94: Microphysiometric Assays.
[1569) Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell. The acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process. The pH
changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, Calif.). The CYTOSE'NSOR is thus capable of detecting the ability of an albumin fusion;
protein of the invention to activate secondary messengers that are coupled to an energy utilizing intracellular signaling pathway.
Example 95: Extract/Cell Supernatant Screening.
[15701 A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be included within the ligands banks as identified to date. Accordingly, the albumin fusion proteins of the invention can also be functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify natural ligands for the Therapeutic protein portion and/or albumin protein portion of an albumin fusion protein of the invention. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.
Example 96: ATP-binding assay., 15711 The following assay may be used to assess ATP-binding activity of fusion proteins of the invention.
[1572] ATP-binding activity of an albumin fusion protein of the invention may be detected using the ATP-binding assay described in U.S. Patent 5,858,719.
Briefly, ATP-binding to an albumin fusion protein of the invention is measured via photoaffinity labeling with 8-azido-ATP in a competition assay.
Reaction mixtures containing 1 mg/m1 of ABC transport protein are incubated with varying concentrations of ATP, or the non-hydrolyzable ATP analog adeny1-5'-imidodiphosphate for minutes at 4 C. A mixture of 8-azido-ATP (Sigma Chem. Corp., St. Louis, MO.) plus 8-azido-ATP (32P-ATP) (5 mCi/u.mol, ICN, Irvine CA.) is added to a final concentration of 100 NI and 0.5 ml aliquots are placed in the wells of a porcelain spot plate on ice. The plate is irradiated using a short wave 254 run UV lamp at a distance of 2.5 cm from the plate for Om one-minute intervals with a one-minute cooling interval in between. The reaction is stopped by addition of dithiothreitol to a final concentration of 2mM. The incubations are subjected to SDS-PAGE electrophoresis, dried, and autoradiographed. Protein bands corresponding to the albumin fusion proteins of the invention are excised, and the radioactivity quantified. A
decrease in radioactivity with increasing ATP or adenly-5'-imidodiphosphate provides a measure of ATP affinity to the fusion protein.
Example 97: Phosphorylation Assay.
[15731 In order to assay for phosphorylation activity of an albumin fusion protein of the invention, a phosphorylatinn assay as described in U.S. Patent 5,958,405 is utilized. Briefly, phosphorylation activity may be measured by phosphorylation of a protein substrate using gamma-labeled 32P-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter. The fusion portein of the invention is incubated with the protein substrate, 32P-ATP, and a kinase buffer. The 32P
incorporated into the substrate is then separated from free 32P-ATP by electrophoresis, and the incorporated 32P is counted and compared to a negative control.
Radioactivity counts above the negative control are indicative of phosphorylation activity of the fusion protein.
Exam I le 98:
Detection of Plies hor lotion Activi Activation of Albumin Fusion Protein of the Invention in the Presence of Polyneptide Ligands, ,15741 Methods known in the art or described herein may be used to determine the phosphorylation activity of an albumin fusion protein of the invention. A
preferred method of determining phosphorylation activity is by the use of the tyrosine phosphorylation assay as described in US 5,817,471.
Example 99: Identification Of Signal Minsduction_ Proteins That Interact With An albumin fusion protein Of The Present Invention.
(1575] Albumin fusion proteins of the invention may serve as research tools for the identification, characterization and purification of signal transduction pathway proteins or receptor proteins. Briefly, a labeled fusion protein of the invention is useful as a reagent for the purification of molecules with which it interacts. in one embodiment of affinity purification, an albumin fusion protein of the invention is covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as carcinoma tissues, is passed over the column, and molecules with appropriate affinity bind to the albumin fusion protein. The protein complex is recovered from the column, dissociated, and the recovered molecule subjected to N-tenninal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oliganucleofide probes for cloning the relevant gene from an appropriate eDNA
library.
Example 100: 1L-6 Bioassay.
115761 A variety of assays are known in the art for testing the proliferative effects of an albumin fusion protein of the invention. For example, one such asssay is the 1L-6 Bioassay as described by Marz et al. (Proc. Natl. Acad. Sci., U.S.A., 95:3251-56 (1998)).
After 68 hrs. at 37 C, the number of viable cells is measured by adding the tetrazolium salt thiazolyl blue 04rn and incubating for a further 4 hrs. at 37 C. B9 cells are lysed by SDS and optical density is measured at 570 nrn. Controls containing 11,6 (positive) and no cytokine (negative) are Briefly, 11,6 dependent 139 murine cells are washed three times in 1L-6 free medium and plated at a concentration of 5,000 cells per well in 50 p.1, and 50 pl of fusion protein of the invention is added.
utilized. Enhanced proliferation in the test sample(s) (containing an albumin fusion protein of the invention) relative to the negative control is indicative of proliferative effects mediated by the fusion protein.

xample 101: Support of Chicken Embryo Neuron Survival.
11577] To test whether sympathetic neuronal cell viability is supported by an albumin fusion protein of the invention, the chicken embryo neuronal survival assay of Senaldi et al may be utilized (Proc. Natl. Aced Sci., U.S.A., 96:11458-63 (1998)), Briefly, motor and sympathetic neurons are isolated from chicken embryos, resuspended in L15 medium (with 10% FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine, and insulin; Life Technologies, Rockville, MD.) and Dulbecco's modified Eagles medium [with 10% FCS, glutamine, penicillin, and 25 mM
Hepes buffer (pH 7.2); Life Technologies, Rockville, MD.], respectively, and incubated at 37 C in 5% CO2 in the presence of different concentrations of the purified fusion protein of the invention, as well as a negative control lacking any eytoldne. After 3 days, neuron survival is determined by evaluation of cellular morphology, and through the use of the colorimetric assay of Mosmann (Mosmann, T., .1. Immune!. Methods, 65:55-63 (1983)).
Enhanced neuronal cell viability as compared to the controls lacking cytoldne is indicative of the ability of the albumin fusion protein to enhance the survival of neuronal cells.
Example 102: Assay for Phosphatase Activity.
[1578) The following assay may be used to assess serine/threonine phosphate (PTPase) activity of an albumin fusion protein of the invention.
[1579] In order to assay for serine/threonine phosphatase (PTPase) activity, assays can be utilized which are widely known to those skilled inc the art. For example, the serine/threonine phosphatase (PSPase) activity of an albumin fusion protein of the invention may be measured using a PSPase assay kit from New England Biolabs, Inc. Myelin basic protein (MyBP), a substrate for PSPase, is phosphorylated on serine and threonine residues with cAMP-dependent Protein Kinase in the presence of [32P]ATP. Protein serine/threonine phosphatase activity is then determined by measuring the release of inorganic phosphate from 32P-labeled MyBP.
Example 103: Interaction of Serine/Threonine Phosphatases= with other proteinS, [15801 Fusion protein of the invention having serine/threonine phosphatase activity ( e.g., as determined in Example 102) are useful, for example, as research tools for the identification, characterization and purification of additional interacting proteins or receptor proteins, or other signal transduction pathway proteins. Briefly, a labeled fusion protein of the avention is useful as a reagent for the purification of molecules with which it interacts. In one embodiment of affinity purification, an albumin fusion protein of the invention is covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as neural or liver cells, is passed over the column, and molecules with appropriate affinity bind to the fusion protein. The fusion protein -complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
Example 104: Assavina. for Heparanase Activity.
[1581] There a numerous assays known in the art that may be employed to assay for heparanase activity of an albumin fusion protein of the invention. In one example, heparanase activity of an albumin fusion protein of the invention, is assayed as described by Vlodavsky et al., (Vlodavsky et at, Nat. Med., 5:793-802 (1999)). Briefly, cell lysates, conditioned media, intact cells (1 x 106 cells per 35-mm dish), cell culture supernatant, or purified fusion protein are incubated for 18 his at 37 C, pH 6.2-6.6, with 35S-labeled ECM or soluble ECM derived peak I proteoglycans. The incubation medium is centrifuged and the supernatant is analyzed by gel filtration on a SepharoseCL-6B column (0.9 x 30 cm).
Fractions are eluted with PBS and their radioactivity is measured. Degradation fragments of heparan sulfate side chains are eluted from Sepharosiem6B at 0.5 <K < 0.8 (peak II). Each experiment is done at least three times. Degradation fragments corresponding to "peak II," as described by Vlodavsky et at, is indicative of the activity of an albumin fusion protein of the invention in cleaving heparan sulfate.
Example 105: Immobilization of biomolecules.
115821 This example provides a method for the stabilization of an albumin fusion protein of the invention in non-host cell lipid hilayer constucts (see, e.g., Bieri et at, Nature Biotech 17:1105,4108 (1999), which can be adapted for the study of fusion proteins of the invention in the various functional assays described above. Briefly, carbohydrate-specific chemistry for biotinylation is used to confine a biotin tag to an albumin fusion protein of the invention, thus allowing uniform orientation upon immobilization. A 50uM solution of an albumin fusion protein of the =

