CA2332338A1 - Hybrid polypeptides with enhanced pharmacokinetic properties - Google Patents

Abstract

The present invention relates to enhancer peptide sequences originally deriv ed from various retroviral envelope (gp41) protein sequences that enhance the pharmacokinetic properties of any core polypeptide to which they are linked. The invention is based on the discovery that hybrid polypeptides comprising the enhancer peptide sequences linked to a core polypeptide possess enhanced pharmacokinetic properties such as increased half life. The invention furthe r relates to methods for enhancing the pharmacokinetic properties of any core polypeptide through linkage of the enhancer peptide sequences to the core polypeptide. The core polypeptides to be used in the practice of the inventi on can include any pharmacologically useful peptide that can be used, for example, as a therapeutic or prophylactic reagent.

Description

HYBRID POLYPEPTIDES WITH ENHANCED
PHARMACOKINETIC PROPERTIES
1. INTRODUCTION
The present invention relates to enhancer peptide sequences originally derived from various retroviral envelope (gp41) protein sequences that enhance the pharmacokinetic properties of any core polypeptide to which they are linked.
The invention is based, in part, on the discovery that hybrid polypeptides comprising the enhancer peptide sequences linked to a core polypeptide possess enhanced pharmacokinetic properties such as increased half life. The invention further relates to novel anti-fusogenic and/or anti-viral, peptides, including ones that contain such enhancer peptide sequences, and methods for using such peptides. The invention further relates to methods for enhancing the pharmacokinetic properties of any core polypeptide through linkage of the enhancer peptide sequences to the core polypeptide. The core polypeptides to be used in the practice of the invention can include any pharmacologically useful peptide that can be used, for example, as a therapeutic or prophylactic reagent. In a non-limiting embodiment, the invention is demonstrated by way of example wherein a hybrid polypeptide comprising, for example, an HIV
core polypeptide linked to enhancer peptide sequences, is shown to be a potent, non-cytotoxic inhibitor of HIV-1, HIV-2 and SIV infection. Additionally, the enhancer peptide sequences of the invention have been linked to a respiratory syncytial virus (RSV) core polypeptide and a luteinizing hormone receptor (LH-RH) core polypeptide. In each instance, the hybrid polypeptide was found to possess enhanced pharmacokinetic properties, and the RSV hybrid polypeptide exhibited substantial anti-RSV activity.

2. BACKGROUND OF THE INVENTION
Polypeptide products have a wide range of uses as therapeutic and/or prophylactic reagents for prevention and treatment of disease. Many polypeptides are able to regulate biochemical or physiological processes to either prevent disease or provide relief from symptoms associated with disease. For example, polypeptides such as viral or bacterial polypeptides have been utilized successfully as vaccines for prevention of pathological diseases.
Additionally, peptides have been successfully utilized as therapeutic agents for treatment of disease symptoms. Such peptides fall into diverse categories such, for example, as hormones, enzymes, immunomodulators, serum proteins and cytokines.
For polypeptides to manifest their proper biological and therapeutic effect on the target sites, the polypeptides must be present in appropriate concentrations at the sites of action. In addition, their structural integrity must generally be maintained. Therefore, the formulation of polypeptides as drugs for therapeutic use is directed by the chemical nature and the characteristics of the polypeptides, such as their size and complexity, their conformational requirements, and their often complicated stability, and solubility profiles. The pharmacokinetics of any particular therapeutic peptide is dependent on the bioavailability, distribution and clearance of said peptide.
Since many bioactive substances, such as peptides and proteins, are rapidly destroyed by the body, it is critical to develop effective systems for maintaining a steady concentration of peptide in blood circulation, to increase the efficacy of such peptides, and to minimize the incidence and severity of adverse side effects.

3. SUMMARY OF THE INVENTION
The present invention relates, first, to enhancer peptide sequences originally derived from various retroviral envelope (gp41) protein sequences i.e., HIV-1, HIV-2 and SIV, that enhance the pharmacokinetic properties of any core polypeptide to which they are linked. The invention is based on the surprising result that when the disclosed enhancer peptide sequences are linked to any core polypeptide, the resulting hybrid polypeptide possesses enhanced pharmacokinetic properties including, for example, increased half life and reduced clearance rate relative to the core polypeptide alone. The present invention further relates to such hybrid polypeptides and core polypeptides, and to novel peptides that exhibit anti-fusogenic activity, antiviral activity and/or the ability to modulate intracellular processes that involve coiled-coil peptide structures. Among such peptides are ones that contain enhancer peptide sequences.
Core polypeptides can comprise any peptides which may be introduced into a living system, for example, any peptides capable of functioning as therapeutic, prophylactic or imaging reagents useful for treatment or prevention of disease or for diagnostic or prognostic methods, including methods in vivo imaging. Such peptides include, for example, growth factors, hormones, cytokines, angiogenic growth factors, extracellular matrix polypeptides, receptor ligands, agonists, antagonists or inverse agonists, peptide targeting agents, such as imaging agents or cytotoxic targeting agents, or polypeptides that exhibit antifusogenic and/or antiviral activity, and peptides or polypeptides that function as antigens or immunogens including, for example, viral and bacterial polypeptides.
The invention further relates to methods for enhancing the pharmacokinetic properties of any core polypeptide through linkage of the core polypeptide to the enhancer peptide sequences to form hybrid polypeptides.
The invention still further relates to methods for using the peptides disclosed herein, including hybrid polypeptides containing enhancer peptide sequences. For example, the methods of the invention include methods for decreasing or inhibiting viral infection, era., HIV-1, HIV-2, RSV, measles, influenza, parainfluenza, Epstein-Barr, and hepatitis virus infection, and/or viral-induced cell fusion events. The enhancer peptide sequences of the invention can, additionally, be utilized to increase the in vitro or ex-vivo half-life of a core polypeptide to which enhancer peptide sequences have been attached, for example, enhancer peptide sequences can increase the half life of attached core polypeptides in cell culture or cell or tissue samples.
The invention is demonstrated by way of examples wherein hybrid polypeptides containing an HIV core polypeptide linked to enhancer peptide sequences are shown to exhibit greatly enhanced pharmacokinetic properties and act as a potent, non-cytotoxic inhibitors of HIV-1, HIV-2 and SIV infection. The invention is further demonstrated by examples wherein hybrid polypeptides containing an RSV core polypeptide or a luteinizing hormone polypeptide are shown to exhibit greatly enhanced pharmacokinetic properties. In addition, the RSV
hybrid polypeptide exhibited substantial anti-RSV activity.
3.1. DEFINITIONS
Peptides, polypeptides and proteins are defined herein as organic compounds comprising two or more amino acids covalently joined, e. ., by peptide amide linages. Peptides, polypeptide and proteins may also include non-natural amino acids and any of the modifications and additional amino and carboxyl groups as are described herein. The terms "peptide," "polypeptide" and "protein" are, therefore, utilized interchangeably herein.
peptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows:

A (alanine) R (arginine) N (asparagine) D (aspartic acid) C (cysteine) Q (glutamine) E (glutamic acid) G (glycine) H (histidine) I (isoleucine) L (leucine) K (lysine) M (methionine) F (phenylalanine) P (proline) S (serine) T (threonine) W (tryptophan) Y (tyrosine) V (valine) X (any amino acid) "Enhancer peptide sequences" are defined as peptides having the following consensus amino acid sequences:
"WXXWXXXI", "WXXWXXX", "WXXWXX", "WXXWX", "WXXW", "WXXXWXWX", "XXXWXWX", "XXWXWX", "XWXWX", "WXWX", "WXXXWXW", "WXXXWX", "WXXXW", "IXXXWXXW", "XXXWXXW", "XXWXXW", "XWXXW", "XWXWXXXW", "XWXWXXX", "XWXWXX", "XWXWX", "XWXW", "WXWXXXW", or "XWXXXW", wherein X can be any amino acid, W represents tryptophan and I represents isoleucine. As discussed below, the enhancer peptide sequences of the invention also include peptide sequences that are otherwise the same as the consensus amino acid sequences but contain amino acid substitutions, insertions or deletions but which do not abolish the ability of the peptide to enhance the pharmacokinetic properties of a core peptide to which it is linked relative to the pharmacokinetic properties of the core polypeptide alone.
"Core polypeptide" as used herein, refers to any polypeptide which may be introduced into a living system and, thus, represents a bioactive molecule, for example any p°lypeptide that can function as a pharmacologically useful peptide for treatment or prevention of disease.

"Hybrid polypeptide" as used herein, refers to any polypeptide comprising an amino, carboxy, or amino and carboxy terminal enhancer peptide sequence and a core polypeptide. Typically, an enhancer peptide sequence is linked directly to a core polypeptide. It is to be understood that an enhancer peptide can also be attached to an intervening amino acid sequence present between the enhancer peptide sequence and the core peptide.
"Antifusogenic" and "anti-membrane fusion," as used herein, refer to a peptide's ability to inhibit or reduce the level of fusion events between two or more structures e.a-, cell membranes or viral envelopes or pili, relative to the level of membrane fusion which occurs between the structures in the absence of the peptide.
"Antiviral," as used herein, refers to the peptide's ability to inhibit viral infection of cells via, era., cell fusion or free virus infection. Such infection can involve membrane fusion, as occurs in the case of enveloped viruses, or another fusion event involving a viral structure and a cellular structure, e.a., fusion of a viral pilus and bacterial membrane during bacterial conjugation).
4. BRIEF DESCRIPTION OF DRAWINGS
FIG. 1. Hybrid polypeptides. Enhancer peptide sequences derived from putative N-terminal and C-terminal interactive regions are depicted linked to a generic core polypeptide. Conserved enhancer peptide sequences are shaded. It is to be noted that the enhancer peptide sequences indicated may be used either as - terminal, C-terminal, or - and C-terminal additions. Further, the enhancer peptide sequences can be added to a core polypeptide in forward or reverse orientation, individually or in any of the possible combinations, to enhance pharmacokinetic properties of the peptide.
FIG. 2A. Enhancer peptide sequences derived from various envelope (gp41) protein sequences, representing the N-terminal interactive region observed in all currently published isolate sequences of HIV-l, HIV-2 and SIV. The final sequence "WXXWXXXI" represents a consensus sequence.
FIG. 2B. Enhancer peptide sequence variants derived from various envelope (gp41) protein sequences, representing the C-terminal interactive region observed in all currently published isolate sequences of HIV-1, HIV-2 and SIV. The final sequence "WXXXWXWX" represents a consensus sequence.
FIG. 3. Comparison of HIV-1 titres in tissues of HIV-1 9320 infected SLID-HuPBMC mice as measured by P24 Levels in HuPBMC co-culture assays. The figure shows a comparison of in vivo T20 and T1249 viral inhibition.
FIG. 4A-4B. Plasma pharmacokinetic profile of T1249 vs.
T1387 core control in CD-rats following IV injection for up to 2 hrs (FIG. 4A) and 8 hrs (FIG. 4B). The T1387 polypeptide is a core polypeptide and the T1249 polypeptide is the core polypeptide linked to enhancer peptide sequences.
FIG. 5. Plasma pharmacokinetic profile of T1249 vs. T20 control in CD-rats following IV administration. The T1249 polypeptide is a hybrid polypeptide of a core polypeptide (T1387) linked to enhancer peptide sequences. T20: n=4;
T1249: n=3.
FIG. 6. Comparison of T20/T1249 Anti-HIV-1/IIIb activity and cytotoxicity.
FIG. 7. Direct Binding of T1249 to gp41 construct M41~178. l2sl-T1249 was HPLC purified to maximum specific activity. Saturation binding to M41~178 (a gp41 ectodomain fusion protein lacking the T20 amino acid sequence) immobilized in microtitre plates at 0.5 mg/ml is shown.

FIG. 8. Time Course of T1249 Association/Dissociation:
The results demonstrate that 'z5I-T1249 and 'z5I-T20 have similar binding affinities of 1-2 nM. Initial on and off rates for 'z5I-T1249 were significantly slower than those of 125I-T20. Dissociation of bound radioligand was measured following the addition of unlabeled peptide to a final concentration of 10~,m in 1/10 total assay volume.
FIG. 9. Competition for T1249 Binding to M41~178.
Unlabeled T1249 and T20 were titrated in the presence of a single concentration of either 'z5I-T1249 or 'z5I-T20. Ligand was added just after the unlabeled peptide to start the incubation.
FIG. l0A-lOB. Plasma pharmacokinetic profile of RSV
hybrid polypeptides T1301 (l0A) and T1302 (lOB) vs. T786 in CD rats.
FIG. 11A. Plaque Reduction Assay. Hybrid polypeptide T1293 is capable of inhibiting RSV infection with an ICso 2.6 ~g/ml.
FIG. 11B. Plaque Reduction Assay demonstrates the ability of RSV Hybrid Polypeptides T1301, T1302 and T1303 to inhibit RSV infection.
FIG. 12A and 12B. Plasma pharmacokinetic profile of luteinizing hormone hybrid polypeptide T1324 vs T1323 in CD
male rats. The T1323 polypeptide is a luteinizing hormone core polypeptide and the T1324 polypeptide is a hybrid polypeptide comprising a core polypeptide linked to enhancer peptide sequences.
FIG. 13. Hybrid polypeptide sequences derived from various core polypeptides. Core polypeptide sequences are shown shaded. The non-shaded amino and carboxy terminal sequences represent enhancer peptide sequences.
_ g _ FIG. 14A-B. Circular Dichroism (CD) spectra for T1249 in solution (phosphate buffered saline, pH 7) alone (10 ~cM at iQC; FIG. 14A) and in combination with a 45-residue peptide from the gp41 HR1 binding domain (T1346); the closed square represents a theoretical CD spectrum predicted for a "non-interaction model" whereas the actual CD spectra are represented by the closed circle (~).
FIG. 15. Polyacrylamide gel electrophoresis showing T1249 protection of the gp41 construct M41~178 from proteinase-K digestion; lane 1: primer marker; lane 2:
untreated M410178; lane 3: M41~178 incubated with proteinase-K; lane 4: untreated T1249; lane 5: T1249 incubated with proteinase-K; lane 6: M410178 incubated with T1249; lane 7: incubation of T1249 and M41~178 prior to addition of proteinase-K.
FIG. 16A-C. Pharmacokinetics of T1249 in Sprague-Dawley albino rats; FIG. 16A: pharmacokinetics of T1249 in a single dose administration by continuous subcutaneous infusion; FIG. 16B: Plasma pharmacokinetics of T1249 administered by subcutaneous injection (SC) or intravenous injection IV); FIG. 16C: Kinetic analysis of T1249 in lymph and plasma after intravenous administration.
FIG. 17A-B Pharmacokinetics of T1249 in cynomolgus monkeys; FIG. 17A: plasma pharmacokinetics of a single 0.8 mg/kg dose of T1249 via subcutaneous (SC) intravenous (IV) or intramuscular (IM) injection; FIG. 17B: Plasma pharmacokinetics of subcutaneously administered T1249 at three different dose levels (0.4 mg/kg, 0.8 mg/kg, and 1.6 mg/kg).
5. DETAILED DESCRIPTION OF THE INVENTION
Described herein are peptide sequences, referred to as enhancer peptide sequences, derived from various retroviral envelope (gp41) protein sequences that are capable of enhancing the pharmacokinetic properties of core polypeptides to which they are linked. Such enhancer peptide sequences can be utilized in methods for enhancing the pharmacokinetic properties of any core polypeptide through linkage of the enhancer peptide sequences to the core polypeptide to form a hybrid polypeptide with enhanced pharmacokinetic properties relative to the core polypeptide alone. The half life of a core peptide to which an enhancer peptide sequence or sequences has been attached can also be increased in vitro.
For example, attached enhancer peptide sequences can increase the half life of a core polypeptide when present in cell culture, tissue culture or patient samples, such as cell, tissue, or other samples.
The core polypeptides of the hybrid polypeptides of the invention comprise any peptide which may be introduced into a living system, for example, any peptide that can function as a therapeutic or prophylactic reagent useful for treatment or prevention of disease, or an imaging agent useful for imaging structures in vivo.
Also described herein are peptides, including peptides that contain enhancer peptide sequences, that exhibit anti-fusogenic and/or anti-viral activity. Further described herein are methods for utilizing such peptides, including methods for decreasing or inhibiting viral infection and/or viral induced cell fusion.
5.1. HYBRID POLYPEPTIDES
The hybrid polypeptides of the invention comprise at least one enhancer peptide sequence and a core polypeptide.
Preferably, the hybrid polypeptides of the invention comprise at least two enhancer peptide sequences and a core polypeptide, with at least one enhancer peptide present in the hybrid polypeptide amino to the core polypeptide and at least one enhancer peptide sequence present in the hybrid polypeptide carboxy to the core polypeptide.