invention in washed membranes is incubated with 20 mlvl NaI04 and 1.5 mg/m1 (4mM) BACH or 2 mg/ml (7.5mM) biotin-hydrazide for 1 hr at room temperature (reaction volume, 150u1). Then the sample is dialyzed (Pierce Slidealizer Cassett, 10 kDa cutoff; Pierce Chemical Co., Rockford IL) at 4C first for 5 h, exchanging the buffer after each hour, and finally for 12 h against 500 ml buffer R (0.15 M NaCI, 1 mM MgC12, 10 In.M
sodium phosphate, pH7). Just before addition into a cuvette, the sample is diluted 1:5 in buffer ROG50 (Buffer R supplemented with 50 mM octylglucoside).
Example 106: Assays for Metalloproteinase Activity.
[15831 Metalloproteinases are peptide hydrolases which use metal ions, such as Zn2+, as the catalytic mechanism. Metalloproteinase activity of an albumin fusion protein of the present invention can be assayed according to methods known in the art. The following exemplary methods are provided:
Proteolysis of alpha-2-macroglobulin [1584] To confirm protease activity, a purified fusion protein of the invention is mixed with the substrate alpha-2-macroglobulin (0.2 unit/m1; Boehringer Mannheim, Germany) in lx assay buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl,- 10 mM CaC12, 25 M
ZnC12 and 0.05% Brij-35) and incubated at 37 C for 1-5 days. Trypsin is used as positive control. Negative controls contain only. alpha-2-macroglobulin in assay buffer. The samples are collected and boiled in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol for 5-min, then loaded onto 8% SDS-polyacrylamide gel. After electrophoresis the proteins are visualized by silver staining. Proteolysis is evident by the appearance of lower molecular weight bands as compared to the negative control.
Inhibition of alpha-2-macroglobulin proteolysis by inhibitors of nzetalloproteinases [1585] Known metalloproteinase inhibitors (metal chelators (EDTA, EGTA, AND
HgC12), peptide metalloproteinase inhibitors (TB4P-1 and TEMP-2), and commercial small molecule MMP inhibitors) may also be used to characterize the proteolytic activity of an albumin fusion protein of the invention. Three synthetic MMP inhibitors that may be used are: WAY inhibitor I, [IC50 = 1.0 1AM against MMP-1 and MMP-8; IC50= 30 pM
against MMP-9; IC50 = 150 pNI against MNIP-3}; MMP-3 (stromelysin-1) inhibitor I [IC50 = 5 NI
against MIVIP-31, and MMP-3 inhibitor II [Ki = 130 n1\4 against MivfP-31;
inhibitors available through Calhiochem, catalog It 444250, 444218, and 444225, respectively).
Briefly, different concentrations of the small molecule MMP inhibitors are mixed with a purified fusion protein of the invention (50pg/m1) in 22.9 p1 of lx HEPES buffer (50 inM HEPES, pH
7.5, 0.2 M
NaC1, 10 m11/1 CaC12, 25 KM ZuC12 and 0.05%Brij-35) and incubated at room temperature (24 C) for 2-hr, then 7.1 n1 of substrate alpha-2-macroglobulin (0.2 unit/m1) is added and incubated at 37 C for 20-hr. The reactions are stopped by adding 4x sample buffer and boiled immediately for 5 minutes. After SDS-PAGE, the protein bands are visualized by silver stain.
Synthetic Fluorogenic Peptide Substrates Cleavage Assay [15861 The substrate specificity for fusion proteins of the invention with demonstrated metalloproteinase activity may be determined using techniques lmonw in the art, such as using synthetic fluorogenic peptide substrates (purchased from BACHEM
Bioscience Inc). Test substrates include, M-1985, M-2225, M-2105, M-2110, and M-2255.
The first four are MMP substrates and the last one is a substrate of tumor necrosis factor-a (TNF-a) converting enzyme (TACE). These substrastes are preferably prepared in 1;1 dimethyl sulfoxide (DMSO) and water. The stock solutions are 50-500 M.
Fluorescent asSays are performed by using a Perkin Elmer LS 50B luminescence spectrometer equipped with a constant temperature water bath. The excitation is 328 urn and the emission A. is 393 mn. Briefly, the assay is carried out by incubating 176 pl lx HEPES buffer (0.2 M NaCI, 10 niM CaC12, 0.05% Brij-35 and 50 niM HEPES, pH 7.5) with 4 tal of substrate solution (50 pM) at 25 C for 15 minutes, and then adding 20 pl of a purified fusion protein of the invention into the assay cuvett. The final concentration of substrate is 1 n.M. Initial hydrolysis rates are monitored for 30-min, Example 107-Identification and Cloning of V11 and VI, domains. , [15871 One method to identfy and clone VH and VL domains from cell lines expressing a particular antibody is to perform PCR with VH and VL specific primers on cDNA made from the antibody expressing cell lines. Briefly, RNA is isolated from the cell lines and used as a template for ].T-PCR designed to amplify the VH and VL
domains of the antibodies expressed by the EBV cell lines. Cells may be lysed in the TRIzol reagent (Life Technologies, Rockville. MD) and extracted with one fifth volume of chloroform. After addition of chloroform, the solution is allowed to incubate at room temperature for 10 minutes, and the centrifuged at 14,000 rpm for 15 minutes at 4 C in a tabletop centrifuge.

The supernatant is collected and RNA is precipitated using an equal volume of isopropanol.
Precipitated RNA is pelleted by centrifuging at 14,000 rpm for 15 minutes at 4 C in a tabletop centrifuge. Following centrifugation, the supernatant is discarded and washed with 75% ethanol. Follwing washing, the RNA is centrifuged again at 800 rpm for 5 minutes at 4 C. The sup6Tiatant is discarded and the pellet allowed to air dry. RNA is the dissolved in DEPC water and heated to 60 C for 10 minutes. Quantities of RNA can determined using optical density measurements.
cDNA may be synthesized, according to methods well-known in the art, from 1.5-2.5 micrograms of RNA using reverse transciptase and random hexarner primers. cDNA
is then used as a template for PCR amplification of VII and VL domains. Primers used to amplify VH and VL genes are shown in Table 7. Typically a PCR reaction makes use of a single 5' primer and a single 3' primer. Sometimes, when the amount of available RNA
template is limiting, or for greater efficiency, groups of 5' and/or 3' primers may be used. For example, sometimes all five VH-5' primers and all JH3' primers are used in a single PCR
reaction.
The PCR reaction is carried out in a 50 microliter volume containing IX PCR
buffer, 2mM of each dNTP, 0.7 units of High Fidelity Taq polymerse, 5' primer mix, 3' primer mix and 7.5 microliters of cDNA. The 5' and 3' primer mix of both VII and VL can be made by pooling together 22 pmole and 28 pmole, respectively, of each of the individual primers. PCR
conditions are: 96 C for 5 minutes; followed by 25 cycles of 94 C for 1 minute, 50 C for I
minute, and 72 C for I minute; followed by an extension cycle of 72 C for 10 minutes. After the reaction is completed, sample tubes are stored 4 C.

Table 7: Primer Sequences Used to Amplify VII and VL domains Primer name SEQ 1D NO Primer Sequence (5'-3') VH Primers Hu VH1-5 1056 CAGGTGCAGCTGGTGCAGTCTGG
flu VH2-5' 1057 CAGGTCAACTTAAGGGAGTCTGG
Hu VI13-5' 1058 GAGGTGCAGCTGGTGGAGTCTGG
Hu VH4-5' 1059 CAGGTGCAGCTGCAGGAGTCGGG
Hu VH5-5' 1060 GAGGTGCAGCTGTTGCAGTCTGC
Ha VH6-5' 1061 CAGGTACAGCTGCAGCAGTCAGG
Hu J111,2-5' 1062 TGAGGAGACGGTGACCAGGGTGCC
Ha J113-5' 1063 TGAAGAGACGGTGACCATTGTCCC
Ha Y114,5-5' 1064 TGAGGAGACGGTGACCAGGGTTCC
Hu .1116-5' 1065 TGAGGAGACGGTGACCGTGGTCCC
VL Primers Hu Vkappa1-5' 1066 GACATCCAGATGACCCAGTCTCC
Ha Vkappa2a-5' 1067 GATGTTGTGATGACTCAGTCTCC
Hu Vkappa2b-5' 1068 GATATTGTGATGACTCAGTCTCC
Hu VIcappa3-5' 1069 GAAATTGTGTTGACGCAGTCTCC
Hu Vkappa4-5' 1070 GACATCGTGATGACCCAGTCTCC
Hu Vkappa5-5' 1071 GAAACGACACTCACGCAGTCTCC
Hu Vkappa6-5' 1072 GAAATTGTGCTGACTCAGTCTCC
Hu Vlambdal -5' 1073 CAGTCTGTGTTGACGCAGCCGCC
Hu Vlambda2-5' 1074 CAGTCTGCCCTGACTCAGCCTGC
Ha Vlambda3-5' 1075 TCCTATGTGCTGACTCAGCCACC
Hu Vlambda3b-5' 1076 TCTTCTGAGCTGACTCAGGACCC
Hu Vlarribda4-5' 1077 CACGTTATACTGACTCAACCGCC
Hu Vlambda5-5' 1078 CAGGCTGTGCTCACTCAGCCGTC
Ha Vlambda6-5' 1079 AATTTTATGCTGACTCAGCCCCA
Hu Rappa1-3' 1080 ACGTTTGATTTCCACCTTGGTCCC
Hu Jkappa2-3' 1081 ACGTTTGATCTCCAGCTTGGTCCC
Hu Jkappa3-3' 1082 ACGTTTGATATCCACTTTGGTCCC
Hu jkappa4-3' 1083 ACGTTTGATCTCCACCTTGGTCCC
Hu Jkappa5-3' 1084 ACGTTTAATCTCCAGTCGTGTCCC
Ha Jlambda1-3' 1085 CAGTCTGTGTTGACGCAGCCGCC
Hu Jlambda2-3' 1086 CAGTCTGCCCTGACTCAGCCTGC
Hu Jlambda3--3' 1087 TCCTATGTGCTGACTCAGCCACC
Hu Jlambda3b-3 1088 TCTTCTGAGCTGACTCAGGACCC
Hu Jlambda4-3' 1089 CACG'FTATACTGACTCAACCGCC
Hu Jlambda5-3' 1090 CAGGCTGTGCTCACTCAGCCGTC
Hu Jlambda6-3' 1091 AATT1TATGCTGACTCAGCCCCA

PCR samples are then electrophoresed on a 1.3% agarose gel. DNA bands of the expected sizes (-506 base pairs for VII domains, and 344 base pairs for VL domains) can be cut out of the gel and purified using methods well known in the art. Purified PCR
products can be ligated into a PCR cloning vector (TA vector from Invitrogen Inc., Carlsbad, CA). Individual cloned PCR products can be isolated after transfection of E. coli and blue/white color selection. Cloned PCR products may then be sequenced using methods commonly known in the art.
115881 The PCR bands containing the VH domain and the VL domains Can also be used to create full-length 1g expression vectors. VII and VL domains can be cloned into vectors containing the nucleotide sequences of a heavy (e.g., human IgG1 or human IgG4) or light chain (human kappa or human lambda) constant regions such that a complete heavy or light chain molecule could be expressed from these vectors when transfected into an appropriate host cell. Further, when cloned heavy and light chains are both expressed in one cell line (from either one or two vectors), they can assemble into a complete functional antibody molecule that is secreted into the cell culture medium. Methods using polynucleotides encoding VH and 'VL antibody domain to generate expression vectors that encode complete antibody molecules are well known within the art.
EXAMPLE 108: Construct ID 2672, HSA-T20, Generation.
[1589] Construct ID 2672 (SEQ ID NO:1186), pSAC35:HSA.T20, comprises DNA
encoding a T20 albumin fusion protein which has full length HSA fused to the amino-terminus of the HIV-1 inhibitory peptide T20, i.e., Y643-F678, in the yeast S.
cerevisiae expression vector pSAC35. The T20 peptide is derived from the ectodomain of the HIV-1 transmembrane protein gp41 and is shown to have inhibitory activity on HIV-1 infection.
Cloning of T20 cDNA
115901 The polynucleotide encoding T20 was PCR generated using four overlapping primers T20-1, T20-2, T20-3, and T20-4, described below. The sequence was codon optimized for expression in yeast S. cerevisiae. The PCR fragment was cut with Bsu 3611.4sc L and ligated into Bsu 3611Ase I cut pScNHSA. A Not I fragment was then subcloned into the pSAC35 plasmid. Construct ID #2672 encodes an albumin fusion protein containing fall length HSA and the HIV-1 inhibitory peptide 120, i.e., Tyr-643 to Phe-678 (SEQ
ID
NO:1188).