The enhancer peptide sequences of the invention comprise peptide sequences originally derived from various retroviral envelope (gp 41) protein sequences, including HIV-1, HIV-2 and SIV sequences, and specific variations or modifications thereof described below. A core polypeptide can comprise any peptide sequence, preferably any peptide sequence that may be introduced into a living system, including, for example, peptides to be utilized for therapeutic, prophylactic or imaging purposes.
Typically, a hybrid polypeptide will range in length from about 10 to about 500 amino acid residues, with about 10 to about 100 amino acid residues in length being preferred, and about l0 to about 40 amino acids in length being most preferred.
While not wishing to be bound by any particular theory, the structure of the envelope protein is such that the putative a-helix region located in the C-terminal region of the protein is believed to associate with the leucine zipper region located in the N-terminal region of the protein.
Alignment of the N-terminal and C-terminal enhancer peptide sequence gp41 regions observed in all currently published isolate sequences of HIV-1, HIV-2 and SIV identified consensus amino acid sequences.
In particular, the following consensus amino acid sequences representing consensus enhancer peptide sequences were identified (the consensus sequences are listed below in forward and reverse orientations because said enhancer peptide sequences can be utilized either in forward or reverse orientation): "WXXWXXXI", "WXXWXXX", "WXXWXX", ny,~XWX", "WXXW", "WXXXWXWX", "XXXWXWX", "XXWXWX", "XWXWX", "WXWX", "WXXXWXW", "WXXXWX", "WXXXW", "IXXXWXXW", "XXXWXXW", "XXWXXW", "XWXXW", "XWXWXXXW", "XWXWXXX", "XWXWXX", "XWXWX", "XWXW", "WXWXXXW", or "XWXXXW", wherein X can be any amino acid, W represents tryptophan and I represents isoleucine.
Forward orientations of consensus amino acid sequences are shown in FIGS. 1 and 2.

Typically, an enhancer peptide sequence will be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues in length, with about 4 to about 20 residues in length being preferred, about 4 to about 10 residues in length being more preferred, and about 6 to about 8 residues in length being most preferred.
In a preferred embodiment of the invention, enhancer peptide sequences which may be used to enhance the pharmacokinetic properties of the resultant hybrid polypeptides comprise the specific enhancer peptide sequences depicted in FIGS. 2, 13, and Table 1, below. Among the most preferred enhancer peptide sequences are ones comprising the following amino sequence: "WQEWEQKI" and "WASLWEWF".
By way of example and not by way of limitation, Table 1, below, lists amino acid sequences that represent preferred embodiments of the enhancer peptide sequences of the enhancer i5 peptide sequences of the invention. It is to be understood that while the forward orientation of these sequences is depicted below, the reverse orientation of the sequences is also intended to fall within the scope of the present invention. For example, while the forward orientation of the enhancer peptide sequence "WMEWDREI" is depicted below, its reverse orientation, i.e., "IERDWEMW" is also intended to be included.

WMEWDREI
WQEWERKV
WQEWEQKV
MTWMEWDREI
NNMTWMEWDREI
WQEWEQKVRYLEANI
NNMTWQEWEZKVRYLEANI
WNWFI

WQEWDREISNYTSLI
WQEWEREISAYTSLI
WQEWDREI
WQEWEI
WNWF
WQEW

WQAW

WQEWEQKI

WASLWNWF

WASLFNFF

WDVFTNWL

WASLWEWF

EWASLWEWF

WEWF

EWEWF

IEWEWF

IEWEW

EWEW

WASLWEWF

WAGLWEWF

AKWASLWEWF

AEWASLWEWF

WASLWAWF

AEWASLWAWF

AKWASLWAWF

WAGLWAWF

AEWAGLWAWF

WASLWAW

AEWASLWAW

WAGLWAW

AEWAGLWAW

DKWEWF

IEWASLWEWF

IKWASLWEWF

DEWEWF

GGWASLWNWF

GGWNWF

In another preferred embodiment, particular enhancer peptide sequences of the invention comprise the enhancer peptide sequences depicted in FIGS. 2, 13 and Table 1 exhibiting conservative amino acid substitutions at one, two or three positions, wherein said substitutions do not abolish the ability of the enhancer peptide sequence to enhance the pharmacokinetic properties of a hybrid polypeptide relative to its corresponding core polypeptide.
Most preferably, such substitutions result in enhancer peptide sequences that fall within one of the enhancer 1~ peptide sequence consensus sequences. As such, generally, the substitutions are made at amino acid residues corresponding to the "X" positions depicted in the consensus amino acid sequences depicted above and in FIGS. 1 and 2.
"Conservative substitutions" refer to substitutions with amino acid residues of similar charge, size and/or hydrophobicity/hydrophilicity characteristics as the amino acid residue being substituted. Such amino acid characteristics are well known to those of skill in the art.
The present invention further provides enhancer peptide sequences comprising amino acid sequences of FIGS. 1, 2, 13 and Table 1 that are otherwise the same, but, that said enhancer peptide sequences comprise one or more amino acid additions (generally no greater than about 15 amino acid residues in length), deletions (for example, amino- or terminal- truncations) or non-conservative substitutions which nevertheless do not abolish the resulting enhancer peptide's ability to increase the pharmacokinetic properties Of core polypeptides to which they are linked relative to core polypeptides without such enhancer peptide sequences.
Additions are generally no greater than about 15 amino acid residues and can include additions of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 Or 15 COrisecutiVe amino acid residues. Preferably the total number of amino acid residues added to the original enhancer peptide is no greater than about 15 amino acid residues, more preferably no greater than about ten amino acid residues and most preferably no greater than about 5 amino acid residues.
Deletions are preferably deletions of no greater than about 3 amino acid residues in total (either consecutive or non-consecutive residues), more deletions preferably of 2 amino acids, most preferably deletions of single amino acids residues. Generally, deletions will be of amino acid residues corresponding to the "X" residues of the enhancer peptide consensus sequences.
Enhancer peptide sequences of the invention also comprise the particular enhancer peptide sequences depicted in FIGS. 2, 13 and Table 1 exhibiting one, two or three non-conservative amino acid substitutions, with two such substitutions being preferred and one such substitution being most preferred. "Non conservative" substitutions refer to substitutions with amino acid residues of dissimilar charge, size, and/or hydrophobicity/ hydrophilicity characteristics from the amino acid residue being replaced. Such amino acid characteristics are well known to those of skill in the art.
In addition, the amino acid substitutions need not be, and in certain embodiments preferably are not, restricted to the genetically encoded amino acids. Indeed, the peptides may contain genetically non-encoded amino acids. Thus, in addition to the naturally occurring genetically encoded amino acids, amino acid residues in the peptides may be substituted with naturally occurring non-encoded amino acids and synthetic amino acids.
Certain commonly encountered amino acids which provide useful substitutions include, but are not limited to, ~-alanine (~i-Ala) and other omega-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; a-aminoisobutyric acid (Aib); E-aminohexanoic acid (Aha); b-aminovaleric acid (Ava);
N-methylglycine or sarcosine (MeGly); ornithine (Orn);
citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-guG); N-methylisoleucine (Melle); phenylglycine (Phg);
cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 4-chlorophenylalanine (Phe(4-C1));
2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); (3-2-thienylalanine (Thi); methionine sulfoxide (MSO);
homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab);
p-aminophenylalanine (Phe(pNH2)); N-methyl valine (MeVal);
homocysteine (hCys), homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline (Hyp), homoproline (hero), N-methylated amino acids and peptoids (N-substituted glycines).
y,~hile in most instances, the amino acids of the peptide will be substituted with L-enantiomeric amino acids, the substitutions are not limited to L-enantiomeric amino acids.
Thus, also included in the definition of "mutated" or "altered" forms are those situations where an L-amino acid is replaced with an identical D-amino acid (ela., L-Arg ~ D-Arg) or with a D-amino acid of the same category or subcategory (era. , L-Arg -- D-Lys) , and vice versa.
It is to be understood that the present invention also contemplates peptide analogues wherein one or more amide linkage is optionally replaced with a linkage other than amide, preferably a substituted amide or an isostere of amide. Thus, while the amino acid residues within peptides are generally described in terms of amino acids, and preferred embodiments of the invention are exemplified by way of peptides, one having skill in the art will recognize that in embodiments having non-amide linkages, the term "amino acid" or "residue" as used herein refers to other bifunctional moieties bearing groups similar in structure to the side chains of the amino acids. In addition the amino acid residues may be blocked or unblocked.
Additionally, one or more amide linkages can be replaced with peptidomimetic or amide mimetic moieties which do not significantly interfere with the structure or activity of the peptides. Suitable amide mimetic moieties are described, for example, in Olson et al., 1993, J. Med. Chem. 36:3049.
Enhancer peptide sequences can be used to enhance the pharmacokinetic properties of the core polypeptide as either N-terminal, C-terminal, or - and C-terminal additions. While it is preferable for the enhancer peptide sequences to be utilized in a pairwise fashion, that is, preferably hybrid polypeptides comprise an enhancer peptide sequence at both the amino- and carboxy-termini, hybrid polypeptides can also comprise a single enhancer peptide, said peptide present at either the amino- or carboxy- terminus of the hybrid polypeptide. Further, the enhancer peptides can be used in either forward or reverse orientation, or in any possible combination, linked to a core polypeptide. It is noted that any of the enhancer peptides can be introduced at either the N-terminus or the C-terminus of the core polypeptide. Still further, multiple enhancer peptide sequences can be introduced to the N-, C-, or - and C-terminal positions of the hybrid polypeptides. Multiple enhancer peptide sequences can be linked directly one to another via the same sorts of linkages as used to link an enhancer peptide sequence to the core polypeptide (see below). In addition, an intervening amino acid sequence of the same sort as described below can also be present between one or more of the multiple enhancer peptide sequences. Multiple enhancer peptide sequences will typically contain from 2 to about 10 individual enhancer peptide sequences (of the same or different amino acid sequence), with about 2 to about 4 being preferred.
It is understood that the core polypeptide is generally linked to the enhancer peptides via a peptide amide linkage, although linkages other than amide linkages can be utilized to join the enhancer peptide sequences to the core polypeptides. Such linkages are well known to those of skill in the art and include, for example, any carbon-carbon, ester or chemical bond that functions to link the enhancer peptide sequences of the invention to a core peptide.

Typically, an enhances peptide sequence is linked directly to a core polypeptide. An enhances peptide sequence can also be attached to an intervening amino acid sequence present between the enhances peptide sequence and the core polypeptide. The intervening amino acid sequence can typically range in size from about 1 to about 50 amino acid residues in length, with about 1 to about l0 residues in length being preferred. The same sorts of linkages described for linking the enhances peptide to the core polypeptide can be used to link the enhances peptide to the intervening peptide.
As discussed for enhances peptide sequences, above, core and intervening amino acid sequences need not be restricted to the genetically encoded amino acids, but can comprise any of the amino acid arid linkage modifications described above.
The amino- and/or carboxy-termini of the resulting hybrid polypeptide can comprise an amino group (-NH1) or a carboxy (-COOH) group, respectively. Alternatively, the hybrid polypeptide amino-terminus may, for example, represent a hydrophobic group, including but not limited to carbobenzyl, dansyl, t-butoxycarbonyl, decanoyl, napthoyl or other carbohydrate group; an acetyl group; 9-fluorenylmethoxy-carbonyl (FMOC) group; or a modified, non-naturally occurring amino acid residue. Alternatively, the hybrid polypeptide carboxy-terminus can, for example, represent an amido group; a t-butoxycarbonyl group; or a modified non-naturally occurring amino acid residue. As a non-limiting example, the amino- and/or carboxy-termini of the resulting hybrid polypeptide can comprise any of the amino- and/or carboxy-terminal modifications depicted in the peptides shown in FIG. 13 or Table 2, below.
Typically, a hybrid polypeptide comprises an amino acid sequence that is a non-naturally occurring amino acid sequence. That is, typically, the amino acid sequence of a hybrid polypeptide, does not consist solely of the amino acid sequence of a fragment of an endogenous, naturally occurring polypeptide. In addition, a hybrid polypeptide is not intended to consist solely of a full-length, naturally occurring polypeptide.
Core polypeptides can comprise any polypeptide which may be introduced into a living system, for example, any polypeptide that can function as a pharmacologically useful polypeptide. Such core polypeptides can, for example, be useful for the treatment or prevention of disease, or for use in diagnostic or prognostic methods, including in vivo imaging methods. The lower size limit of a core polypeptide is typically about 4-6 amino acid residues. There is, theoretically, no core polypeptide upper size limit and, as such a core polypeptide can comprise any naturally occurring polypeptide or fragment thereof, or any modified or synthetic polypeptide. Typically, however, a core polypeptide ranges from about 4-6 amino acids to about 494-500 amino acids, with about 4 to about 94-100 amino acid residues being preferred and about 4 to about 34-40 amino acid residues being most preferred.
Examples of possible core polypeptides, provided solely as example and not by way of limitation, include, but are not limited to, growth factors, cytokines, therapeutic polypeptides, hormones, era., insulin, and peptide fragments of hormones, inhibitors or enhancers of cytokines, peptide growth and differentiation factors, interleukins, chemokines, interferons, colony stimulating factors, angiogenic factors, receptor ligands, agonists, antagonists or inverse agonists, peptide targeting agents such as imaging agents or cytotoxic targeting agents, and extracellular matrix proteins such as collagen, laminin, fibronectin and integrin to name a few.
In addition, possible core polypeptides may include viral or bacterial polypeptides that may function either directly or indirectly as immunogens or antigens, and thus may be useful in the treatment or prevention of pathological disease.
Representative examples of hybrid polypeptides which comprise core polypeptides derived from viral protein sequences are shown in FIG. 13, wherein the core polypeptide sequences are shaded. Core polypeptides also include, but are not limited to, the polypeptides disclosed in U.S. Patent No. 5,464,933, U.S. Patent No. 5,656,480 and WO 96/19495, each of which is incorporated herein by reference in its entirety.
Core polypeptide sequences can further include, but are not limited to the polypeptide sequences depicted in Table 2, below. It is noted that the peptides listed in Table 2 include hybrid polypeptides in addition to core polypeptides.
The sequence of the hybrid polypeptides will be apparent, however, in light of the terminal enhancer peptide sequences present as part of the hybrid polypeptides.