[15911 The 5' and 3' primers of the four overlapping oligonucleotides suitable for PCR amplification of the polynucleoticle encoding the HIV-1 inhibitory peptide T20, T20-1 and T20-4, were synthesized:
T204: 5'-AAGCTGCCITAGGCTTATACACTAGITTGATTCATAGTTTG-3' (SEQ ID NO:1189) T20-2: Y-TACACTAGITTGATTCATAGITTGATTGAAGAAAGICAAAATCAACAAGAAAA
GAATGAACAAG-3' (SEQ ID NO:1204) T20-3: 5'-AAACCAATTCCACAAACTAGCCCATTTATCCAATTCCAACAATTCTTGTTCATT
CT __ ITTCTTGTTGAT-3' (SEQ Me NO:1205) T20-4: 5*-1TG6CGCGCCTTAAAACCAATTCCACAAACTAGCCCATITATCC-31 (SEQ ID NO:1190) [15921 T20-1 incorporates the Bsu 361 cloning site (shown underlined) and nucleotides encoding the last four amino acid residues of the mature form of HSA (SEQ ID
NO:1038), as well as 24 nucleotides (shown in bold) encoding the first 8 amino acid residues of the HIV-1 inhibitory peptide T20, i.e., Tyr-643 to Leu-650. In T20-4, the Asc I site is underlined and the last 31 nucleotides (shown in bold) are the reverse complement of DNA
encoding the last 10 amino acid residues of the 111V-1 inhibitory peptide T20, Asp-669 to Phe-678. The T20-2 and T20-3 oligonucleotides overlap with each other and with T20-1 and T20-4, respectively, and encode the HIV-1 inhibitory peptide T20. The PCR
product was purified (for example, using Wizard PCR Preps DNA Purification System (Promega Corp)) and then digested with Bsu361 and AscL After further purification of the Bsu361-Ascl fragment by gel electrophoresis, the product was cloned into Bsu3611Ascl digested pScNHSA.
After the sequence was confirmed, the expression cassette encoding this T20 albumin fusion protein was subcloned into pSAC35 as a Not I fragment. A Not I fragment was further subcloned into pSAC35 to give construct ID # 2672.
[1593J Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected HSA sequence (see below).
[1594J T20 albumin fusion proteins of the invention preferably comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to either the N- or C- terminus of the HIV-1 inhibitory peptide T20, i.e., Tyr-643 to Phe-678. In one embodiment of the invention, T20 albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature T20 albumin fusion protein is secreted directly into the culture medium. T20 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAP, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant FISA leader sequences including, but not limited to, a chimeric FISA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, 120 albumin fusion proteins of the invention comprise the native HIV-1 transmembrane protein gp41 signal sequence. In further preferred embodiments, the 120 albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
, Expression and Purification of Construct ID 2672.
Expression in yeast S. cerevisiae.
[15951 Construct 2672 can be transformed into yeast S. cerevisiae by methods known in the art (see Example 3), Expression levels can be examined by immunoblot detection with anti-HSA serum as the primary antibody.
Purification from yeast S. cerevisiae cell supernatant.
[15961 The cell supernatant containing the secreted T20 albumin fusion protein expressed from construct ID #2672 in yeast S. cerevisiae can be purified as described in Example 4. N-terminal sequencing of the albumin fusion protein should result in the sequence DAHICS (SEQ ID NO:2143) which corresponds to the amino terminus of the mature form of HSA.
The &cavils' R').20 can hi masa win an in vitro, irOctivity Away andifor a Cell-Cell Fusion Inhibition Assay.
[15971 The in vitro infectivity and cell-cell fusion inhibition assays are described in Wild et al., "Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection", Proc. Natl.
Acad. Sci. USA, 91: 9770-9774 (1994)).
Method [15981 High-titered virus stocks may be prepared in CEM human leukemia cells as described previously (see Wild, C., et al., "A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition", Proc. Natl. Acad. Sci. USA 89: 10537-10541 (1992)). Infectious titers may be estimated by end-point dilution on AA5 and CEM continuous cell-lines. Reverse transcriptase (RT) activity present in the supernatants may be taken as criteria for successful infection. The 50% tissue culture infection dose (TCID50) may be calculated by using the formula of Reed and Muench (see Wild et at., "Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection", Proc. Natl. Acad, Sci. USA, 91: 9770-9774 (1994)). Primary 11IV-1 isolates may be expanded in activated peripheral blood mononuclear cells, "PBMC", from normal donors.
[15991 The ability of the T20 albumin fusion protein to inhibit infection with prototypic cell-free virus, i.e., FIW-1 um or HIV-1 MHZ, may be evaluated by incubating serial dilutions of cell-free virus with AA5 or CEM target cells containing various concentrations of the T20 albumin fusion protein. The T20 albumin fusion protein may be tested against prhnary isolates and the prototypic H1V4 LAI isolate in a similar assay using PBMC as target cells. Both assays are carried out as described in Wild et at., 1992.
116001 The ability of the T20 albumin fusion protein to block virus-mediated cell-cell fusion may be assessed as described previously in Wild et at., 1992. Briefly, approximately 7 x 104 MOLT-4 cells may he incubated with 104 CEM cells and chronically infected with the HIV-1 isolates in 96-well plates (half-area cluster plates; Costar) in 100 111, of culture medium. The T20 albumin fusion protein may be added in 10 jiL and the cell mixtures may be incubated for 24 hrs at 37 C. At that time, multinucleated giant cells may be estimated by microscopic examination at x40 magnification.
The activitRof 720 albumin faision encoded by construct ID. ji 2672 can be assayed using an in vitro infectivity Assayptd/or Fusjon inhibition Asa*, Method [1601] The T20 albumin fusion protein encoded by construct 2672 can be tested in the in vitro infectivity bioassay as well as the cell-cell fusion inhibition assay as described above under subsection heading, "The activity of T20 can be assayed using an in vitro Infectivity Assay and/or a Cell-Cell Fusion Inhibition Assay".
EXAMPLE 109: Construct ID 2673, T20-HSA, Generation.
[1602] Construct ID 2673, pSAC35:T20.11SA, comprises DNA encoding a T20 albumin fusion protein which has the HSA chimeric leader sequence, i.e., the HSA-kex2 signal peptide, followed by the H1V-1 inhibitory peptide T20, i.e., Y643-F678, fused to the amino-terminus of the mature form of HSA in the yeast S. cerevisiae expression vector 552.

pSAC35.
Cloning of T20 cDNA
[1603] The DNA encoding the HIV-1 inhibitory peptide was PCR generated using four overlapping primers. The sequence was codon optimized for expression in yeast S.
cerevisiae. The PCR fragment was digested with Sal IICla I and subcloned into Xho IICla I
digested pScCHSA. A Not I fragment was then subcloned into the pSAC35 plasmid.