r TABLE 2 ~qwpo~ _ .
t a110~UlYBtYI,lQ~1 t NN~RAIFJIQ.IVW
i N~~DKIAfASI.WNWF
4 YT6L~l~QN44EK
a Aoxw~aaa~.~aAwmv~44u~G
6 ~J~.tVVNCiaC41A1AWUlYEHYIJCD4 T tR,AtEJl44faLQl.T~iaC41~1ARILAY
E V444HN11,ARIEA44~1~TilYfKiaC4L
9 RQI16GNQQQNNLLRAlEAQ4HLLQLT
IIITI.TiI4ARQLL6GMQQQNNURAlEIl4 12 11VS1.8NGV5V1.T8KVl.DLJQ~tIfiDKQU.
ti U 6TtIKAVY8L6NGYSV1.T8KVLDLICNY
16 Ao-~llilLEGEYNfatG&ALLSTNKAWSL6NG.NH2 19 Ac-U.SINKIIWSLSNGVSYLTSKVI.DIJWY.NH2 ZO Ao-YISUHSL1FFS4N4Q,EtQIE~Q~ r ~r nICVyABLWNWi'--NH2 r AC.NNU~u~4o~a.t~oLTVwoacamuuY~mruaD4~N2 a ActeswKa~accNGrDaucvra~cc4o~KnwAVrn.4u~uc4sT.N~u t3 Ac.~LSwKEwCCNGTDAIMa.IKQF3DKY-Mi2 I4 Ac~NKCNGTOAIMCLdK4O.DKYiQtHYTE4wi2 25 Ao-DAIMa.IKQO.OKYIWAViF~.QLLMQST.HHZ
I6 Ac.~CNGTDAKVta.IKQE1DKY1WAV1E1~U~HH2 tl AoawIGTi7~llMG.aCQQ.OKYKNAYtELQ,UrNH2 :8 Ao-~ISGVAVSKVI~IIFGEVNKaC6ALL6TNKAVYSLSHGY.HH2 ~9 Ao~6GVAVStM~iLEGEVNMKBAIIbT'HKAYYSI.SNG.NH2 80 AoY~ILE~iEVwQKSAtj~IHKAMt 6NGVGVLT8K~NHZ
i1 Ao,ARW~QRWGO,~OKYEEI1SKNYHYLFNEYARUQa.V-NttZ
=t Ao~R~AICQLmIiVB~I~KNYtnLI~VEYARLIGG.VGER-wt2 ii A~oNQ44NNLitWFJl4Q~I.TiIWriat4lrw;2 A~o~RIl1EA44MJA1t.'IVW~iaGalrtiARaJIY.wt2 ss Ao.4HU~LTirwcaaYawruao4.itt~
is Ao~ibGMq44NNURAtEJl4QE~t~QLT.wtx n A~'n-Ti~ARQLI~GtY~QQ4NwjRAIFJIQ~IHZ
i~ Ao~4lOVARSOa9adG='JIIRDTIit~AV~4SV~1S8.lMt :9 Ao,11AY11t.VEALOOAitSDa=lalG=JItEtOTNKAV4SV~S~iW2 ~t0 A~o,AICQARSDa~~Jlai~01'TtKAV~ISY4SSIGwNA~Ntt2 <t Ao~illAt~GVAT$A4tTAAYALVEAiG~IiSD.Mtt d2 AaATaA4ITAAYALYFJ4lO4AR80~~
Ao.MVAL
A~Q~JIIRQiMUlv4SY4S8Ki~I/A~M~
46 A~.~IRDIw~AV~QSVGIS8KiNttVAaCSVQD~Y~w12 d6 Ao-AV48vpS8IGMNMCSV4bYVlaSOlf.~ait TYW~GtK4LARtUWERYU~4~NElZ
GM44QNHlt.RAlP~l4QE8LQ~2 ASLWNVYRNHZ
4QEbIa:QEIIEr~Htt2 St IbiNNYZBUGSQ4QQ~t6G~19~wNA6t~wtt ~1-_. _ a wo~o~.~tcr~eant~a~nts~tz 66 AatTAIlFFA~I~IppWtiAYQr4lCWSWD'~i~Ht tS~tiH2 .60 Ao.DKWASLWt~NIIF~W2 61 A~NEQELLEx.pt~lfYABLVYNIM'rNH2 52 AaEJGIEQE<L,Et~IMfASLH2 63 AN~l4Qt~IGQF11~~K1AfASLYVNWFNH2 6~ Ac+~QNQQEfWEQEU.<ZDKHfASLWNYYF~NH2 65 AWHSLIEESQHQQEKbIEG~.OKWASLHf~NVF-HH2 66 Ao-NOQKt~SNNV~QNRQQSYSINISIIKF.E-Ntt2 6T Ac.0El~7AStSQVNElQN4SI~lFIRfGSOt3L~NH2 6E Ac.~V'SKGYSALRTGWYTSVITIEL6NIKEH-NH2 69 Ae-WSi.8I~IGVSVLTSKVLDUWYIOKQUrNH2 TO Ae~MINKIKSALLbTNKAYYStfiNGVSVLTSK~i~H2 T1 Ac~IINF~IDPLVFPSDEFOAStSQ'~NQSLAFIR.NH2 T2 Ac~II.VYAQWFIYOnRGYiNtiIILAQIAEA.NH2 TZ AN.NGVOLTFTIERYQARL1~ITYALVSKOASYRS.NH2 T4 Ac~LI.VLWUQLNRfiSYLKOSOFIl)MLO~IH2 16 Ac.LAE~IGEF.SYTmTEREmEEEREDEEE-NH2 T6 Aa.AI.IJIFaIGEFSYTEDTEREDTf~REDEEt~Nt:ART~HHZ
Tf AofTERSVDLYAAUaFAGEE~Vi~l1=REO1EEERE~H2 » Ao~ESYiEDIERED~tF~F.ART-NH2 19 Ao-VDLVAAUJIEAGEE$YTEDTER~TEE6.HH2 a0 Ao~HSEi:?iSWLYMII,AEAGt~SYT6~Nit2 i1 A~o~SYAAL~QFIYDVLImIfIHDALRMidO~A.~lH2 a AasNVFs a esoan.Yrac~st~sctN
ao- ~~
es ~toen.~a~eLe~weAaaHU~cu.T~auro~wLwE~ruaDaicH2 as osa~a~a~Aa~aa~ar~oL,~aaa~au~c.wE~m.taD~tHZ
a Awt~tcLwaduwnEraEt~AGNwA~au~ctwtcavA.r~tu W AoiIMTII,Qt:IIGtAIL~tRIOAtJIYRaMJRY'DtGIGl4~t2 f0 Aai.BNLI~SNNSOEIMFJILa~t(xiNKI.TqWGIgYEOE'.~2 9h L1NHAP.NH2 Aa' t.I~HINi'~ltEt2 n A,o.
99 AG~YrSUHSLIQQ~Iq~.I,~LOK~fASL~VNHIi'~t~iH2 t00 Aye.RWGQ,LFDEfY~IWYft~EYAItL~.YGiR~H2 - ~o~ Ao~aa~o~.n~aac~t~ow,RarwE~ruwn~a~
tat A~o~EtN:tJBptQKA5L1Mi1NRNH2 1as tas A~o.~r~ow.
tat Ao+~rot~, toe Ao.YOwvr~asoa~AStSavEt~asu~D~x~
106 Ac~DP~.VFPSOEF~SlS4YNEIaHCSIJD~RISSOEL4NH2 110 Ae.PLYI1'SOE~A~SISQVHQDI~tCSLAARK6D
tt1 AaI.VFPSDER7A81SQVHEKDI4SLAlIRiGSDnIJW.NH2 t12 AaVFPSOEFOASISQYNE~I~1SLJD~RICSOE~J~WWHH2 11s Ao.r~so~oAStsav~tacwasoee~uvH.~Hz 114 Ac-PSDF~AStSqVHp9HqSLAEIRID;OELU~INVHA,NH2 116 AcSDEfDASISQY~HQStAFiRICSpE~tWYNAG~
t16 Ac.DEFDAStSpVttEKIHQSLAFIRtGSDEV31NVNAGK~i2 11T Ac.E~ASiSpVNpaHqSLAFIRKSDE~HNVNAGi~.HH2 118 Ac.fDASISQYHEIQNqSLAFIRKSDE1L~1NVHAGIGST.HH2 119 Ac.OASISQVNEbNQSI~IFIRfCSOELLHNVNAGtGSTI'.NH2 1Z0 Ar..ASGYAVSKVLHLEGEVNb~AIl~'HKAyyg(~H~
ttt AeaGVAVSKYLHl~3EVMat~Atl6T~CAW5L6I~IG~IH2 1Z2 Ac~GVAYSKYU1LEGEYHb(GiA~bTHKAyyS~HGV-NH2 12s Ae VAVSKVLM~GEVNKtIf~AII~THKAWSLSHG11S~1H2 1Z< Ac,IWSKVLHtFGEVNIaKSA~tb'1NKAWSLSNGVSY.HH2 1t5 Ae~VSKYUiLEGEYNWKSALf.STHKAYY8LSNGVSVL.~IH2 tZ6 Ac.~SKYt~t.EQEYNK11CSAU.S11i1GlYYSLSNC,YSVLT-NH2 1a Awcvu~G~n~aaKSAUS~HKAVVSCSHGVSH.ts~
1t6 AcEVMQI~SAU~TNKAVYSLFNGVSVL.TBK~lH2 1Z9 Ac~UtL~GEVHKIf~AiI~NKAWSL6NGVSYLTSKY.HH2 130 Acct.EGE11t~1KdCSALtS7HKAWSL6NGySyLTSKVLrHH2 u1 Ao~G~vHwxsAU~auws~cw~.TSKV~D~Hz to A~GEV~aaKSAU srwuvvscsHGVSVt.TSK~
1:s A~o.GEVIiK~KAYiISLbHGVBVL'fSlM 0llG~NH2 ti4 I~o~EYMaIBALL.6'fHKAWSLSHGYBVL"I~KYLDLJW~E12 1:6 Acart~dmCSALIb~tCAVYSLSNGIfSy~,TStMatlWY~Hfit 1s6 Ao.MaICSAIIb~HKAYY8t~NGYSVLT8KVl~LtOYYi.Ntl2 1sT Ao.IOI~At~~VyS~T8KYL.0llQtYID.lt~tt tie AoiC6Atl~ttiluWStfittGY8V~.'~IM~lWY1014HHZ
1s8 AoiCBAV,b~puVYSL8HGV8YL.T8KV~OLJWY1DEGC~~llit Ao.6AU.STfIKAWS~tGY5H,T8KYl,DL~WYIWSGLrt~lH2 u1 Ao.AU s~rnuvvsvsHGVSUttsK~coumrrotaou.~H=
to Ao.
1~
.' ~M~ M~'R1i ~~Z
Y~NH2 t4T Ao. ~ Yl8~Gl2 ~d~
~~~~~~NAY~4-HH2 160 A~N.sNaø~CHq"~E~O,A~IMWAYIEI~rHHt 16t . 1H2 to Ibi09~10CllQtW1lM01i0E1B~IMGIAYfB~.IJIQ~t N0.

ti~6 oo,wsc t6T AAIIYALVFJ1l0 0JIRS~~IEAIRp~a1!

t6Q Ao~YIITBACUTAIIYAL.YFJ1l94AR~S~~IIIRt'.lalt 169 Ao~YIlT6AGtTAAYALYEAI~QARSWA~tRDTN.M~t t60 AaVAT6~t~TMYAWEJUGIaIIRSD~iRp?N[~.pHx t6t Ao,AT5AQ1TAAYAI.VEAICnARSOtE!ll,ICFJUR~~IMU.NHx 162 Aa'~A01'1'AAYALYEAICQARSOfEta~11RD7NKAY.NHZ

163 AcSAQ(TMYALYEJUC~AR~SDIE~FJIIRDTN((Ayr~f2 16~ Ac~AQtTIUWALVEAKQARSDtEtDJ~J~iRD1'HEUy~.~

165 Ae~lt1'MVALVEIIICpARSpIEIQiD'JURDTNKAV~1SV,NHx t66 AatTAAVALVEAKQAItS0IE14JCFJ11RDTNWViqSY~.NHx 16T Ac.TMVALVEAKQARSWEM.tSEAiRDTNKIIYQSY4S.NH2 the Ao,MVALVEAKQARSDIE~.IffJItRDTHIUypS</qSS~IHx 169 A~.AVALVEAKAARSD(E1UJ~URDTHKIIVCSV~QSSf~Ntix tT0 Ar.~VALVEAICAARSOIE~J~IIRDTHiuYQSV~SS~2 tT1 Ao,AI.VEJ~ICCAR~SOa3QJ~A1RDTNI~IlY4SV~1SSIGlYlatx tTt Ac~LVEAiCGARSDIFJQ~JURDTrIKAV~SynsStG~2 1T3 AaYF~IKpARSO(p~AIROTHKIIVK~SV~QSS1GMJ~NHx 1T4 AcfJUCOARSDa3aJCFJURDTItKAVrI,SYGISSt~3NLN.~2 1t6 At~ICGARSOfE't~JCEA1RDTNKAV~L1SVQSSIGMJYAl~al2 n6 Ao~IAR,SDa~Q.KF~ROT~iKAV~y~S(~y t>? Ao ARSOaxi(a' , Y~iSSICiHUYAaGS~~l2 JIfRflTNKAVQS

1Te Ao.RSOa~,~F,AiRpTNtuINQSV~SSKiNUYAaCSV~i2 tTa Aa~DtO~JIfRDnqCAV~QSVr ~ SSIGNINAa~Hx tQ0 Ao.t7a~QJ~JitRD'traCAIPC~VG1SSK3NtNAp~p.(~x tat AaA~rau~asvnsstc~anrAacsv~aa~rraix yea.~~tR~arHtuvr~sv~asscc~uvAacs~rr.>Iflnrwr~wx tss Ao~auc~aurrwuv~asv~ta~rAacswao~rvN,NHx ts~ ~~o~auv~asvrxsstc~rlwAacsva,~rvN~Hx _ us Ao.mauvasvasstam vAacsvrxoYV~aas~lHx tas Ac~Ratrauv~sv~asstcNwAace.~atx tar A
~uRa o, a~uaavrwx nauvr~vrxss~c~NwA

tae A
i ~RDnauvnsv~ass cavt~wx c~Nw~n tag Ao-Y~nan~svA,,oPto~sa~.~awcsocm9xas~~

teo Ao-tpNOCrcea~sYAt oc~s t>it Ao~PNOrtLNNSYAI.OPa7lSa~lIWC60L~S1 t1<t A~o~~tl~tSlfIlLOp~Sa3MWC6DL~ESta~Y~tH2 t!s IhyOflll~a~ISY

~e~ Ao~>~NSVAe~oaa3.~awceot~aewa:RS.~aix tf6 Ao-ILtWSI/

t% A,oV

t9T Ilo~l~81/

the Ao.Ngy >e00 I~o.~VAL1~1O1Sa3t~aWCS0L~S1~MRRSNmp,OS~Hx >001 A~,AI.OPIO~SalHICAICSOI~StarlNail~HCl~Sf~ai2 . . . .a~s~

T
~0! Ao~iOLF~IaINl~N4l~8tOMAM4SST.HHt !tO Ao.EiNIWC80L~SKE1MRRSH~tGMM~QSSTT~~tH2 !11 AaEIRAIRGQRALRGExRAUtGEIRAIRGEt~IRGK-Nti2 Z>= AaYT6LIHSL1E~4HQ4Q(WFd~KWAS1.VYHWF~iHx tis Ae~YTSI.SiSt~QNQQEKH~~r r ar u~yyA8LifYN~YRt~IH2 !t4 Ac.YTSLIHSLS~SCNQQEKNEQEL.LELDKIAfASLWHWF NH2 t16 AaYTSUHSUQaQNQQE~Q~IECELIEI~KWASLIfYMNF-HH2 :16 Ac-YTSUHSUQQSQN44GKNQQQU.QLJ~lKWA6LHrtJWf'~-HHZ
!17 AaC~IF~l.,EI.OKWA8LxYNHIf'~.HH2 !ta Ac~QEU.ELOKWASLWNWFNHt =t1 A~E~KIAfA8LV4N1M'-~IH2 t20 Ac~I.ELDtMfASLWNW~iHZ
~ ttt Aca.EIDKIAtASLWMfYF~NH2 ~ ttt Ac-A.OKIAfABt~fYNHrt'-.tlH2 -tt6 Ac~WASL~Mlflff'~NH2 ~?Zl Ae,ASLWHWF~HH2 t29 Ac~YTSUHSLS~SCNQQE~Q~IEQ~LLELDKWASt.ANM~HH2 =30 Ac.YTSLIfiSLfE~QNQQElWF4QL1a0KWA8LWNWF.NH2 ~t:1 Ac.Yi5U4SUE~4MQaFxWQCEti~.DKWASLtIYNWF.NH2 ~tS6 Ao.PSLRDP1SAOSICALS1CA~GGOWiMF~~Y~G.NH2 bT Ao-StRDPISAEE;IMIbYAUGGOWtCVL6~iYSGG~HEt2 ~ >tiE Ao~I.ROPISAASIQAL~YAI.~aGWI~IKVLFJC~tiYSa(iD.l~lH2 ti9 Au~R~PISA~IOAI~Y
~llfS A~OP~SAOSIAALbY
~!S1 Ao~fSABSiQAL6Y
' Ztt Ao~SAEfSIGAtBY
!~ A~o.6AASIMtSY .
tA~ Ao~A~tQAL6Y
u5 Ao~3SlOIltBY
f~t6 I~o~StOAI~Y
Ao~6IGAL6Y
u3 Ao.l~
Ao.~tBY
>e61 AoitY
I~o.POJI
:~
Ao-»T
» Ae.E~11~t3PP~,S
SSo -~b-T~ _ t66 Ao.tSL~Rl~lfriTtil.~GNAW~tlAl~6SDQIRS.NHt t6T Ao.6t.~LD~i~NU~3NAWlaFDAtCEI.IFSSpOa.RSf~tJH2 >D6a II~aFRLDI1~QTHUGNAW~DAKF11ESSD4llliSWC~HH2 !69 . AC~1MRRSNQf~.OSt~Nt~
t10 Ac~FI.DKWASLANARtrH2 Ili Ac.l~3.OKWASLFHFF~IfiZ
tT2 Ac~3.OKWASIJIHIfVF~iH2 tT3 AaI~tDKWA8L1AMA~iH2 n4 Ac~LGNVNHSISNAIDl4.E~SHStU.OKVHVKLTSTSA-NH2 tT6 Ac-TEt~HVHHSISHAt.O~ESHStQ.OKVHVKLTSTS-NH2 !T6 AcSTELGHVKHSISNALDtQ~ESHSKtDKVHVKLTST.NH2 tTf Ac-ISTE1~GHVHHStSHALDf4~ESNSK1.DKVNVKLTS~IH2 tT8 Ac~Ot,STELGNVNNSISNA~Db.EE,SHSIa.DKVHVia.T-NH2 I~1S1~1~GHYHHStSHAI.OIG,FFSHSKLDKVt~M~rH(i2 ta0 Ac.t~~T~L~GNYHHStSNAIDIG~SHSICI.OKVHVK~IH2 tat Ac-GHLOtSTE~GNVHNStSNAL~OIQ.E1~HSIMKVNY~HH2 tat Ao-TGNt~G~HYNHSISNALOta.IF~SHSta.dKVH-NH2 tEa A~IITGt~tL~tSTII~GHVNHStSNAL0IaFFSHSta.DKV~HH2 ta4 AWVtGNt~tS'lEl~(iHVNHS1SNM~I~NSfa.DK NH2 ta5 AcNIVI~GHI,D~GHYNHS1SNAL0fa~SHSlCD~2.
tab Ac.QHVI~GM.01ST1:1GHVHNStSNAt~I~HSt~NH2 taT A NALDta.EESHSi~rNli2 ma AaDSsQVNTGt~d.OiSTELGHVNNSISNAI.OIS~ESHS.HH2 i69 AdLDSQYIVrf'at~STEt~GMIHHSISNAIDWgSN~Ii2 t~0 AoILOSQYJV1~GHLDISTEJJGHVNHStSNIILDi~~i~W2 !a6 Ao-~T Aa A~
tlta A~DA
t00 Ao~E~DA
>b1 II~o.~pA
»
t03 Ao~tiET~l »4 . Ao~Gt~DA .~
>I05 Ao~RL6GH~A
:0't' AaTLR~GEEDA ' Ao~TI.RL6G~DATY4lQ~Slt~SQYrVniM.DIS~.HH2 i09 A~ifilliL~SGE~CAItD.SQYIYIxiW.J~,T~t~t i1a Ao-TA'iIFJIYEiEYIflGt~LAYAV~4QFYliD~T~!Wt _26_ WO 99/59615 PCT/tJS99/11219 T ~ -iii Aoi~lA?IFJIYtiAV~DOL84LAYAVrilalQ~~1'I~tZ
!ti tTA7IEJlYHEYTDOL~LJlYAW3l~QMYN~t i1T Ao~.RI~~StTAtIEIIYHEY~D~iL.BGIJIYAVI3itIiQQ~Y.Mi2 !ts ANI.RIJCESfTA'tIEAYHEYTDGL601~lYAV'~12 i19~ Ao-Hit.RLI~SITA11EAVHEYtDGL6GUlYA44~1H2 i20 Ac,AHn.RLICES~1'A'IIEJIVHE'~fTDGL~GAYAVCi~4-HH2 iZt Ac~AANA.WJ~EStTATIEAVHEYTDGL64lJlYAYOtW~t~lH2 i2t Ao~tIKCDDECI~tISYKN(3TYDYPKIfEi~KLI~IRNEtICGV.NH2 iZt AcaCCODECIdHSVIWCIYDYPKYEEESKIIIRt~KQVfC~Ili2 its Ac~DOEI'~NSVtWGTYDYPf(YEEESIa.HRt~IEIKGVWrtiH2 u5 Ac-0DEC1AHSVKNaTYDYPKYEEEStd.NHH~KGVK<,Sd~lH2 u6 Ac.~OEC~ANSVKHCiTYDYPKYEEESKU~IRHEIKGVW.SS~iH2 rn A~cxuwsvtwc~r~rowK~sKU~NEUCCiwass~.~nt2 t2a At~NINSVKNGTYDYPKYEEESKLHRHEUCGYfa.~SSMt3~1H2 i29 Ac~tNSVfM(i'iYDYPKYEFEStD.tIRHEtKaYKt.SSIAGV-Wi2 ii0 Ac~tSVIWCiTYO'YPI~YF~SKLNRH6KGVI~L6SfdGVY~HH2 i31 AcSYKNGtYDYPKYEEaKUtRHE3iCGVKt.SSfItGYYQ.NH2 is2 AcrVIWG?YDYPKYEEFSKilrRHEtKaVtQ~S~IIGYYQf-NH2 i33 AcaWf3TYOYPKYE~E&KLHRNEIKaVi(I~SIiI6VlfQlt,.NH2 is4 Ac~AFIRKSDELIiMLHH2 !I6 Ac~WIJIGAALGVATMMA sC'IALHaSWJiSGAlON4~W2 ii6 A~lll~lGML~tiYATAAatTAGUIUiGtSW.t~IS4AlONtR-NH2 iS7 AN.AGAAL~sVATAIIQfTAG~L,HD~VI~IS4A1DNLRA~IH2 i3S Ac~AaAA(~GYATAAQiTAGIALHQSIdUIS~IA1DNLRAS~H(i2 i39 Ac~iAALiGVATIUIQITAGtAUiQSIIIWSGNDNLRASi~NH2 710 Ae,AAId'sYATAIIG(TA~LHQS~SQNONt.RA~SL~2 itt Ao,AL~GIIATIIAGrt'AGW.fi~SWJiSQAIOb.RAStF'GHHZ
itt Ao~GYATAAQfi'A~AI~t~W~SQA1DMRASt~IT.NH2 l~.t Ao~iYATAIIC~TA
f~ AWATIIAAfI'A ~
it6 Ao,ATAA41TA
i~6 AaTAAGtTA
i~tT Ao~MMA
:~
i~9 Ae~ITAGIAUWS
i60 Ao~tTAGIAL~tCS~pNpNLRAgL~fT~IGIIIEAIR~IH'Z
!51 AaTACiIAIJtaSW.I~SCIIIVlIf.RAStE11H4AtEN~Q~Et2 i6t »
i5t >~
i6T
. ., i68 A,o.ASW~CAIDNLitASt~T~iGAIE~~L~W2 i69 I1M.SNLHSOAIDNI.RIISI~TTH4lllEA~lG~E141tJl~t~IH2 Y~HHZ
!6f T .
" » Ao~ll~llt~L,El~O~Rl~~luVi~Nt » AaNLRASLE1THQAlEAIR~IIGQI~YQGYI~pYtH~it i69 AcIRASLEtItICAIG~CAGQE~.I~YQGy~pyWN.NH2 i'10 AaRASLETTNQAIEAIRQAG4E»uYQGYQO~YINN~ti2 n1 Ae-YTEVITIEL6NQ~NKUNt31DAY14.JKQEI~lM4NH2 :» AaTSHTIEL6Ht~MNN(iTWIVKI.IiCiqgpKYIWdYH2 iTJ AcSV~WI~INGTpAyb,p~qapKYKHA,1,1H2 i7< Ac~NU~NKUNGTDAIMMWEf.OKYIWAYTELAL4NH2 n6 Ae~HKUN(3TDAK111Q.IKCEIDKYKNAYfELQLIJKQS.NHt ' n6 Ac.d.E~KWASLWNWFC.i~lH2 m A~cxaoKwasuNw~.NH~
m A~KwAS~~u i19 AaYTSLNiSt.lEES4NQQE3WGQElI.ELDKHIASL~f~tF~IH2 sat w iat Ao~(VEQJ.SK~N~pKWASI.WNHT~~IH2 ia3 Ac~tMtCpLmKYE~l~t~.E~RRSN4lCL.DS~2 i84 Ac.~~AIGQtEDKYIEFIt.SIDJIFtfiICSDEIU1NVV NH2 ses A~uxcEC~A~.a~E~KwAS~wNw~-.r~Hz ia6 ~p~a~KAKSOLgStCEVNHR~SNpIq,pSI-NHZ
iaT AaCN~I.SOSFP1~PQY.NH2 iaa Ao~A~OpyLGRPEQA~.DPS4HEi2 ia9 Ao~F'SSWD~dDiq~S~H2 i90 AaIWQEWEEit(YDt~d1'~4lqqpQ,l~i~tH2 iS't Ao~WCE:'WE~ti(VDR~11'ALl~lGt44E1WwYELtSC HN2 ~~o~IEW~YOR~TAU~A0,1440WIlYELOitQrNH2 i~ Ao~YIfERINDE~TAt~,FFAqlqp~~al~l~IN2 tND~TAI~FJIGIICQEt~Y~,~tQ~g~
i~5 Ao~RIND~AU~JlCI4QE2WWYQrt>l~l.'iW~tElt !!6 Ao~tiICYD~~~q~WD,~2 !~T Ao~KVDF~ttTALt~EAWYBAII~SWDY~Wt2 AO~IIDH,~~TALLEF~Q09W VYBAfIad~SWDVE"~~f~
i99 Ao~H.~ltT~qlq~,l~q~Ht Ao~A.EF~~ff1' Ao~i~
Ao~'AV.~ACI4QElQ~tVYBAII~tSWD'YF~iMAIE'~W~t E'~tB.riIfAI~.OLIRdR~IGiV~iIIWOIQ.NHt ~i~6 Ao~IQa~'VVIQbQ~3J~L.~W~~~i~IN't Ao.4CM10YhCt~CIQBI.RL1V~VCiIiWU~TEt'Y~'AtEiM.l~.tiH2 d0r . Ao~IC~riMIRLIVW~iPE~tI~RIItAtpMl~~EIZ
~tO~pIM.~MQ41<GlLqLIL~'N~WYRt~IRY~Mi2 AaQQqLl~yy~qqgl,RL.IYWC~IIQ~t~IRIItAIEKY~NEt2 ~tt0 Ao-4CLtaNYfQi~qCAIRI.TV~TIQtU4TR11fAfACYL~NH2 41t Ao~"~,OWISIbQQaJ,RLtVYV~iTiQ~TiiVi'AfEIMaGNH2 su AouwHaRaae~uutY~taAmrcrc~ao~
~t: aaaa~eu.~rtvw~na~a~rRVrAracYUa~~a~z ~e~s ~o.o~wtarax,~aeus~.tvwr~na~~oc»YrA~arnuaoo~ea~e~

1'.
ItT Iboi~LtL~YW~iTt~AVfAIAM.l~141~N.Hi~tt 41i lb~EbQGBj~'iLTYW~i11W1Ja11tYTAtA~YLIGDQIIQLItA,~NHt 41i Ao4~~TVYVri~tlQiLQTRVTIIfEKYLKDC~ICLHAYW~Ht 4t0 A~1LLRLtVWCiTIQII~QTRVTAfEIM.I~QIICLNIlHKW2 ~1 Ac.HIRI.ZYW~i'fIMLQTRYfAIEKYLKDMCWAH~GC.~W2 4tt Ao.HNURAlEAQQtdlrQt.TYIIIKiPKQtJGARIUIYERYUCt7Q.tdH2 413 AcSELEtICRYKNRYASRKCIiAKI~CGLL~QHYEtEVAAAK.NH2 424 Act.EItSRYIQrItVASRKCRA1Q-ICQILQHYREYMAK&~NH2 4Z5 Ac4.EIKRYiWRVASRKCRIIItFiCQ~L~iYREIIAAIUCSS-NH2 tZ6 Il~tQtYIQ~iRVA8RKCRAtCFiCQU~QttYRF~YAAAKSSE~NH2 42T AaI~YtWRVASRICCRAKFiCQtI~QHYREYMAK&SEN-HH2 4za Ac~arnwRVASwCCru~cau cHYC~YA~AtcssEND.rrH2 4Z9 Ac~RYIWRVASRKCRA1CFKQtLQHYitEYAAAKSS1:TI0R-NHZ
430 Ac.YIQiRVASRKCRARFKQLI~tittYREIfAAAICSSENORirNH2 4l1 Ac4WRVASRKCRAKFfCQtJ.~GHYREVAAAKSSENDRLR.~IH2 43Z A~HRVASRKCRJIIWWtL4ltlfREYMA1CSSE?iDRLEt4~IN2 433 Ac.RYASRKCRAtWCG1LU41tYREVMAKSSENDRLRlIrHHZ
4S4 Ac~VASRtCCWIICFiQAtI~CHYREVMA1GSSEHORtR111rNHZ
~S Ac.ASRKCRAtfFKGl3~HYREI/AAAKSSENDRtR111.K-HH2 X1.16 Ac.SRKCRAtWC4tl~GtiYR~yAAAKSSEHDRlR111XQ~1H2 4n AbRIC~CRA!(i~CQf,L4HYREIfAAAKSSENORL.R1ILCQaII~lH2 433 ANCCRI1l~10Qt.L0,E~lYEt1':yMAKSSE~IOfLRLI~.ICQAdC~t~WZ
~t39 Ao4;RAlCFKALLiQkfYREVAAAtGSSEt~RiRLIl.IWIIACP.~tf2 41o AcaiAtCFIC~QL1~0E11fitEVMAKSSENOftIRLUJ44lidCPS~H2 441 Ao,MC~iC4L1.~1fIYREYAAAKSSfI~IDRLRLIJ.ICQIIICPSL~i2 44Z AoiQ~CCLJ,~tfHYREYMARSSENDRi~uJLCQItCP8~D.NH2 s4i IbfICALLAEtYREVMAICSSENORLRIILCtWCP8i~V-NH2 ~4 Ao.IG~fIJCiIYRE:If 146 A~o.QIl~iIfR~Y
44T IIYo~WHYREVAMI~SE~DRLRLtlJCAIICPSt,DV~I.HH2 449 A~o~ttlfREV
460 Ao-YREY .~q~
461 Ao.REIfM~ICSSEHDRLRiJ.UC~CPSt~VDSiPRTPWH2 46t A~.EYENDEiIRLLiJGCIKCPSL0VOS4PRTP04tH3 A~
46t 461 A~OtBRUJJCWI
VLLDY~QC1114N1t2 ii6 ~ AaOYRWIiG~LRREi3H.61fL~Ut~.YLL0YQG111RHH2 T

. as ~ou~a~cr~ouruw ~N=

. s~o Ao.cu~xFaiucuRr ~

.w U.N~
rnaH

. ~t A~uLCUFU.vu~Ynaw.PVrx uw~Hz i4t Ac~RWFLFaILCtJFII.YLI.DYQGW.pV~CpI,IP3.NH2 6t3 Ao~iHtR.RIJLCUf~.LVLLDY4G~PV~I~iS~tti2 6~4 Ao~FLFaJI
CUf~
w OYQG~

~ SS.HH2 .
PUPG

i45 ActIft.RI.ILCiJFt.I.YLLOY4Ga~Y~CPUPGSST.Htix i46 Ao.tFLFIU.I~CLtFI.LYLIDYQGWLpY~UPGSSn'NH2 6R AG~~ ~ ~ ~~ ~ ~ VLt~YQGIdt.~MCpUpGSSTI~IH2 i~ta ActFILlJ~CUA1.VLLDYQGMLPVGPI~I~GSST1~'1'NHZ

i~t9 Ac.FILLIrCtJRI.VLLDYQGM1.PIICi~RGSSTTIi'~G.NH2 650 AoIL.I.LCLIFLLVLLDYQGI~PGSSTTSTGp.HH2 651 AcaJ~I,IFtJ.wl7YCGMLPV'CPLIPGSST15TGPC.NH2 652 A~Il~FIlYLLDYQGMLPVCPLJPGSSTISTGpCR.NHt ' 663 Ac.~UFU.w,~DYG(Si~.PYCPLJP~iSSTISTGPCRT.HH2 65< Ac.aJRawpy 655 Ac~lFiL.YLLDYQG

656 Ao.lHlVII~YCIGPUPGSS'1T5TGPCRT~IT.NH2 65T AcrFU.VtIOYQGMt.PY~CPtJPGSSTTS1'GpCRTCM1T-NH2 iss Ae~LLVI~OAGFFLLTRILT1PQSL~SW4YTSlNi~GTHH2 66! AaU.VI.QAGl3U.TR!(.TtPQSLD,StlYW1'Sf.NFLGGT'f'NHZ

i60 Aei.VI~OA s~.L'fRtLTt~LDSIfY~Y1'6LJ~GGZTVHH2 66t Ao~IIGR~L,T~LT6~LOSWWTSLHR~GGT1;V~CHHZ

662 AoI~QA~tI.TEm.TIPQSLGSWWI~I~JFt~IsGmC4HHZ

i6s Ao.pAG~.,TRIt.T(PQSLDS~VYYfSLNt~iGITYG~G~2 Ao,AG~.TR6.

f66 Ao~.I.TRIL

i~66 Ao6~llTit6.

A~lTt~.

Aoai.TR0.

669 Ao.i.TRll. _ iT0 h~o~AfNWUiAWICDLE~LLF~

1 Mit 1mEL~IWitr i'f'f RI~N1RAI~iLIrQL.TYW~It srz Ao.oGG~a~atw~t~,nau~.uu.T~ucaua~a~AVa~YUCOa-NH:

iT4 Clit~O

Ao~

6 .

aQYlitiLl.ORt~tPI.YDG1.R~~DYIYBN~li#f~

iTe IbIfSA.?~OGNIGSLAEICGII~QOtA8t.1fR1f61'A.~Wt AollN~IRLpLLtWIHiI~YRypS~q~yyY.~2 iE1 I~o~6YPlVL.6lA .

662 AolJCENRL!'iNKAVr~SV~QSSfGMJVAnC6.NHZ

6E3 NMJ.RAIPJl44E~.tVllVril~Q~Q,I~tRILAVERYLKO~lH2 i6s WttJ
RNEA40HUA1L?VNKi69~

. YUt2 pI~RIUIVER

664 A1041~ISB.YPL'lSl.

~sa ~rwa~naa~tu~a~T

-so-Z' I~aRPaNIf~0H
~INYABGYVNVVPC~cycac) C~ItIAfASIJINIIYYFC.(ayaao) sst ctaotcvYASt~N~.c~uc) s9s AaNNU.Ew~Qa~aH~.ivwGaca~aAwmvam~
i95 Ac-0GGYTBt~IiSLIQQEJWE4tILELD(i1MA81,WNHIF~lH2 i96 Ae.~lVLIt.IAGFFIITRIL~PQSLOSWW1'SI,HF~G(i1'~NN2 69T Ac~LLV1~11lGfFla.TRILTa~QSLaSWYYTStIiFUG~(iTT~NlI2 f93. AN.YL~RiIGFF~i11R1LT1PCSL~SWWTBUtR~GGITN~NH2 689 Ae~Vt~QAGFFLLTR1LTIPCStDSWYYI'SL~IF~GGT1Y~'..t~IH2 iQ0 A~i~AGFFLI.TRtI.TIPQSL~SVWYTSt~GTIVGlrNH2 i01 . AG~4AGFFLi.TRtLTIPQSIDSWWTSLNf~.~'sGITYaG~~1H2 i02 Ac~AGFRt.TWLtIPQSLOSWWT6WFLGGTNCL~GQ~iH2 i0s Ac..GFFL,L?RILTIPQSLDSWW~SLNFLGGTNC:GGQN~IH2 i0< Ao-FFIITRiLtIP4SLDSVYVY1BIJ~IFLrGGTZV'Ct~GOHS.NH2 605 ACfILTRa.TIPQSLDSV1NYISUdFUGGTTY~t~('aQNSQ~IHZ
E06 AN1.TRILTIPQSLDSWWfsLIJI~GGTTV~GQNSQS~NH2 60T Aci.?Rn.TIPQS~DSHNVTSU~1FLGG?TYGI.GQNSCSP.NH2 608 Ac.~EIOKWASI.VYNWA~tH2 609 A~tEL.OKIAfASAHINWF~HH2 i10 Ac~I.OKAASLrYNYYF~NH2 i11 Aoa.KLDIMfASLWHHfF.NHZ
i12 Ao~7lS1dYA81.HrttiNF~NH2 t1= AaOEft.EWVNAG1CST-Htt2 6th ActCSOEtI~tiVNAGICST.NH2 itb Ao.a'i~pEVliNVHAGICST.NHZ
S16 Ao,ARRlC60EtJ~HVNA st'ECST.NH2 M
i16 Ao-Y VGIDS~
H9 Ao.6HA0~1l~A
i20 A~o.~INV1'YNABI.YlS4FHE~TLOE~SNVIWLYOr~VR16Q4NH't Stt tti ASLWNWF'~Ntt2 i~6 Ao~Sa3MWC6 ~ttt Ao.NQ~30H6013L8.DlMlASLWNWR~1t11M~AfYa~i~IHZ
~3t Ao~,Sqr( aI
fS<
_$1-T
i41 AaHHYTSt.fEiSIJ~nHnnDW6nEZL,Et~KWAHLWMi~tt2 .~H2 t~3 IIe.EWMf'fSt.IEiSLI~nNQQAQ~IB~E~.OIMfASI.~W2 i~t4 Ac~NNYIStaIiSLI~nHQnEtW80E71ELOKWA~SJ~tH2 i~S Ac.DRE~WYT8tJHSt~SnNQQFJQ~EQEU~LOKHfA-HN2 N6 Ac.WDREtHHYrSUHSLIEFSnNQQE~~IC-nELLELOKIfIf.I~lH2 i67 Ac.~lfWR6HHY~SUNSL~N4QEKH8QE31F.i~K~lH2 i~ Ae.H<ESNORE,IHHYISt~iSLtEESnH~EIWE4E"LL~ ~.NH2 i48 A~V~WpEtEtHHYTSUHSL~SnHQQEIC~lE4E'LI~LrHH2 ib0 Ac.TYYMEWDREiNtiYTSUEiSUEE&nNQQEiWEQEIIF~iH2 i61 Ac.idISNWIEWDtiE7t~Y~SI~HSIIE~nHQOEfWEQELL,Ht(Z
65Z Ac.t~IMIYNfAEWDRE1NHYTStItiSL.1~ESnHnQEfWEQELrlIH2 653 Ac~IHMTVYWiEiNDRE~HNYI~LIf~nHQnAWEnE-HH2 A QHQnEtWEn.NH2 ib5 A qH~~~
656 A~Git4~IHI~VYIdEWDREWHYTSt~tiSLI~QHQQ6lQi.NH2 657 Ac~QMMH1~1WMEVWREI~iHYTStJEISL.a:ESnNQQE~tH2 66a A LlEESQHQQErNH2 659 qq~
~SIna~W2 i6t 662 AcSt~iCSOEI~iHVNA~S'fi~Wt i6; /Io~DASISnYHEXINnStJIHRKeHH2 LWHWF~HH2 ids ~~t GICBT~f~
67s IIc.QVNEJaltnSIJI~DA.1.HHYNAGlC81'.t6;2 iT4 llo~li~aHnStJIl~iiCSD9.I.lWVHA~ST-!Wt ~1~2 iT6 Ao~IInSLA~OAt~WVWIIitCBT~~t 67t A~e~N~A~LB.YP0846~1MfAlr~WNHfP~HHt AaCQGI~IEJIQCH~t.TWYri40~1ARfLIIV~tYI~QiQ.HH2 ~w~a~Ww~.n ~Anc~a~.Tw~uaneJa~wuve~uaon nvt~a~
s>a ..
..... ...

'p.
N0.
T<1 t~
t~.t Ao.N~~INNCUiJIIFJl44~tr~1LZW1~ia0~rGIlRtUIYFR~IHt t~' Ao-0MQQGWN~J.RAtEAQRI~L~Rt.TiIYN~iIIWtrRARILAY8.NH2 T~6 Ao.6GN~4QGNMIRAIEJlQ4HLLQLTtpL,iqJIRlIJIY.NH2 T6a AetiSf~lLtY~QARO~l.~6GIVb44NNl1RAlFJl4Gl~LQt,N.HH2 760 Ac~iARSf,AILT1~IARQlI6GMQaQNNLLRAIEAQ~ILLQlrHH2 76~ Ac.GST~IGARS~1'I,TiKIAR~GNnQQNNLiRAIEIIQ~i~IHt 765 Ac~STI6GARSWTLTYCARpL.~GIVGqqHNq~H2 7ss A~GSnkGARSWmtva~ARau.~GN~A,EAaa-NHI
T6T Ae-RA~KQLLqHYREYMAKSSE~IORtRIIrNH2 T68 Ac,IUDGKQLL~HYRLVMAIGSSp~tDRt~NHI
769 AcaQ~KQLt~QHY(tEyAAA~SENORtRI,LLK.HHZ
770 Ac.F'KCU.~QHYREyA~AKSSENDRtR11LC4NH2 7Tt Ao.RAtOFtCQELQHYREYMAKSSEHDWRVI.tCQMC~S.tiHZ
7T2 OKWASt,WINHO~J~2 TT3 Bladn.FOASSSQVNElCINQSIJIRRICSD~NVNAGfGST~IH2 TT4 Ar.~YDAStSQVHEIpNCSIJIFIRKSDELLHNVNAGICST~Mi2 Tf6 Ao-Y'DASiSqVNpDHQSIJIYIRKS0E11~E1NVNAG1GST~NH2 Tl6 Ac~OASISQYNEl4NQStAYiWCSOEIIHNyNp(3tGST~NH2 m A~SSS4V~QE'IGQQSLAARICSDB.WGqy~GIGbT~NH2 7'Ta A