Construct ID #2673 encodes for the chimeric leader sequence of HSA fused to the HIV-1 inhibitory peptide T20, i.e., Tyr-643 to Phe-678, followed by the mature form of }ISA.
[1604] The 5' and 3' primers of the four overlapping oligonucleotides suitable for PCR amplification of the polynucleotide encoding the HIV-I inhibitory peptide T20, T20-5 and T20-6, were synthesized:
T20-5: 5 "-AGGAGCGTCGACAAAAGATACACTAGITTGATTCATAGTTTG.3 ' (SEQ ED NO:1192) T20-6: 5*-CGCGCATCGATGAGCAACCTCACTCri GTGTOCATCAAACCAATICCACAAA
CTAGCCCA1TTATCC-3' (SEQ ID NO:1193) -T20-5 incorporates a Sal I cloning site (shown underlined), nucleotides encoding the last three amino acid residues of the HSA chimeric leader sequence, and the DNA
encoding the first 8 amino acids (shown in bold) of the HTV-I inhibitory peptide T20, i.e., Tyr-643 to Len-= 650. In T20-6, the underlined sequence is a Cla I site; and the Cla I
site and the DNA
following it are the reverse complement of DNA encoding the first 10 amino acids of the mature HSA protein (SEQ 13) NO:1038). The bolded sequence is the reverse complement of the 31 nucleotides encoding the last 10 amino acid residues Asp-669 to Phe-678 of the HIV-1 inhibitory peptide T20. The T20-2 and T20-3 oligonucleotides (as in Example 108) overlap with each other and with T20-5 and T20-6, respectively, and encode the HIV-1 inhibitory peptide T20. Using these primers, the HIV-1 inhibitory peptide T20 was generated by annealing, extension of the annealed primers, digestion with Sal I and Cla I, and subcloning into Xho I1Cla I digested pScCHSA. After the sequence was confirmed, the Not I
fragment containing the T20 albumin fusion expression cassette was subcloned into pSAC35 cut with Not I to generate construct ID 2673. Construct ID #2673 encodes an albumin fusion protein containing the chimeric leader sequence, the HIV-1 inhibitory peptide T20, and the mature form of HSA.
[1605] Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected T20 sequence (see below).
(16061 T20 albumin fusion proteins of the invention preferably comprise the mature form of HSA , i.e., Asp-25 to Len-609, fused to either the N- or C- terminus of the HIV-1 inhibitory peptide T20, i.e., Tyr-643 to Phe-678. In one embodiment of the invention, T20 albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a -further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature T20 albumin fusion protein is secreted directly into the culture medium. T20 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, T20 albumin fusion proteins of the invention comprise the native HP/-I transmembrane protein gp41 signal sequence. In further preferred embodiments, the T20 albumin fusion proteins of the invention further comprise an N-terminal metlaionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Purification of Construct ID 2673.
Expression in yeast S. cerevisiae.
116071 Construct 2673 can be transformed into yeast S. cerevisiae by methods known in the art (see Example 3). Expression levels can be examined by immunoblot detection with anti-HSA serum as the primary antibody.
Purification from yeast S. cerevisiae cell supernatant.
[16081 The cell supernatant containing the secreted T20 albumin fusion protein expressed from construct ID #2673 in yeast S. cerevisiae can be purified as described in Example 4. N-terminal sequencing of the expressed and purified albumin fusion protein should generate YTSLI (SEQ ID NO:2151) which corresponds to the amino terminus of the HIV-1 inhibitory peptide T20.

The activity of atbantin fitsion encoded by con trgct ID # 2673 can be agaped rising an in vitro htfectiViti, 43SaIl and/or a cell-Cell,Fasjon IrthiaitioP A.C.CaY.
Method (16091 The T20 albumin fusion protein encoded by construct 2673 can be tested in the in vitro infectivity bioassay as well as the cell-cell fusion inhibition assay as described above in Example 108 under subsection heading, "The activity of T20 can be assayed using an in vitro Infectivity Assay and/or a Cell-Cell Fusion Inhibition Assay".
EXAMPLE 110: Indications for T20 albumin fusion proteins [16101 Based on the activity of 120 albumin fusion proteins in the above assays, T20 albumin fusion proteins are useful in treating, preventing, and/or diagnosing HIV, AIDS, and/or SW (simian immunodeficiency virus) infections.
EXAMPLE 111: Construct ID 2667, EISA-T1249, Generation.
[16111 Construct ED 2667, pSAC35:HSA.T1249, comprises DNA encoding a T1249 albumin fusion protein which has the full length HSA protein, including the native HSA
leader sequence, fused to the amino-terminus of the second-generation fusion inhibitor peptide, "11249", Le., Wl-F39, in the yeast S. cerevisiae expression vector pSAC35. The T1249 peptide is a second-generation fusion inhibitor derived from the 111V-1 transmembrane protein gp4I and is shown to have inhibitory activity on HIV-1 infection.
Cloning of 77249 cDNA
[1612] The polynucleofide encoding TI249 was PCR generated using four overlapping primers T1249-1, 11249-2, T1249-3, and T1249-4, described below_ The sequence was codon optimized for expression in yeast S. cerevisiae. The PCR
fragment was cut with Bsu 3611Asc I, and ligated into Bsu 36I1Asc I cut paNHSA.. A Not I
fragment was then subeloned into the pSAC35 plasmid. Construct ID #2667 encodes an albumin fusion protein containing the full length HSA protein, including the native HSA
leader sequence, fused to the T1249 peptide, i.e., Trp-1 to Phe-39.
[16131 The 5' and 3' primers of the four overlapping oligonucleotides suitable for PCR amplification of the polynucleotide encoding the T1249 peptide, 11249-I
and T1249-4, were synthesized:
T1249-1: 5-AAGCTGCCITAGGC1TATGGCAAGAATGGGAACAAAAG-3' (SEQ ED NO:1181) 11249-2: 5'- TGGCAAGAATGGGAACAAAAGATTACTGCTTIGTTAGAACAAGCTCAAATT
CAACAAGAAAAGAATGAAT-3' (SEQ ID NO:1206) 11249-3: 5'- GAACCATTCCCATAAAGAAGCCCATITATCCAACTITTGCA,ATTCATATTCA
T1'CTTTTCTTGTTGAATTTGAGCTT-3' (SEQ ID NO:1207) T1249-4: 5'-TTGGCGCGCCTTAGAACCATTCCCATAAAGAAGCCCA1 (SEQ ID NO:1182) 116141 11249-1 incorporates the Bsu 361 cloning site (shown underlined) and nucleotides encoding the last four amino acid residues of the mature form of HSA (SEQ ID
NO:1038), as well as 21 nucleotides (shown in bold) encoding the first 7 amino acid residues of the 11249 peptide, i.e., Trp-1 to Lys-7. In 11249-4, the Asc I site is underlined and the last 30 nucleotides (shown in bold) are the reverse complement of DNA encoding the last 10 amino acid residues of the 11249 peptide, Asp-30 to Phe-39. The 11249-2 and oligonucleotides overlap with each other and with T1249-1 and T1249-4, respectively, and encode the 11249 peptide. The PCR product was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corp)) and then digested with Bsu36I
and Ascl.
After further purification of the Bsu36I-Asci fragment by gel electrophoresis, the product was cloned into Bsu3611Asci digested pSeNfISA. After the sequence was confirmed, the expression cassette encoding this 11249 albumin fusion protein was subcloned into pSAC35 as a Not I fragment. A Not! fragment was further subcloned into pSAC35 to give construct LD # 2667.
[1615] Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected HSA sequence (see below).
[16161 T1249 albumin fusion proteins of the invention preferably comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to either the N- or C-terminus of the HIV-1 inhibitory peptide T1249, i.e., Trp-1 to Phe-39, hi one embodiment of the invention, 11249 albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypepfide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature T1249 albumin fusion protein is secreted directly into the culture medium. 11249 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, 1g, Fibulin B, - Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, hut not limited to, a chimeric IISA/MAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, 1I249 albumin fusion proteins of the invention comprise the native HIV-1 transmembrane protein gp41 signal sequence. In further preferred embodiments, the T1249 albumin fusion proteins of the invention further comprise an N-terrninal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
Expression and Purification of Construct ID 266Z
Expression in yeast S. cerevisiae.