1SCVHEIaNGAI~IF~DE~NVNAGtCST~NH2 GtCS'f-NIi2 1M AaYWISISqY~Q~paqp~~p~~eqy~GKST~~2 A~S1SQ'~EIQNQSIJIpRICSD01ENVHAGI~T.HHZ
GtCS1'~
>6s Ta6 A~a~VYPSOEYD4l51S4VHF~NCA~LAYI~UIOA~V~
OS4VN~-2I~i4S0B1J1NV~t~W2 auna~Ri~liZ
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b~irliKZ
t~~ b~~~
oaxw~s~yc ... ww",s 71sT Ito.TA
vs~nn Tll6 Ao-TTA
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H0.
~ ~

HHt 1~

i1o Ao~S0ET~6tSQYNh~IqSIJII~RlG60U1AANWNli2 H! Ae~11FP8~F.FOJ1SISQVti~7ENQSlAf~t~0Et1J1AANN2 H2 A~VYPSOEI~SISQYHII4SlJl~itC60FUEiNVHH2 i!s Ac.JIAAA ItiSL~SCNQCE'~tWEQELL~1.DKVYASt~VNWf'~iN2 H< AaY'I~L~SLIElE;44Q4tEWE4alE~KWAS1.WNWRNH2 i!6 AoYTSLMSL~ESQNaQFJ~~QEGEI ~ ~ nKWASLWNWFNH2 i16 A~QIWHNMTIN~AEWOR~NNYi'SLIEiSLJ~QNQQEKQ.NH2 i!T Ac~QNIMNMIWIAEVYORE7NNYTSUHSLt~QQQQEiW.NH2 Ha AeiiMMNMTYYWIEWDRE1NNYT8LtIiSLIEESQQQQEKQ.NH2 it9 Ac.NKSLQNYhINMfYYIA~NDREINHYIiUHSI~EESQ4NN2 a0 AcfDASISGVNEKiNQSLJIFIEESDELLHNVNIIGKSTHH2 i2! ACJ1C(R(CSDE1JC4HH=' a3 AaYISt~S ASI.IAMW!'Mt2 1?~ IIerYTSUfiSLI~SQD4QEKNF:QE1J.ELDKWAS~VYHHrt'HH2 iZ5 Ae-YTSLJtiSLIEI~QDQQFJmEQFI,I.EI~IMfASLWhNYf'~NH2 tt6 Ac.YlSUt~SLI~SQNQQF~QJ~E4EL~
~KWASLWOWF.NNZ

f41 Ae~.E~IHtTQSI,EQACI

tit 14~J~NGSA8t~QA~.lt t~ Ao~NIT

i46 Ac.Lt~aM'fASLEAAQI

N6 Aa~i PJWITASLEQA

.

i16 AaRMW00LlJQHYREYMAICSSENDRLRLLL~G4~dlJPB~lIHZ

L
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Q
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ip Aa ~t2 16~ Ao-T

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16a Ao-11! Ao-iT0 Ao-Ao- AB~VMNF~I~lH2 m Ao-sn Ao-IT< Aa i16 AoYTSi~LJ~/1M

i16 AaY.IEiSLIAAIICN

iTT AaYIELIE1M

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;
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ie.,x T
N0. g.
a~i ~ I~o.N~SW~t~iLWV~iI!
aas 8foan.YDPWFPSDEFDAStSQVNFJDN4SlJlFtRK8DE4~~t2 ia5 ~ Blofln.Pl.YI~DE~SISQVH~tQSLADELI~it~IH2 tab 8loun Yt'PBDE~SISQVNEt~I4SLDE71~WV~NH2 iaT BIotIn~OE'F~StSQVNEIGHQSLAt~OBIJ~iMVNA(iK~IHZ
iaa Btodn-VYPSDE~DA~ISQYH~IQSIJI~RICSOF~JJ~fV.NN2 tag Btotin~VYPSDE~fOASISQVNt~IQAtJIYIRtCADFU.EHV~IVli2 ~90 Ac-VYPSDE~AStS4V~4~1A1JlF(tilGlOEtLFQV.trHt 19t Ar.NYTSiJHStJEI~QNQQE~Q~iE~4EI ~ ~ .~KWASI.WNWI'~~NH2 t9Z Ao-t~IHYISLIttSUEESQNQQEtW~Ft mr n~yA8LIfYN~Yt~hIH2 a9s Ac-tMMfTSUtiSUEESAtIQQE'IQ~IEQE1 ~=~KWASLWNWf.,NH2 sae A~.at~t~HSUE~saHaaEtnaEat~aKwASLHZ
i95 Ac~YTSUHStJEESQNQQEtCHiEQEI! ~ ~KWA8t.VYHIf4Ert~t~It~2 ass Ac-YrsuHSt~EESaemaawE-aEU~rorcwASLwt~ls.~Hi esr Ac.Yrscmsurfsnsaaoa~t~ua.DKwAS~.v~(r.~at~
sss Ac.YrsLa~st~ESat~taaEtnt~Ea,~KwASU~uHVU~t(trt.t~atz t89 Ae.YDPLVFPSDEFOAStSQVN~laNQSIJIFt~SDELLHHVHAGtC-llti2 !00 Ac~tYTSUHSUtSCI~IQQ~QrEQaLELOKWASI.HtitINFtl~lHt 10t Ae-NNYZSUHSI~&GNQQEIQtE4E71FLOtCWASLWNWFH1.NH2 t05 Ar.~ICCi:AlCPK4Ll~4lfYREYMAICSSt~fORLRL1LCQNICPSt.DYpStIPRTPD.NHt t06 IIo.RAtQ:KqIJ~pEIYRI:yIIAAKSSt~IORLRLLUCQMCPSLDYDSEPRIpD.ttH2 110T AcaIYPSDE~fDASIS4YNt~IQAI~IYIAAADdI.EIYV~NtIZ
t09 AG.YOA8t8QVllEFFINQAIJIYIWtAO<xJrNti2 .
aH0 11<FSQbtQQEIWE~.L1~4NH2 aH1 Ao~HGIYDYPKY»SblII~KGYt~SIKGW01~
aHt 'fl4St~V~M.1~1H2 Hi tt6 LY~NWf'~
a!t6 El.I.~KWASt~fVfHHlt'~H2 t1T
~ta h>a Ao-ao Aanaoa~eaeisatcwasc~c a>; Ao-su sts Lwst~t ..
l4o~AllYALipAHIJIUJIP$A.~tQiYiWRY/lSRt4CRJ11~I94LJ~QMfR~/AH2 fH A~o,AI~YN1PAVLLJVtJIPCRII!øi04Lt~ElYREVMI1K~AYpRi.Rt~~d Nt VYPSOEYDJ~SISQYII~qAt~Y~i~AOBi~Mf~~t -as-T
N0.
A~o~W~E~iKAIIYA~H2 15f Ik~~DEL~W2 154 OecanoylaRiC8p~t~2 !55 Ilodt.Aca.IRtCSaELI~NN2 156 Ao-YDAS(SQV~HH2 !ST Ac.~(EIQH4S4~H2 !5a IIo-StSQVHE~IAA1~1Y1RiCADEL4NH2 169 Ao-tlVllEEtNMIJIY1WCADE(1,~NH2 160 Ac-EDHOAIJI~RaCApEI.4NH
!61 A~~H4AtJlYIRIUpEVrt~(HZ
!62 ANJIYiRIUIGEL.LrHHZ
16' FDAStSCVNEIflHQAIJIE(IiKSDELIrt~lH2 A~'t'1~RE~INYTSUHSUEESCNQQEKNEQELf.EL~HH2 165 IIc.ASRIC~'Vita'ICqwqHYltEyMAKSSENORiRIJJJCQAdCPSLDVDS-HH2 tl6T JI~VKE1NDRElNHYTSUHSI~SQI~IQQEEWEQ~L~HH2 !sa ~.wKCEpaN~ros~.vFrsoEFUas~savHE~aHast~H2 ti69 Ac~IIYPSDEYDASISQYHEEWQSLAYIRiCA0E1U1MLHH2 ft0 ~Ic.YD~ISISQVHEEINDALIIyfRKI~pE~yt~IH2 17t ~Io-YO~ASISpVHEEWppLAy~p~H2 Ao~IIYPSOEYDASISqyHEEINCAIJIyW"I.~HV-HlH2 f7; I1o~11YPSDEYD~ISIS~V~pNAAIJIYiRICAL~IJtM1~41H2 lT~t Oeanoyf.Y~CNQQE:IQiEQ6~.pKWps~yy~.HH2 as Ao~rrnsoEYaAS<sctvrtEE~tatu~waoEU~nr.HC~
srs ~o.DEm~AS~sam~tEtaEtasu~sa m weysaHOacs~rn~aW~ctrHKVHSVtEKn~tr.HHz ~'i A~o~8lblGAFaGrtHKVHSVIEKIN~IVKiI~GN~f.EI~NHH2 17! A~~3lTHKVHSH~An'lt~Qi~JIVDf~QNLEI~LNK HH2 .
~~~YID'a.YDKVRSQLtiDHYfC~7~GNGAFE"~itG~tt GmDYPKYi~H2 fdZ II~o~YEdW~,INSVKNaTYD~YPKY~NH2 ~AYAW'IWLLJW~IPAAaI~W~.~IHt let A~o~AAYIItJ.PIIYLLAIL~MDSNYKNt.YDKVRSQ~tROI~i~IH2 tab Aoit~l~IlEt~I~tNNBV.H~
~AI~FQHt ~QtVEOQFLaWYIYNAELI.VAt~B~t~IH2 !ss AaBHYKHt.YDKYR,gqUipH~#;t II~o~IVOR6NNYT6LIfiSLI~Q~IQqpW6pH~W2~
...r..w~.~Yp~y~~
AO~WDRAHHYTSLIHStarESpHqQEtWBQB.l~H2 I~o~IIWEtAHHY'r6UHSLIqqEIQ~IEqE~H2 196 Ilo"y~.~SG1N44EiW60k3l.QDKWl~6l.WNWF~HH2 !1't I~o~Yl4l~qlJipgl~IqHt ,8B-T
N0.
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loot t00i Ao.YOISIF~IK~UCBDLE~8i~5fNIQC6NQI~StaNW11.N1iz 1004 BlWlnyf~0~SIEIHKAK60LEEStCEIMKK6HQla.DS~IiMAM~NH2 1005 AaYTSU-0H
1006 fmoc.tiS4l~.OH
1007 Fmoc~CN4QEK-0H
100E , Fmoc.t~lEQ611E40H
1009 Fmoo-OKWA840H
1010 Ftnoc~lYNWF.OH
1011 Ac.AKTLEKiVYDTWHLI.FiSSAUffaNIJCSVACtiI.Sl~iH2 1012 Ac~NftI~QAIaKCFWM1MQEVC'sKAMYA-HH2 1013 Ac-I.ENER1LDEHOSNVKNI.YDKYRI~QI.RDN~IH2 101< Ac-~ENERTLDFHOSNVKNLYOKVRL~QLRDNVKEL~GN(i.NH2 1016 Ac~TL.DFHOSNYKNLYOKIfRI~QLROHVftFI~GN~'sAFFF~JH2 _8~_ T ' ~MIIMIM
Iett eto~rl~N~I~iGI~LIIEft l4zZ 81ot1tqA.6~VHl~IQBtJIYI~OBLt~dlt t4ts AaSGSQYNmHQSUIYIREC60E11.#IZ
1~t AotDISIB.NIWCSOLFESt~NI~NQE~S1QH~VE.W2 t4zb Ac~SIEI~IiWCSDigSI~~NpQCSHnB~StGHWti~NH2 t4z6 Ao~iDtStEJ htKAICSDLEFJItCEIMiCKAN4lQ~SKiNVYfi~iH2 toZT ANWSfELttKAKSDLEESICEINnCICANnI~SIGMMi.~IHz t4z6 Ilo-lD(S(E7~IKAtSSDIFEA!('S1MICKSNntCi.DSK3HWH.HH2 t~ BIoSVALJOpIDtSIEWIWC&DIgSI~IMWGSNQIarNHz t~0 BIoHnyf,AL.OP10(SIELI~IKIItGSD~I~EIMIQCSNQIQDSLNN2 t431 desAmtcwl~most~s.t~ISYAiDPt01S1El.NIWGSDIgStC~YfKiCSNQK4Nii2 to3z desAmtttoTyrosIne,AL.OPIOIStELNfUIItSDLEEStCEVYIICKSNnI~SI~lH2 t433 IIo-YDASISQVHE'EINQAt~IFiRKADEL,NH2 1434 IIo-YDASISQVHmNnSlJlYIRiCADEL1r8W2 toss e~oa~.~D~s~snvc~mNnu~~wuo~NHz toss e~os~.mAS~uvNmNnsDa.t~c~tz t431 Ao-YDASISaVHmHQSIJIF~SDE~Nt t433 Ae~~INL~EWDRE7NNYT6tlEiSUE~SQNQQEIQ~IEnFI~NHz 1439 BIoNnFIHKAKSDtEE&IC~IMRRSHQ~DSiGNHM.NHz 1444 Ilo-IIESTQICAi-~Gt1'NKVNSYIFxINrnFEIIYGIøFGN~~t~tH2 t446 Btoat~-0EIfDA,S~~QVNAQHnSIJIFtRICSDEUrHHt t4~6. A~.~EWDRAtiNYIStIEiSU~QHn~EQELrt~lH2 t4~T . Ao~MQEINE,QIQIRYIFJWlS4SL~C~AQtQQQWz t4fa A,o.YV~WE-QKYRYIFJlt4SQSL,EQ~InIQQI:IWGYE4NH2 t41<9 IIo~INGEHIBOIiVRYI:FJWrt'ALL,6nAnt4Q~EYEW2 t~ Ao~V~lVB4lMilfl~TALI.E~lllptn4EEQi~AYEx.Nitz ~~nc t4ss Aoan~at~lcvrs~u~rrA»cu~o~na~la~nu~~ua~a.~tz YALJrN6~Ntiz tO66 Ao-»i'~Y1'niI~AJ.VU~tAtTL,DE~DSHYtWLYOKVRI~IQt~HEtz t46T
t46a d~wmtn~tSQY~t4SlJlF~ICSDE~I~tz 1466 II~a81SQV1~t~iNnSLJIYII:~OE7~lWt ' to6o A~.anur»ncianaa.RC.~w~,ouilrrA~cn,con.~~z tah tosz Ao~o~usesav~Nean~tos~~asoat~t~ .
toss Ao.
t464.
w~c~r~anc t4B6 wr~rvrnc t s~.~~
1~4 I~o~AIIIPBOEYDN6~fliEl~HaNJIYIRIC~OBlt~Y~t~
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ta76 Ao~Y~3IMDY~3YCi~tt IOTI AaYOIIVKiVK3HKiF.NEi2 tQT6 Aa~fVaESIYF~CIKYRYI.PJINITAt~QG4AGi4AEECAEW ala..NH2 lor9 AwncvmnfAQrAe~Aau~xcA~a.au~
Coal A~.vrsuHSt~sac~Qaowe~a~anAS
1061 Ao~IYVPSOEFDAS1,SQVttE~IQStJIHt;~OEtlli~N.NH2 1066 AeStWtSEQIDQttQCDEQt~GT~4NfitdiCiKINYYT'SpWr3V.t~iH2 1064 Ac~SKNiS 1CVWYTSa9IKi.NEl2 1066 Ac-0L.StWISEQtOQiICKOEGICEC3TGVNGL~tiGKIfVyVTSDW.t~IHZ
1066 Ac~E~t~ICNISECIOQIICImEQKEf~fOiV~ldsGKINHfi'SD-NH2 106T AcaEDL.SICNISEQIppItCKpECICC-GTGI~V~GI~GGKVYVYI'S-HH2 1068 Ac.GtEaLSIWISEQIOQItCKOEQI~GTGIMGGGGICWWT-NH2 1069 Aa~GIEDLStWISEQIDQtt~EQIa:GTGINGI~GCiKWW~trHZ
1090 t-Haputoyf.~S .N~
1091 A~VYPSOEYDAStSCYHEtaNQALJIYtRICApELLEM/~NH2 1091 AeaIYPSDEFDASiSQVHE~NAA1J1E1RECADOJ.ENV.Ntl2 1093 AcaIYPSOEYDAStSaVhtEICtNQAIJIYIRFJ~pEIIFJiWNEl2 1094 8lodeyHYDA&ISQVNEICiHGISLAFIRE~OEL4NH2 1095 AeaUG1E0t5lCHtSEQIOQtiCKDEG11S>;GT~SVIKii~fsGKW~IH2 1016 Aa~AAKa1E0L8EWIS
lOIT Ac4aAAIGIED(.SICHIS
1066 Ac4'OAAtGtF~LStWtSEC1m411C1m6tSa~(iHK'sVG~41Ei2 1069 Aa~N(iDECOQdtiDFYDKn~'OQGD~NMfYYYT~s'WRi4Wf~l2 1100 AoaQi~lCtOC~ftDF<ID
1t0! Ao-tt06 ttt0 lttl Aoi.BPIV~ BYIWWIINYYVCiPSLIfg~PFi.PU~III;.NH2 ttlt YS~PF~PLJ.PfROJHt 1tt= xgq,SpE~I,LP~fI~
ltt4 ygp,SpRpu~
1t16 ~p~p~,~2 Itt6 ~~p~p~~
1111~
1116 ~q,S2 1119 y 11!! Ate. ySq,~.~Wt t~
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ltaz s~.coEmASESavN~rosto 11f3 2.Napt~hoyf~OEEDASESQQHEKWQSLAFIRtC50Et.LrNH2 flat t-tiapt~ttvoyLGOE~DASESQQNEKQNCSLA~KSDExI.~NH2 ltss z~~oE~DASESa~cawcsEo 1136 Ac~IVKiOEFDEStSQVNE~I~KSOEU~lH2 ttaT IIo-YiSLGGOEFDESISQVME~ESSI.AFIRiGSOELLrGGWHWF.trH2 1136 A~.YTSUH.SL(3GDEfDESISQVH~4E~SLAFIRtCSDELL~,a~3WASLWNWF.NH2 1139 Z~lapt~oyf~OEFDES~SQYNEIG~St.AFIRKSOEVrNil2 1140 24taphll~oyE40EEDESISQVHE7CEEStDEIIrHN2 1t41 2~iaph~hoyf.GDEEDEStSQYCII-~SLJIf~CSOEIIrNH2 1142 Z.t~lapl4hoyt~0EE0ESlSQYQEfflEESLZF~DEV~ti2 1143 BIoHn.GDEYDEStSQVNEbEEStJIFIRtGSDEIL.NH2 1144 Z-Naptdtt0yl~GOEYDEStSQYHE~SIJ1FIRIGSDELIrNH2 1146 Ao-YTSt.~SUO~EIQEE~fIRiGSDEU~,DKWH1~IH2 lli6 VYPSOGYO~IStSGVNEEIN4AlJlYiRKADAlE~I~IY.~ft2 tl4T A,a~G~luRAtEA4GIHLir4ltY4VDSK4l~Q~lRILIIYERYLtmCI~HtIZ
1146 GGGYYPSOEYDA~S~SQYNEEINGALAY,iRKMEU.EHWNtt2 1t49 Ate(NU,RIUEIl4GEILl~QLZYHl~f3EKCLAIIRIIJWERYUCDQ.tIH2 ltso wo~v~mt~c~nvt~mv~av~uDVHU.~tHz nst ~rRVtmcsc~H. -itsz ~o.pecrnu.~mc~nv~umvnavR~o~HU.~o:
rtes ~ ~a~c~DE~r.~rratz 1166 Ao-1t66 Ao.
ltsr ~o.
ltao 11E1 11,0.
ltit I~o.
tree lts4 lte6 ~tss tier ...,....
!ta ..".,..
rtes rs~",~.o,~t,~~
t1'J~0 VYNWF~NH2 !tH I~o. IISL.YItYI'llEtt ltit ~o YiSC61SL1~Sf~N4061a1t6G~'J~KYAYLYNY~it T.