Construct 2667 can be transformed into yeast S. cerevisiae by methods known in the art (see Example 3). Expression levels can be examined by irnmunoblot detection with anti-HSA serum as the primary antibody.
Purification from yeast S. cerevisiae cell supernatant.
(16181 The cell supernatant containing the secreted T1249 albumin fusion protein expressed from construct ID #2667 in yeast S. cerevisiae can be purified as described in Example 4. N-terminal sequencing of the albumin fusion protein should result in the sequence DAHICS which corresponds to the amino terminus of the mature form of HSA.
The activity of TI249 allnaninfitsien encoded bp' =tract ID 2667 can = ass, ed in.
an in vitro Infectivity Assay and/pr a Cell-Cell FilSiOtt inhibition Assay.
Method 116191 ' The T1249 albumin fusion protein encoded by construct 2667 can be tested in the in vitro infectivity bioassay as well as the cell-cell fusion inhibition assay as described above in Example 108 under subsection heading, "The activity of 120 can be assayed using an in vitro Infectivity Assay and/or a Cell-Cell Fusion Inhibition Assay".
EXAMPLE 112: Construct ID 2670, T1249-HSA, Generation.
11620) Construct ID 2670, pSAC35:T1249.HSA, comprises DNA encoding a TI249 albumin fusion protein which has the HSA chimeric leader sequence, i.e., the HSA-kex2 signal peptide, the second-generation fusion inhibitor peptide, "T1249", i.e., WI-F39 fused to the amino-terminus of the mature form of HSA in the yeast S. cerevisiae expression vector pSAC35.

Cloning of T1249 cDNA
116211 The DNA encoding the second-generation fusion inhibitor peptide was PCR
generated using four overlapping primers. The sequence was codon optimized for expression in yeast S. cerevisiae. The PCR fragment was digested with Sal 1lCla I and subcloned into Xho 11Cla I digested pScCHSA. A Not 1 fragment was then subcloned into the pSAC35 plasmid. Construct ID #2670 encodes for the chimeric leader sequence of HSA
fused to the T1249 peptide, i.e., Trp-1 to Phe-39, followed by the mature form of HSA.
116221 The 5' and 3' primers of the four overlapping oligonucleotides suitable for PCR amplification of the polynucleotide encoding the 11249 peptide, T1249-5 and T1249-6, were synthesized:
T1249-5: 5 '-AGGAGCGTCGACAAAAGATGGCAAGAATGGGAACAAAAG-3' (SEQ NO:1184) 11249-6: 5'-ATCGATGAGCAACCTCACTC1'1 GTGTGCATCGAACCATTCCCATAAAG
AAGCCCATITATC-3' (SEQ ID NO:1185) [1623] T1249-5 incorporates a Sal I cloning site (shown underlined), nucleotides encoding the last three amino acid residues of the HSA chimeric leader sequence, and the DNA encoding the first 7 amino acids (shown in bold) of the T1249 peptide, i.e., Trp-1 to Lys-7. In T1249-6, the underlined sequence is a Cla I site; and the C/a I site and the DNA
following it are the reverse complement of DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO:1038). The bolded sequence is the reverse complement of the 30 nucleotides encoding the last 10 amino acid residues Asp-30 to Phe-39 of the 11249 peptide. The T1249-2 and T1249-3 oligonucleotides (as in Example 111) overlap with each other and with T1249-5 and T1249-6, respectively, and encode the T1249 peptide. Using these primers, the T1249 peptide was generated by annealing, extension of the annealed primers, digestion with Sal I and Cla I, and subcloning into Xho I1Cla I
digested pScCHSA.
After the sequence was confirmed, the Not I fragment containing the T1249 albumin fusion expression cassette was subcloned into pSAC35 cut with Not Ito generate construct ID 2670.
Construct ID #2670 encodes an albumin fusion protein containing the chimeric leader sequence, the T1249 peptide, and the mature form of HSA.
[16241 Further, analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected T1249 sequence (see below).
[1625] T1249 albumin fusion proteins of the invention preferably comprise the mature form of IISA , Le., Asp-25 to Leu-609, fused to either the N- or C-terminus of the 11249 peptide, i.e., Trp-1 to Phe-39. In one embodiment of the invention, 11249 albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression.
In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature T1249 albumin fusion protein is secreted directly into the culture medium. T1249 albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MAF, INV, Ig, Fibulin B, Clusterin, Insulin-Like Growth Factor Binding Protein 4, variant HSA leader sequences including, but not limited to, a chimeric HSAJMAF leader sequence, or other heterologous signal sequences known in the art. In a preferred embodiment, 11249 albumin fusion proteins of the invention comprise the native HIV-1 transmembrane protein gp41 signal sequence. In further preferred embodiments, the T1249 albumin fusion proteins of the invention further comprise an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and/or variants, are also encompassed by the invention.
rie,iwiliv of 11.2* albumin ,fitiion encoded by construct ID # 2670 can be assayed using an in vitro In/em vuy Assay and/or z Cell-Cell Fusion Inhibition AsSay.
Method [16261 ..The11249 albumin fusion protein encoded by construct 2670 can be tested in the in vitro infectivity bioassay as well as the cell-cell fusion inhibition assay as described above in ExtUrip. -re 108 under subsection heading, "The activity of T20 can be assayed using an in vitro infectivity Assay and/or a Cell-Cell Fusion Inhibition Assay".
EXAMPLE 113: Indications for 11249.õalbutniu fusion protejns [1627j Based on the activity of 11249 albumin fusion proteins in the above assays, T1249 albumin fusion proteins are useful in treating, preventing, and/or diagnosing HIV, AIDS, and/or SW (simian immunodeficiency virus) infections.
EXAMPLE 114: Construct ID 2702IISA-GCSF,T314201, Generation [16281 Construct ID 2702, pSAC35:HSA.GCSF.T31-L201, comprises DNA encoding a GCSF albumin fusion protein which has mature IISA fused downstream of the ITSA/kex2 leader sequence and upstream of amino acids T31 to L201 of GCSF, in the yeast S. cerevisiae expression vector pSAC35, Cloninz of GCSF cDATA
[16291 The polynucicotide encoding the GCSF C-terminal deletion mutant was PCR
amplified using primers GCSF-5 and GCSF-6, described below. The amplimer was cut with Bsu36I and Asa and ligated into pScNHSA. Construct ID #2702 encodes an albumin fusion protein containing mature HSA fused downstream of the HSA/kex2 leader sequence and upstream of amino acids T31 to L201 of GCSF.
[16301 Two oligonucleotide primers, GCSF-5 and GCSF-6, suitable for PCR
amplification of the polynucle,otide encoding the GCSF C-terminal deletion mutant, were synthesized:
GCSF-5: 5'- AAGCTGCCTTAGGCTTAACCCCCCTGGGCCCTGCCAG (SEQ ID NO:
1197) GCSF-6: 5'- GCGCGCGGCGCGCCTCAAAGGTGGCGTAGAACGCGGTACGAC (SEQ
ID NO: 1198) 116311 GCSF-5 incorporates the Bsu36I cloning site (shown underlined), and nucleotides encoding the last six amino acids of HSA as well as the first six amino acids of mature GCSF (amino acids T31 through A36). GCSF-6 contains an Awl cloning site (shown underlined) and the last 25 nucleotides are the reverse complement of DNA
encoding the last eight amino acid residues of the GCSF C-terminal deletion mutant (S194 through L201). The PCR product generated with these primers was purified (for example, using Wizard PCR
Preps DNA Purification System (Promega Corporation)) and then digested with Bsu36I and AscL After further purification of the Bsu3611Ascl PCR fragment by gel elelctrophoresis, the product was cloned into Bsu36UAscl digested pSeNITSA. After the sequence was confirmed, the expression cassette encoding this GCSF albumin fusion protein was subcloned into pSAC35 as a Nod fragment 116321 Further analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing can confirm the presence of the expected HSA sequence (see below).
[16331 GCSF albumin fusion proteins of the invention preferably comprise the mature form of HSA, i.e., Asp-25 to Leu-609, fused to either the N- or C-terminus of the C-terminal deletion mutant of GCSF, i.e., T31 to L201. In one embodiment of the invention, GCSF
albumin fusion proteins of the invention further comprise a signal sequence which directs the nascent fusion polypeptide in the secretory pathways of the host used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed, and the mature GCSF albumin fusion protein is secreted directly into the culture medium.
GCSF albumin fusion proteins of the invention may comprise heterologous signal sequences including, but not limited to, MFa-1, Invertase, 1g, Fibulin B, Clusterin, Insulin-like growth factor binding protein 4, K. lactis killer toxin, and variant HSA leader sequences including, but not limited to, a chimeric HSA/MFa-1 (HSA/kex2) leader sequence, a chimeric Klactis/M:Fa-1 leader sequence, or other heterologous signal sequences known in the art. In a further preferred embodiment, GCSF albumin fusion prteins of the invention comprise the native GCSF signal sequence. In further preferred embodiements, the GCSF
albumin fusion proteins of the invention further comprise and N-terminal methionine residue.
Polynucleotides encoding these polypeptides, including fragments and/or variants are also encompassed by the invention.
Expression and Purification of Construct ID #2702 Expression in yeast S. cerevisiae 116341 , Construct #2702 was transformed into yeast S. cerevisiae by methods known in the art (see Example 3) and as previously described for construct II) #1642 (see Example 19). Expression levels were examined by immunoblot detection with anti-HSA
serum as the primary antibody (data not shown).
Purification from yeast S. cerevisiae cell supernatant [1635j A general procedure for purification of albumin fusion proteins is described in Example 4. The cell supernatant containing GCSF albumin fusion protein expressed from construct 11.) #2702 in yeast S. cerevisiae was purified as described in Example 20. N-terminal sequencing of the albumin fusion protein should result in the sequence DAFiKS
which corresponds to the amino terminus of the mature form of HSA.
The activity of GCSF albumin fusion encoded by construct ID # 2702 can be assayed using an in vitro 1VFS-60 cell proliferation assay Method 116361 The GCSF albumin fusion protein encoded by construct 2702 was tested using the in vitro NFS-60 cell proliferation bioassay previously described in Example 19 under subsection headings "The activity of GCSF can be assayed using an in vitro NFS-60 cell proliferation may" and "The activity of GCSF albumin fusion encoded by construct ID #
1642 can be assayed using an in vitro NFS-60 cell proliferation assay".