itlT AoaKVIMFJ~HIT
II

I 9~tBYEt~I~rMl2 j,~pIQQ

lira Aocuc~~ar AUeoAmaa ~xw~av.NHz tt~'! AoaiVnEVYEpKYRyIfLF
AN~rAti . El~lEYELQ~Q.~NH2 .6G1A,4lQ4 lla0 Ao~4~V~:79QY~RYtF.ANfTAtI~GIJ1~Q[Q4pWGYE't~tp~lH2 ltal Aoif4G~E'WEtIQYRYI.FJIt~tf1'A1J.6~lQIQQEI~IEYF~QKt,.NH2 ltat Ac~W~WEEIIMiYLt=JWITALa.ECApiQQaWEYElqtq~lpi2 1136 Ao~EVVfOR~IRYLEANIT'AU

.~4AGfQA EIQ~IEYE~Qi~l2 11x4 AC-IN~Q IT/UJ.ECAQIQG EIWEYE101Qrt~tH2 ttab AeJIMQEYYERQVEtIfL.EANfTAIJFMGIQQOWEY~QfC4t~IH2 11x6 AcaA~QEINEf~tMCnF~V~itrAtJ.EpIl611QQOWE~fELqtCt,i~lH2 llat Ae~MQEINEQKVRt-LEANtTAtl.EOAOtQQ~-aWEYELGIt~IN2 llEa Ae 11Na1PSDEYOwsISQVNEEINQAUIYtRiCADEI.LE,NVNfi2 tta9 AG~HafP&DENalOA~SISQyNEEWQAIJIYIRiUIDEL1~NV,t,~H2 ttao Ao-vtew~soEYOAS~saA~auwu~oE~NV.~u ttll A

1112 At~VYPSOE~fDAS~SQyNEEtNCALAYIW~AOEIIFNFFNH2 1193 Ao-lf'I~UTALtFMQIQCE1WEYE10fa.01MfAS~WNHf~.HH2 1114 Ae..YISUrALLEQA4tQQEfWEYEIQIaoKWA8LVYE~N~NEi2 1195 Ao-YTSUTALLEpA4IQQE1WEYEl~tllCi.DGWAS~YY~NFaVH2 1196 Ao-lrTSUTAI1.EAACI

l1ST Ao-YIEUTA~tF~Aqt ~Y~a.Ana,Aua-TAtI~QACItQ4AQtt'YFLOIaJIw,llua,Ataw-NH2 !1!9 AoaKMMWEQICYEtYLEANITAIIt I

QAG ~IWEYELOIarNH2 QQ

1100 Ao~fNQEAIIpKVRYCEJINfTALLEqA4IQQAWEYE1~Q1~4NH2 1m1 AoaN~ElIYAAKyRYL
EIU~ITAt LE

. . E1~E11fEL~0lQrNH2 , ~OIlGI4Q

tiDt AoaN0AA60tMRYlFJWITAUZ3 lQIQC 99YEY0.Afi4NEt2 tai AO~N~A~E11AVRYLt:AMrALI

~L1A~IQ~ WE~fdQl~lr~al2 tmt A
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~

~ IWFYB~II~fi2 .
OAAIQ~

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AIMtYlFJllffr ~WNFNH2 . T~

TNHZ

ttta T,~

lilt TNEt2 ltli T.~,a~
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~111M11M
t:t0 Ao~fVH9VAEESDE~i~fNWF.NHt t~t ttst Ao~IVFIEESDF~IHf.HHt 1tu ?.t~lapls~hoyf~iFHFHEESDEZIFt~iFRt~lti2 list Z.NapI~hoyt~iESDEI..W.NH2 1135 ANfVHWFGOEFDESISQVnEQEESi~FI~0E1L~~',yyNyyF~H2 t?x6 Ac~WNWFiHSUEESQNQC~iWEpE.IW~ ..nKIIyASLIM~NVFNHt tt37 AaYTSUTAIFAQIQQEETIEYEI~ELDEWASLHfEIAfFIVH2 lt3a AcrYTSLifiSLGGDEFDEStSQVHE~ESlA~OEILGGWASIIfVNYYFtrH2 tb9 Z.Na~tf~oyf.GDEFOFSISQIIiQE~IaFtEESDEIl~IH2 tl~i0 H.OARQU.SSIIdQQQbIHLLiAIEAQQHLI~QI.TVHf~GfKQtAIARILJIVERYLICDQ-0H
t?M Ae.CPKYVKQNTLICIJITGMEt~iVPE~CqTR~ti2 tt~tt Ae~LFGIUAG~IGWEGWDGWYGFRHQHSC.NH2 t?~t3 Aot~tFLGGT~Mi2 t?~t4 Ac~t,OSWWISt~IFrJGCi1'.Mtt t?~t3 Ae.~tLTIPQSLiISVWV1SLJ~1FLGGT.Nil2 t?rt6 AerG~I.TWLTIPQSLDSWW15U1FLGGT~~NHZ
lZt7 _ Ao.WI~EWEQfQTALLECAQtQQEiCMEYE1plCl.Df(WA8tVYNVYF.NH2 t>Na Ao~VYNWHTALIFpAp~
tZt9 Aa.~VC~IV6QK11AIiFCACIQQQp~E~a.DKWAS1.WE1NFNHZ
t?S0 Ao~VVaG~YEQIMtYiFJINITALt~Il4I44Q4EYEL~QID.ri~lfl2 , tl6t Ao.~EINEqKHRn,FJlql1'Atl~GIIGIQQEtCEYF~~qIa.~t~tHZ
V1EL~U.~~-M~
ti6J Ao~IIG~CAhtGTDAiMCLJISW~KYfWAVtBiqla.W~lft2 t~ ~Ytst~t~EESGNQQDaIEQEIJ.~J~JKWABLWHWF.NH2 -wwa~carrvw m~letz wr~aw t~
w ~t E~t4~IW64B~IMfA
tS6t tS63 A~o.GYAIRLFJ1ACNWIRt3AUpL~RDR,S6~P.NHZ
tS6< A~o~CYR6GNIl8RAWYAV1'PZYATRDGta.t~T'~llHt ~5 A~PiWIIHfTI~ppAlIIISIYPQ~ft tS66 Aod~li~y t~ II~oVDR9SNYT6UT
tS~ AoCW~NpRBSNY18f1T
A~o~IV~INDRF~SMfTStJt t~ AoCfNGIEWDR9SNYTSU1' t?Tt Ao.~i4NSQSPIBMiSPI~APPTAPaYRWA~
ttTt Ao~iS811~iPARTALTTAQGTSt.IfP8Ar~~
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t!~ I~o~111~IIIPEtE3tAU.BMlt~4f>pQ~1f0~11~WF~HHt t><E! AaIT
fiat Ao~~YI~WEJi'AtJ~QAGIQQI~EY6rQtGtJEIN~HIFNNt t?,63 A~lNEItAlIF4AQtQC~4BYAJAIaDEINE1NF1~IH2 t?~ A~.W~QE1N61T
lte5 A YIREAOA.W~INF~f~lH2 tte6 YiRFJIOELiIVEINF~NH2 tteT Ac~IV~IEWEtDEYDAS1,S4V~d~A~A~EL~IYEWI'~H2 tme A
1239 Ao~fffrlEINFRODEYDAStSGVHF~IOALJIYIREJIDEL.INE1NF1~IH2 tt90 AcaAA4EIAlEIDEYOASiSQVN~IOAIJIYIREADELVYE~YFMH2-tt91 AG~fII~QE1NDEYDASISQVNEWNCAUIYIRFJIDEI.WEVYF~NH2 lteZ A~HfQEWDEYDASISQV~IQAUYtEIJ~IDELVYGWFNH2 tt93 ANHQEWEQIQTALt~QACIGQEbEYEIJQtD.I'~
1294 AOIQTALIF~AQIQQE1CEYBJCIIDJEWASLWEINFNHZ
195 A~f~QIINEITAIIFCA
tma ~rrPSa~ro~sr.Sav~tmwatAtr~~c~D~r.~z tms Ac.~rv~rnsD~ro~s~savHmeta~u~AO
tioo YTSUHSU~snH
tiol Ao~fV~EIAfDEYDAS~QV~NQAIJIYIREJ~D~IAfAWFNEtZ
t>ot Aa~N~QAWDEYOASJaNOALaIYiE~JIDBaIIfAVY~li2 t» Aa~44r:1IlWDEYDASfS4YHCWH4A~AYIR~aELWEWF~t~iZ
ti04 Sto~n-YOPLVR~SOEFOASISQVH~IQSt~iGS0E3~11H2 . tlo5 BIoBn-YDPLYFPSDEi~A5N4StJIF~Mi2 ~pp8 GICST~M~Gt t>0T Ao~Yi~IVDti6.I~iEtt floe ~o~Ai~WBOIQ.Hli2 lave Ao~IV~WBmQT
ato ~o~vrea~aT
lift AoiNCE~IfERBSIlYtSLIT
tst: Ao~a~ew~esA~rrsur uti Ao~ce~n~asAmsur tats weRas~~rsur.
tsts ' ats 1m Aoanrswaa~eatrs~u;~umvraas~rAVU~t~N:
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tut A~o,~.n.
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t:ti Ao~111Af8YQLRt~3~tt tt~t tits tii6 tiiT IbWSY~Jtl1'rllEti T

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t:~ A
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o tiii A
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~ o WF.NH2 1:u Ao~IMQEWEQ~iaTAtI6~AAt4Q~UtEY9~KLIlKIAfA6LWAVVE'~

1:M Ao.TNKAVYSt~NGVSV~.