?esults 116371 Both the partially purified GCSF albumin fusion protein encoded by construct 1634 (HSA-GCSF) and the GCSF C-terminal deletion mutant albumin fusion protein (L-171) encoded by construct 2702 demonstrated the ability to cause NFS-60 cell proliferation, with the C-terminal deletion mutant exhibiting a more potent proliferative effect (see Figure 19).
Unexpectedly, the fusion protein encoded by construct 2702 exhibited 2-3 times more activity than the fusion protein encoded by construct 1643. Alternate GCSF
albumin fusion constructs comprise albumin fused to amino acid residues 1-169 of mature GCSF
and albumin fused to amino acid residues 1-170 of mature GCSF.
EXAMPLE 115: Construct 11) 2876., HSA-11?Na hybrid [1638] Construct ID 2876, pSAC35:HSA.IFNaA(C1-Q91)/D(L93-B166) R23K,A113V comprises DNA encoding an IFNa hybrid albumin fusion protein which has mature HSA fused downstream of the HSA/kex2 leader sequence and upstream of an IFNa AJD hybrid amino acid sequence, in the yeast S. cerevisiae expression vector pSAC35.
Regarding the composition of the hybrid IFN, the first 91 amino acids are from the subtype IFNa2 (also called IFNaA) and the remaining 75 an are from IFNa1 (IFNaD). We incorporated two point mutations (R231(..., Al 13V). The fusion was generated by PCR and fused downstream of HSA within the yeast expression vector pSAC35.
Results CID 2876 Expression and Purification [16391 The yeast strain BXP-10 was transformed with pSAC35:CID 2876 and a transformant selected for fermentation. A 5-liter fermentation was performed and analysis of supernatant demonstrated high expression (approximately 500 mg/I). A small proportion of the supernatant was processed to pilot purification. Approximately I mg of CID
2876 protein (greater than 95% pure based on N-terminal sequence) was obtained following a purification through Blue-sepharoseT followed by gel filtration, followed by Q-anion exchange. The remaining fermentation starting material is available for further purification if needed.
ISRE Activity [16401 All type I IFNs mediate their activities through engagement of a common rFN
receptor complex and activation of the ISRE signal transduction pathway.
Activation of gene xanscription through this pathway leads to the cellular responses associated with IFNs including anti-proliferation, antiviral and immune modulation. Using a reporter based strategy, the ability of CID 2876 to activate the ISRE signal transduction pathway was determined. CID 2876 was found to be a potent activator of the ISRE pathway, demonstrating an EC50 of 2.7 ng/ml (data not shown). This compares favorably with the potency of CID 3165 in this assay system.
Anti-viral activity [16411 A hallmark activity of IFNs is their ability to mediate cellular protection against viral infection. While most human type I 1FNs display antiviral activity in a species restricted manner, the hybrid IFN employed in this study has been demonstrated to be active on =trine cells. Thus the antiviral activity of CID 2876 was evaluated on the murine cell line L929 infected with EMCV. Results indicate that CID 2876 does demonstrate antiviral activity in a cross species manner (data not shown).
Example 116: Activity of Construct 3070 (GLP4 Albumin Fusion) Measured by its Vitro Stimulation of Insulin mRNA in INS-1 Cells [16421 It has recently been shown that GLP-1 increases the expression of insulin mRNA in pancreatic beta-cells (Buteau et al., Diabetologia 1999 Jul;42(7):856-64). Thus, the ability of the GLP-1 albumin fusion protein encoded by CID 3070 to stimulate insulin mRNA was evaluated using the pancreatic beta-cell line INS-1 (832/13).
[16431 Figure 14 illustrates the steady-state levels of insulin mRNA in INS-1 (832/13) cells after treatment with GLP-1 or GLP-1 albumin fusion protein encoded by construct ID
3070 (CD 3070 protein). Both GLP-1 and the cret 3070 protein stimulate transcription of the insulin gene. The first bar (black) represents the untreated cells. Bars 2-4 (white) represent cells treated with the indicated concentrations of GLP-1. Bars 5-7 (gray) represent cells treated with the indicated concentrations of CID 3070 protein.
[16441 It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
[1645]