Ac.KAWSL6NGVSH.

tiST Ao~INnEYVE4lQTAU~4G1AWEY6~QlQ.IEINEINf'~Mi2 t>M Ac~VCGHfE~QIQTAU~AQI4QEKGEIfd4i4Jl:INEWi'M~2 lis9 A~IAI~QEV~E~iIQT

1140 AoYDPLYFPSOEFDAS1SQVNEtQNGSl.lil~tlZ

1ui ~uo~rnso~roAS~snvNmNaAUmuo~u.~NV.r~

tut Ruof~YTSUHSUE~QNGI4ElWFn~t~! ~t n~LIfVNWF~IHZ

1u4 Ac~GN~lQQNNU.RNFJ1QG1HW0l,.TiNNGtICQLrOARl4NHZ

1145 Ac~QQQNHIIRAIFJIQGtIU~QI.IWVGIKQL~4ARIU1VERYlt~Q~HHZ

1u6 AcSGN~GGNNIIRAlEA44~.Irt~.IW4GlKQL~iARIUIYERYUmQ~NHZ

U~I Ia t AEWA
EIW~ I
~

1uT Ao.W .
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QG

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l:st Ao.IM4~ECiQTALLC-M4144FJGlEIfEWtGJIEWAGt WAW.NH2 t:5: Aa.~EWE~IQTALIEMQtQCEIWEYEI~QICLDIMfAGI.VYEVYFNHt YGLttPGIN
' Hft2 NQINVS

l:u Ao~~AW~IWSY~.RPGWAWFHttl 1=55 Bt ' V~fYEQIQT
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lift A
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lift A
AaSA15GJ111st o, .

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lift A
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1542 Ac-AAAWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-NH2 1543 Ac-WQEAAAKITALLEQAGIIGlQEKNEYELQKLDKWASLWEWF-NH2 1544 Ac-WQEWEQAAAALLEG1AQIGlQEKNEYELQKLDKWASLWEWF-NH2 1545 A~W'QEWEQKtTAAAEQAQ1QQEKNEYELQKLDKWASLWEWF-NH2 1546 Ao-WQEWEQKITALLAAAQIQG1EKNEYELQKLDKWASLWEWF-NH2 1547 Ao-WQEWEQKITALLEQAAAAQEKNEYELQKL.DKWASLWEWF-NH2 1548 Ao-WQEWEG1KITALLEQAQIDAAANEYELQKLDKWASLWEWF-NH2 1549 Ao-WOEWEQKITAU~QAQIQQEKAAAELQKLDKWASLVVEHIF-NH2 1550 Ao-WQEWEQKITALLEQAC~IGiGIEKNEYAAAKLOKWASLWEWF-NH2 1551 Ao-WQEWEG1KITAU.EQAQIQQEKNEYELAAAAKWASLWEWF-NH2 1552 Ao-WQEWEQKiTAU-EQAQI(1QEKNEYELQKLDAAASLWEWF-NH
1553 Ao-111~EWEQKITALLEQAQIQQEKNEYELQKLDKWAAAAEWF-NH
1554 Ac-WQEWEQKITALLE(dAAG~IQQEKNEYELQKLDKWASLWAAA-NH
1556 Ac-YTSLIHSUEESGINQQEKNEQELLLDKWASLWNWF-NH2 1557 Ao-YTSUHSUEESG1NQEKNEQELLELDKWASLWNWF-NH2 1558 Ac~ERTLDFHDS-NH2 1559 ~YTSUHSL1EESQNQQEKNEG~ELLELOKV1~IASLWN(VIr)F-NH2 1563 Ao-YTSLIHSUEESQN(Q)QEKNEQELLELDKWASLWNWF-NH2 1564 Ao-YTSLiHSUEESQNQ(~DKWASLWNWF-NH2 1566 Ac-FYEIIMDIEQNNVQGKKGIQQLQKWEDVWGWIGNI-NH2 1567 AaINQTIV1MHGNITLGEWYNQTKDLQQKFYEIiMDIE-NH2 1568 Ac-WNHGNITLGEWYNQTKDLQG1KFYEIIMDIEaNNV~-NH2 1572 Ao-YTSUHSUEESENQQEKNEQELLELDKWASLWNWF-NH2 1573 Ao-YTSLIHSUEESQDQQEKNEQEU.ELDKWASLWNWF-NH2 1574 Ao-YTSUHSUEESQNEQEKNEQELLELDKWASLWNWF-NH2 1575 c-YTSUHSUEESQNQEEKNEQELLELDKWASLHMWF-NH2 1576 Ac-YTSUHSUEESQNQQEKDEQELLELDKWASLWNWF-NH2 1577 Ao-LGEWYNQTKDLQQKFYEIIMDIEQNNVQGKKG1QQ-NH2 1578 Ao-WYNQTKDLQQKFYEIIMDIEQNNVQGKKGIQQLQK-NH2 1579 Ac-YTSUHSUEESQNQQEKNEEELLELOKWASLHINWF-NH2 1580 Ao-YTSUHSUEESG1NQQEKNEQELLELDKWASLWDWF-NH2 1586 Ao-XTSUHSUEESQNQG1EKNEQELLELDKWASLWNW)C-NH2 1588 Ao-YNGtTKDLG1QKFYEIiMDIEQNNVQGKKGIQQLQKW-NH2 1598 A~YTSUHSUEESQNQGIEKNEQELLELDKWASLWNWF
1600 Ao-TLTVQARQLLSGNf~QGINNU-RAIEAQQHU-QLTWI~GIKQLQAR-NH2 1603 Ar~LQQKFYEIIMDIEQNNVQGKKGIQQLQKWEDWVGW-NH2 1627 Ao-YTSUHSUEESQNQQEKNEGIEIUIi.QKWABLWNWF-NH2 1628 Ao-YTSUHSUEESQNQQEKNEQEI:.if.WAIS4WNWF-NH2 1629 Ao-YTSUHSUEESG1NQQEKNEQELLELAKWASLWNWF-NH2 1630 A~o-YTSUHSUEES~NQQEKAEQELLELOKWASLWNWF-NH2 1631 Ao-YTSUHSUEESG1NQDEKNAQFILi~.DKWA8LWNWF-NH2 1632 Ao-YTSUHSL1EESQNQQEKNEAELLELDKWASLWNWF-NH2 1634 A~Wr.IEWEQKITALLEQAG1IQQEKNEQELQKLDKWASLWEWF-NH2 1635 Ao-Wr3EWEQKITALLEQAG11QQEKAEYELQKLDKWASLWEWF-NH2 1636 A~-WQEWEQKITALLEQAQIaQEKNAYELQKLDKWASLWEWF-NH2 1637 Ac-WQEWEQKITALLEC~AQIQG1EKNEAELQKLDKWASLWEWF-NH2 1644 Ac-EYDLRRWEK-NH2 1645 A~EQELLELDK-NH2 1646 A~EYELQKLDK-NH2 1647 Ao-WQEWECIKITALLEQAQIQQEKNEQELLKLDKWASLWEWF-NH2 1648 A~WQEWEGIKITALLEQAQIDQEKNEC1ELLELDKWASLWEWF-NH2 1649 Ao-W~QEWEG1KITALLEOAQIQQEKNDKWASLWEWF-NH2 1650 Ao-YTSLIHSLIEESQNQAEKNEQELLELDKWASLWNWF-NH2 1651 Ao-YTSLIHSLIEESQNQQAKNEQELLELDKWASLWNWF-NH2 1652 Ao-YTSLiHSLIEESQN~QEANEQELLELDKWASLWNWF-NH2 1653 Ac-YTSUHSLIEESANQ(~EANEQELLELDKWASLWNWF-NH2 1654 Ao-YTSLIHSL1EESQA(~QEKNEQELLELDKWASLWNWF-NH2 1655 Ao-YTSLIHSLiEESQNAQEKNEQELLELDKWASLWNWF-NH2 1656 Ac-YTSUHALIEESQNQQEKNEQELLELDKWASLWNWF-NH2 1657 Ao-YTSLIHSAIEESQNQQEKNEQELLELDKWASLWNWF-NH2 1658 Ac-VYPSDEYDASISQVNEEINQALAYIRKADELLENV NH2 1659 Ao-YTSUHSLAEESQNQQEKNEQELLELDKWASLWNWF-NH2 1660 Ao-YTSAIHSUEESQNQQEKNEQELLELDKWASLWNWF-NH2 1661 Ao-YTSLAHSUEESQNQQEKNEQELLELDKWASLWNWF-NH2 1662 Ao-YTSLIASUEESG1NQQEKNEGIELLELDKWASLWNWF-NH2 1663 AcrATSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-NH2 1664 Ac-YASUHSLIEESQNQQEKNEQELLELDKWASLWNWF-NH2 1665 Ao-YTALIHSUEESQNQQEKNEQELLELDKWASLWNWF-NH2 1666 Ac-RIQDLEKYVEDTKiDLWSYNAELLVALENQ-NH2 1667 ~DLTDSEMNKLFEKTRRQLREN-NH2 1668 Ao-SEMNKLFEKTRRQLREN -NH2 1689 A~VFPSDEADASISQVNEKINQSLAFIRKSDELLHNV-NH2 1670 Ao-VFPSDEFAASISG~VNEKING1SLAFIRKSDELLHNV-NH2 1671 Ao-VFPSDEFDASISAVNEKINQSLAFIRKSDELLHNV-NH2 1672 Ao-VFPSOEFDASISQANEKINQSLAFIRKSDELLHNV-NH2 1673 Ao-VFPSDEFDASiSQVAEKINQSLAFIRKSDELLHNV-NH2 1674 Ao-W~EWEQKITAALEQAQiQQEKNEYELQKLDKWASLWEWF-NH2 1875 A~-W'QEWEQKITALAEQAQIQQEKNEYELQKLDKWASLWEWF-NH2 1676 Ao-Wr.ZEWEQKITALLEQAA1QQEKNEYELQKLOKWASLWEWF-NH2 1877 A~W~QEWEQKITALLEQAQAQQEKNEYELQKL.DKWASL~VEWF-NH2 1878 Ago-WDEWEQKiTAU-EQAQIAG1EKNEYELL1KLDKWASLWEWF-NH2 1879 Ao-W~QEWEQKiTALLEQAQIQAEKNEYELQKLDKWASLWEWF-NH2 1680 Ao-VFPSDEFDASISQVNEKINQSAAFIRKSDELUaNV-NH2 1681 Ao-VFPSDEFDASiSQVNEKINQSLAA1RKSDELLHNV-NH2 1682 A~VFPSDEFDASISQVNEK1NQSLAFIRKSDEALHNV-NH2 1683 Ao-VFPSDEFDASISQVNEKINQSLAFIRKSDELAHNV-NH2 1884 Ao-VFPSDEFDASISQVNEKINQSLAFIRKSDELLANV-NH2 1685 Ac-WQEWEQKITALLEQAQIQQAKNEYELQKLDKWASLWEWF-NH2 1687 A~WG1EWEQKITALLEQAC11QQEKNEYELt~ALDKWASLWE~NF-NH2 1688 Ac~WQEWEQKITALLEQAQl4QEKNEYELQKADKWASLWEWF-NH2 .d WO 99/59615 PCTlUS99/11219 It is to be understood that the peptides listed in Table 2 are also intended to fall within the scope of the present invention. As discussed above, those peptides depicted in Table 2 that do not already contain enhancer peptide sequences (that is, do not represent hybrid polypeptides) can be utilized in connection with the enhancer peptide sequences and teaching provided herein to generate hybrid polypeptides.
Further, the core polypeptides and the core polypeptide of the hybrid polypeptides shown in Table 2 and FIG. 13 can be used with any of the enhancer peptide sequences described herein to routinely produce additional hybrid polypeptides, which are also intended to fall within the scope of the present invention.
It is noted that while a number of the polypeptides listed in Table 2 and FIG. 13 are depicted with modified, eTa., blocked amino and/or carboxy termini or d-isomeric amino acids (denoted by residues within parentheses), it is intended that any polypeptide comprising a primary amino acid sequence as depicted to Table 2 and FIG. 13 is also intended to be part of the present invention.
The core polypeptide sequences, per se, shown in Table 2 and FIG. 13, as well as the hybrid polypeptides comprising such core polypeptides, can exhibit antiviral, and/or anti-fusogenic activity and/or can exhibit an ability to modulate interacellular processes that involve coiled-coil peptide structures. Among the core polypeptide sequences are, for example, ones which have been derived from individual viral protein sequences. Also among the core polypeptide sequences are, for example, ones Whose amino acid sequences are derived from greater than one viral protein sequence (e~ct., an HIV-1, HIV-2 and SIV -derived core polypeptide).
In addition, such core polypeptides can exhibit amino acid substitutions, deletions and/or insertions as discussed, above, for enhancer polypeptide sequences as long as the particular core polypeptide's antiviral and/or antifusogenic 0 activity (either per se or as part of a hybrid polypeptide) is not abolished.