. Applicant's File . InternedMitil p'plitattuti "
Reference Number: PF564PCT . Number: .
Unassigned...
INDICATIONS RELATING TO DEPOSITED BIOLOGICAL MATERIAL
(PCT Rule 13bis) A. The indications made below relate to the deposited biological material referred to on pages 126 and 127 in Table 3 of the description.
B. IDENTIFICATION OF DEPOSIT: ____________________________________ Further deposits are identified I, on an additional sheet: I I
Name of Depository: American Type Culture Collection Address of Depository: 10801 University Boulevard Manassas, Virginia 20110-2209 United States of America Accession Date of Accession = Date of . Number ... . Deposit Number Deposit 1 , PTA-3767 ....05-Oct-2001 : 2 PTA-3766 . 05-Oct-3 PTA-3759 04-Oct-2001 4 PTA-3761 04-Oct-2001 . PTA-3762 04-Oct-2001 6 . PTA-3771 05-Oct-2001 7 PTA-3770 05-Oct-2001 8 .. PTA-3768 , 05-Oct-2001 9 PTA-3757 04-Oct-2001 . 10 PTA-3758 04-04-,2001 11 PTA-3764 . 04-Oct-2001 = 12 PTA-3941 ; 19-Dec-13 PTA-3763 04-Oct-2001 = 14 PTA-3916 = 07-Dec-PTA-3917 . 07-Dec-2001 16 PTA-3918 07-Dec-2601 17 PTA-3760. 04-Oct-2001 18 PTA-3742 28-Sep-2003. =
19 PTA-3773 05-Oct-2001 20 PTA-3940 , 19-Dec-2001--21 PTA-3740 . 28-Sep-2001 22 PTA-3741 28-Sep-2001 23 PTA-3742 19-Dec-2001 24 PTA-3939= 19-Dec-2001, , PTA-3919 . 11-Dec-2001 26 PTA-3924 12-Dec-2001 27 PTA-3923 12-Dec-2001 28 PTA-3943 19-Dec-2001 29 PTA-4671 . 16-Sep-2002 30 PTA-467.0 .. 16-Sei-2002 31: PTA-4770 . 22-Oct-2002 32 .

CANADA
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NORWAY
The applicant hereby requests that the application has been laid open to Public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open inspection, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on the list of recognized experts drawn up by the Norwegian Patent Office or any person approved by the applicant in the individual case.
AUSTRALIA
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FINLAND
The applicant hereby requests that,, until the application has been laid open to public inspection (by the National Board of Patents and Regulations), or has been finally decided upon by the National Board of Patents and Registration without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.

UNITED KINGDOM
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DENMARK
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If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Danish Patent Office or any person by the applicant in the individual case.
SWEDEN
The applicant hereby requests that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of the PCT
Applicant's Guide). If such a request has been filed by the applicant any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Swedish Patent Office or any person approved by a applicant in the individual case.
NETHERLANDS
The applicant hereby requests that until the date of a grant of a Netherlands patent or until the date on which the application is refused or withdrawn or lapsed, the microorganism shall be made available as provided in the 31F(1) of the Patent Rules only by the issue of a sample to an expert. The request to this effect must be furnished by the applicant with the Netherlands Industrial Property Office before the date on which the application is made available to the public under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands, whichever of the two dates occurs earlier.

Claims (12)

THE EMBODIMENTS OF THE INVENTION FOR WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A nucleic acid molecule comprising a polynucleotide encoding a HSA
(human serum albumin)/kex2 leader sequence, a polynucleotide encoding mature human serum albumin, a polynucleotide encoding a mature granulocyte-colony stimulating factor (G-CSF), and a polynucleotide sequence comprising a promoter sequence, a selectable marker sequence, and a region for termination of transcription, wherein said HSA (human serum albumin)/kex2 leader sequence is fused to the N-terminus of mature human serum albumin, wherein said mature human serum albumin is fused to the N-terminus of mature G-CSF, and wherein said albumin fusion protein has G-CSF activity, optionally wherein the nucleic acid molecule is part of an expression cassette.

2. A nucleic acid molecule comprising the cDNA contained in ATCC Deposit No.
PTA-3766.

3. A vector comprising the nucleic acid molecule of claim 1 or 2.

4. A vector according to Claim 3 comprising a promoter and termination region operably associated to a nucleic acid molecule encoding a leader sequence and an albumin fusion protein, the nucleic acid comprising the construct contained in ATCC Deposit No. PTA-3766, wherein said leader sequence is a hybrid HSA/kex2 leader sequence comprising the amino acids of SEQ ID
NO:1111.

5. A vector according to Claim 3 comprising a promoter and termination region operably associated to a nucleic acid molecule encoding a leader sequence and an albumin fusion protein comprising amino acids 1 to 783 of SEQ ID
NO:226, wherein said leader sequence is a hybrid HSA/kex2 leader sequence comprising the amino acids of SEQ ID NO:1111.

6. A vector according to any of Claims 3 to 5, wherein said promoter is a promoter.

7. A vector according to any of Claims 3 to 6, wherein said vector is a pSAC35 expression vector.

8. A host cell comprising a vector according to any of Claims 3 to 7.

9. The host cell of claim 8, wherein said host cell is a yeast cell, optionally wherein the yeast cell is a Saccharomyces cerevisiae and/or wherein the yeast cell is glycosylation deficient and/or wherein the yeast cell is protease deficient.

10. A method of producing an albumin fusion protein comprising:
(a) culturing a host cell according to claim 8 or 9 in suitable conditions for expression of the albumin fusion protein; and (b) isolating the albumin fusion protein.

11. The albumin-G-CSF fusion protein encoded by the nucleic acid molecule according to any one of claims 1 to 7 for use in medicine.

12. The albumin-G-CSF fusion protein encoded by the nucleic acid molecule according to any one of claims 1 to 7 for use in the treatment of inflammatory disorders, cancer, leukaemia, such as myelocytic leukaemia, acute myelogeneous leukaemia and acute lymphoblastic leukaemia, neutropenia, such as primary neutropenia (e.g. Kostmann syndrome), secondary neutropenia, neutropenia in HIV-infected patients and neutropenia associated with chemotherapy, infections associated with neutropenia, myelodysplasia, autoimmune diseases and disorders, psoriasis, wound healing, lymphoma, such as non-Hodgkin's lymphoma, Hodgkin's disease or glycogen storage disease;
or for use in the prevention of inflammatory disorders, neutropenia, such as neutropenia in HIV-infected patients or neutropenia associated with chemotherapy;
or for use in the diagnosis of inflammatory diseases.