With respect to amino acid deletions, it is preferable that the resulting core polypeptide is at least about 4-6 amino acid residues in length. With respect to amino acid insertions, preferable insertions are no greater than about 50 amino acid residues, and, more preferably no more than about 15 amino acid residues. It is also preferable that core polypeptide insertions be amino- and/or carboxy-terminal insertions.
Among such amino and/or carboxy-terminal insertions are ones which comprise amino acid sequences amino and/or carboxy to the endogenous protein sequence from which the core polypeptide is derived. For example, if the core polypeptide is derived from gp41 protein, such an insertion would comprise an amino and/or carboxy-terminal insertion comprising a gp41 amino acid sequence adjacent to the gp41 core polypeptide sequence. Such amino and/or carboxy terminal insertions can typically range from about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid residues amino to and/or carboxy to the original core polypeptide.
The hybrid polypeptides of the invention can still further comprise additional modifications that readily allow for detection of the polypeptide. For example, the hybrid polypeptides can be labeled, either directly or indirectly.
peptide labeling techniques are well known to those of skill in the art and include, but are not limited to, radioactive, fluorescent and colorimetric techniques. Indirect labeling techniques are also well known to those of skill in the art and include, but are not limited to, biotin/streptavidin labeling and indirect antibody labeling.
The invention further relates to the association of the enhancer polypeptide sequences to types of molecules other than peptides. For example, the enhancer peptide sequences may be linked to nucleic acid molecules (e.a., DNA or RNA) or any type of small organic molecule for the purpose of enhancing the pharmacokinetic properties of said molecules:

5.2. SYNTHESIS OF PEPTIDES
The enhancer, core and hybrid polypeptides of the invention may be synthesized or prepared by techniques well known in the art. See, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman and Co., NY, which is incorporated herein by reference in its entirety. Hybrid polypeptides may be prepared using conventional step-Wise solution or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry. (see, e.g., Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., 1997, CRC Press, Boca Raton Florida, and references cited therein; Solid Phase Peptide Synthesis: A Practical Approach, Atherton & Sheppard, Eds., 1989, IRL Press, Oxford, England, and references cited therein). Likewise the amino- and/or carboxy-terminal modifications.
The enhancer, core and hybrid polypeptides of the invention can be purified by art-known techniques such as normal and reverse phase high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion, precipitation and the like. The actual conditions used to purify a particular polypeptide will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, solubility, stability etc., and will be apparent to those having skill in the art.
Hybrid, enhancer and core polypeptides may also be made using recombinant DNA techniques. Here, the nucleotide sequences encoding the polypeptides of the invention may be synthesized, and/or cloned, and expressed according to techniques well known to those of ordinary skill in the art.
See, for example, Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NY.
One may obtain the DNA segment encoding the polypeptide of interest using a variety of molecular biological techniques, generally known to those skilled in the art. For example, polymerase chain reaction (PCR) may be used to generate the DNA fragment encoding the protein of interest.
Alternatively, the DNA fragment may be obtained from a commercial source.
The DNA encoding the polypeptides of interest may be recombinantly engineered into a variety of host vector systems that also provide for replication of the DNA in large scale. These vectors can be designed to contain the necessary elements for directing the transcription and/or translation of the DNA sequence encoding the hybrid polypeptide.
Vectors that may be used include, but are not limited to, those derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. For example, plasmid vectors such as pcDNA3, pBR322, pUC 19/18, pUC 118, 119 and the M13 mp series of vectors may be used. Bacteriophage vectors may include l~gtl0, Agtll, hgtl8-23, AZAP/R and the EMBL series of bacteriophage vectors. Cosmid vectors that may be utilized include, but are not limited to, pJB8, pCV 103, pCV 107, pCV
108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWEl5, pWEl6 and the charomid 9 series of vectors.
Alternatively, recombinant virus vectors including, but not limited to, those derived from viruses such as herpes virus, retroviruses, vaccinia viruses, adenoviruses, adeno-associated viruses or bovine papilloma viruses plant viruses, such as tobacco mosaic virus and baculovirus may be engineered.
In order to express a biologically active polypeptide, the nucleotide sequence coding for the protein may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequences. Methods which are well known to those skilled in the art can be used to construct expression vectors having the hybrid polypeptide coding sequence operatively associated with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA
techniques and synthetic techniques. See, for example, the techniques described in Sambrook, et al., 1992, Molecular Clonina A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Bioloay, Greene Publishing Associates & Wiley Interscience, N.Y., each of which are incorporated herein by reference in its entirety.
The nucleic acid molecule encoding the hybrid, enhancer and core polypeptides of interest may be operatively associated with a variety of different promoter/enhancer elements. The promoter/enhancer elements may be selected to optimize for the expression of therapeutic amounts of protein. The expression elements of these vectors may vary in their strength and specificities. Depending on the host/vector system utilized, any one of a number of suitable transcription and translation elements may be used. The promoter may be in the form of the promoter which is naturally associated with the gene of interest.
Alternatively, the DNA may be positioned under the control of a recombinant or heterologous promoter, i.e., a promoter that is not normally associated with that gene. For example, tissue specific promoter/enhancer elements may be used to regulate the expression of the transferred DNA in specific cell types.
Examples of transcriptional control regions that exhibit tissue specificity which have been described and could be used include, but are not limited to, elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Svmp. Quant. Biol. 50:399-409; MacDonald, 1987, Heaatology 7:42S-51S); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adams et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444): albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276) alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); alpha-1-antitrypsin gene control region which is active in liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); beta-globin gene control region which is active in myeloid cells (Magram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Shani, 1985, Nature 314:283-286); and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378). Promoters isolated from the genome of viruses that grow in mammalian cells, (e. g., vaccinia virus 7.5K, SV40, HSV, adenoviruses MLP, MMTV, LTR
and CMV promoters) may be used, as well as promoters produced by recombinant DNA or synthetic techniques.
In some instances, the promoter elements may be constitutive or inducible promoters and can be used under the appropriate conditions to direct high level or regulated expression of the nucleotide sequence of interest.
Expression of genes under the control of constitutive promoters does not require the presence of a specific substrate to induce gene expression and will occur under all conditions of cell growth. In contrast, expression of genes controlled by inducible promoters is responsive to the presence or absence of an inducing agent.
Specific initiation signals are also required for sufficient translation of inserted protein coding sequences.

These signals include the ATG initiation codon and adjacent sequences. In cases where the entire coding sequence, including the initiation codon and adjacent sequences are inserted into the appropriate expression vectors, no additional translational control signals may be needed.
However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon must be provided.
Furthermore, the initiation codon must be in phase with the reading frame of the protein coding sequences to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of transcription attenuation sequences, enhancer elements, etc.
5.3. USES OF THE ENHANCER PEPTIDE SEQUENCES, CORE
POLYPEPTIDES AND HYBRID POLYPEPTIDES OF THE
INVENTION
As discussed above, the enhancer peptide sequences of the invention can be utilized to enhance the pharmacokinetic properties of any core polypeptide through linkage of the core polypeptide to the enhancer peptide sequences to form hybrid polypeptides. The observed enhancement of pharmacokinetic properties is relative to the pharmacokinetic properties of the core polypeptide alone. Standard pharmacokinetic character parameters and methods for determining and characterizing the pharmacokinetic properties of an agent such as a polypeptide are well known to those of skill in the art. Non-limiting examples of such methods are presented in the Examples provided below.
The enhancer peptide sequences of the invention can, additionally, be utilized to increase the in vitro or ex-vivo half-life of a core polypeptide to which enhancer peptide sequences have been attached. For example, enhancer peptide sequences can increase the half life of attached core polypeptides when the resulting hybrid polypeptides are present in cell culture, tissue culture or patient samples, (e. a., cell samples, tissue samples biopsies, or other sample containing bodily fluids).
The core polypeptides and hybrid polypeptides of the invention can also be utilized as part of methods for modulating (e. a., decreasing, inhibiting, disrupting, stabilizing or enhancing) fusogenic events. Preferably, such peptides exhibit antifusogenic or antiviral activity. The peptides of the invention can also exhibit the ability to modulate intracellular processes involving coiled-coil peptide interactions.
In particular embodiments, the hybrid polypeptides and core polypeptides of the invention that exhibit antiviral activity can be used as part of methods for decreasing viral infection. Such antiviral methods can be utilized against, for example, human retroviruses, particularly HIV (human immunodeficiency virus), eTa., HIV-1 and HIV-2, and the human T_lymphocyte viruses (HTLV-I and HTLV-II), and non-human retroviruses, such as bovine leukosis virus, feline sarcoma and leukemia viruses, simian immunodeficiency viruses (SIV), sarcoma and leukemia viruses, and sheep progress pneumonia viruses.
The antiviral methods of the invention can also be utilized against non-retroviral viruses, including, but not limited to, respiratory syncytial virus (RSV), canine distemper virus, newcastle disease virus, human parainfluenza virus, influenza viruses, measles viruses, Epstein-Barr viruses, hepatitis B viruses and Mason-Pfizer viruses.
The above-recited viruses are enveloped viruses. The antiviral methods of the invention can also be utilized against non-enveloped viruses, including but not limited to picornaviruses such as polio viruses, hepatitis A virus,, enterovirus, echoviruses, and coxsackie viruses, papovaviruses such as papilloma virus, parvoviruses, adenoviruses and reoviruses.
Other antifusogenic activities that can be modulated via methods that utilize the peptides of the invention include, _ 57 -but are not limited to modulation of neurotransmitter exchange via cell fusion, and sperm-egg fusion. Among the intracellular disorders involving coiled-coil interactions that can be ameliorated via methods that utilize the peptides of the invention are disorder involving, for example, bacterial toxins.
The antifusion or antiviral activity of a given core polypeptide or hybrid polypeptide can routinely be ascertained via standard in vitro, ex vivo and animal model assays that, with respect to antiviral activity, can be specific or partially specific for the virus of interest and are well known to those of skill in the art.
The above description relates mainly to antiviral and antifusion-related activities of core and hybrid polypeptides of the invention. The hybrid polypeptides of the invention can also be utilized as part of any method for which administration or use of the core polypeptide alone might be contemplated. Use of hybrid polypeptides as part of such methods is particularly preferable in instances wherein an increase in the pharmacokinetic properties of the core polypeptide is desired. For example, insulin is utilized as part of treatment for certain types of diabetes. A hybrid polypeptide comprising an insulin or insulin fragment as the core polypeptide can, therefore, also be utilized as part of methods for ameliorating symptoms of forms of diabetes for which insulin is used and/or contemplated.
In addition to the above therapeutic methods, the peptides of the invention can still further be utilized as part of prognostic methods for preventing disorders, including, but not Limited to disorders involving fusion events, intracellular processes involving coiled-coil peptides and viral infection that involves cell-cell and/or virus-cell fusion. For example, the core and hybrid polypeptides of the invention can be utilized as part of prophylactic methods of preventing viral infection. -The hybrid polypeptides of the invention can still further be utilized as part of diagnostic methods. Such WO 99/59615 PCT/US99/l 1219 methods can be either in vivo or in vitro methods. Any diagnostic method that a particular core polypeptide can be utilized can also be performed using a hybrid polypeptide comprising the core polypeptide and a modification or primary amino acid sequence that allows detection of the hybrid polypeptide. Such techniques can reflect an improvement over diagnostic methods in that the increased half life of the hybrid polypeptide relative to the core polypeptide alone can increase the sensitivity of the diagnostic procedure in which it is utilized. Such diagnostic techniques include, but are not limited to imaging methods, era., in vivo imaging methods. In a non-limiting example of an imaging method, a structure that binds the core polypeptide of a hybrid polypeptide can be detected via binding to the hybrid polypeptide and imaging (either directly or indirectly) the bound hybrid polypeptide.
5.4. PHARMACEUTICAL FORMULATIONS, DOSAGES
AND MODES OF ADMINISTRATION
The peptides of the invention may be administered using techniques well known to those in the art. Preferably, agents are formulated and administered systemically.
Techniques for formulation and administration may be found in nRemington's Pharmaceutical Sciences", latest edition, Mack Publishing Co., Easton, PA. Suitable routes may include oral, rectal, vaginal, lung (eTa., by inhalation), transdermal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few. For intravenous injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer to name a few. In addition, infusion pumps may be used to deliver the peptides of the invention. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
In instances wherein intracellular administration of the peptides of the invention or other inhibitory agents is preferred, techniques well known to those of ordinary skill in the art may be utilized. For example, such agents may be encapsulated into liposomes, or microspheres then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are effectively delivered into the cell cytoplasm.
Additionally, due to their hydrophobicity, when small molecules are to be administered, direct intracellular administration may be achieved.
Nucleotide sequences encoding the peptides of the invention which are to be intracellularly administered may be expressed in cells of interest, using techniques well known to those of skill in the art. For example, expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses, may be used for delivery and expression of such nucleotide sequences into the targeted cell population.
Methods for the construction of such vectors and expression constructs are well known. See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor NY, and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.
Effective dosages of the peptides of the invention to be administered may be determined through procedures well known to those in the art which address such parameters as biological half-life, bioavailability, and toxicity. In particularly preferred embodiments, an effective hybrid polypeptide dosage range is determined by one skilled in the art using data from routine in vitro and in vivo studies well know to those skilled in the art. For example, in vitro cell culture assays of antiviral activity, such as the exemplary assays described in Section 7, below, for T1249, will provide data from which one skilled in the art may readily determine the mean inhibitory concentration (IC) of the peptide of the polypeptide necessary to block some amount of viral infectivity (e.g. , 50%, ICso; or 90 0, IC9o) . Appropriate doses can then be selected by one skilled in the art using pharmacokinetic data from one or more routine animal models, such as the exemplary pharmacokinetic data described in Section 10, below, for T1249, so that a minimum plasma concentration (Cmin) of the peptide is obtained which is equal to or exceeds the determined IC value.
Exemplary polypeptide dosages may be as low as 0.1 ~,g/kg body weight and as high as 10 mg/kg body weight. More preferably an effective dosage range is from 0.1 - 100 ~g/kg body weight. Other exemplary dosages for peptides of the invention include 1-5 mg, 1-10 mg, 1-30 mg, 1-50 mg, 1-75 mg, 1-100 mg, 1-125 mg, 1-150 mg, 1-200 mg, or 1-250 mg of peptide. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms or a prolongation of survival in a patient.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, ,e.~., for determining the LDso (the dose lethal to 50~ of the population) and the EDSo (the dose therapeutically effective in 50°s of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDso/EDSO. Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDSO with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the ICSO (era., the concentration of the test compound which achieves a half-maximal inhibition of the fusogenic event, such as a half-maximal inhibition of viral infection relative to the amount of the event in the absence of the test compound) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography (HPLC) or any biological or immunological assay capable of measuring peptide levels.
The hybrid polypeptides of the invention can be administered in a single administration, intermittently, periodically, or continuously. For example, the polypeptides of the invention can be administered in a single administration, such as a single subcutaneous, a single intravenous infusion or a single ingestion. The polypeptides of the invention can also be administered in a plurality of intermittent administrations, including periodic administrations. For example, in certain embodiments the polypeptides of the invention can be administered once a week, once a day, twice a day (e. g., every 12 hours), every six hours, every four hours, every two hours, or every hour.
The polypeptides of the invention may also be administered continuously, such as by a continuous subcutaneous or intravenous infusion pump or by means of a subcutaneous or other implant which allows the polypeptides to be continuously absorbed by the patient.
The hybrid polypeptides of the invention can also be administered in combination with at least one other therapeutic agent. Although not preferred for HIV therapy, administration for other types of therapy (e. a., cancer therapy) can be performed concomitantly or sequentially, including cycling therapy (that is, administration of a first compound for a period of time, followed by administration of a second antiviral compound for a period of time and repeating this sequential administration in order to reduce the development of resistance to one of the therapies).
In the case of viral, eTa., retroviral, infections, an effective amount of a hybrid polypeptide or a pharmaceutically acceptable derivative thereof can be administered in combination with at least one, preferably at least two, other antiviral agents.
Taking HIV infection as an example, such antiviral agents can include, but are not limited to DP-107 (T21), DP-178 (T20), any other core polypeptide depicted in Table 2 derived from HIV-1 or HIV-2, any other hybrid polypeptide whose core polypeptide is, at least in part, derived from HIV-1 or HIV-2, cytokines, era., rIFN a, rIFN (3, rIFN y;
inhibitors of reverse transcriptase, including nucleoside and non-nucleoside inhibitors, e.g., AZT, 3TC, D4T, ddI, adefovir, abacavir and other dideoxynucleosides or dideoxyfluoronucleosides, or delaviridine mesylate, nevirapine, efavirenz; inhibitors of viral mRNA capping, such as ribavirin; inhibitors of HIV protease, such as ritonavir, nelfinavir mesylate, amprenavir, saquinavir, saquinavir mesylate, indinavir or ABT378, ABT538 or MK639; amphotericin B as a lipid-binding molecule with anti-HIV activity; and castanospermine as an inhibitor of glycoprotein processing.
The hybrid and/or core polypeptides of the invention may, further, be utilized prophylactically for the prevention of disease. Hybrid and/or core polypeptides can act directly to prevent disease or, alternatively, can be used as vaccines, wherein the host raises antibodies against the hybrid polypeptides of the invention, which then sez've to neutralize pathogenic organisms including, for example, inhibiting viral, bacterial and parasitic infection.

For all such treatments described above, the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e~ct. Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1).
It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions.
Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the oncogenic disorder of interest will vary with the severity of the condition to be treated and the route of administration. The dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient.
A program comparable to that discussed above may be used in veterinary medicine.
Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by subcutaneous injection, intravenous injection, by subcutaneous infusion or intravenous infusion, for example by pump. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Pharmaceutical compositions suitable for use in the -present invention include compositions wherein the active ingredients are contained in an effective amount to achieve WO 99/59615 PCT/US99/i 1219 its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. For oral administration of peptides, techniques such of those utilized by, e.a., Emisphere Technologies well known to those of skill in the art and can routinely be used.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.a., i5 by means of conventional mixing, dissolving, granulating, dragee-making, levigating, spray drying, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, emulsions and suspensions of the active compounds may be prepared as appropriate oily injection mixtures. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, liposomes or other substances known in the art for making lipid or lipophilic emulsions. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium.carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. -pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, trehalose, mannitol, or sorbitol;
cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylceliulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic l0 acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizes, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. in addition, stabilizers may be added.
In instances where an enhancement of the host immune response is desired, the hybrid polypeptides may be formulated with a suitable adjuvant in order to enhance the 3o i~unological response. Such adjuvants may include, but are not limited to mineral gels such as aluminum hydroxide;

surface active substances such as lysolecithin, pluronic polyols, polyanions; other peptides; oil emulsions; and potentially useful adjuvants such as BCG and Corynebacterium parvum. Many methods may be used to introduce the vaccine formulations described here. These methods include but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal routes.
6. EXAMPLE: IDENTIFICATION OF CONSENSUS AMINO
ACID SEQUENCES THAT COMPRISE
ENHANCER PEPTIDE SEQUENCES
The retroviral gp41 protein contains structural domains referred to as the a-helix region located in the C-terminal region of the protein and the leucine zipper region located in the N-terminal region of the protein. Alignment of the enhancer peptide sequence regions contained within gp41 (FIG.
2A and 2B) of gp41 from all currently published isolate sequences of HIV-1, HIV-2 and SIV identified the consensus amino acid sequences shown in FIG. 1.
As described in detail in the Examples presented below, such sequences represent enhancer peptide sequences in that linkage of these peptide sequences to a variety of different core polypeptides enhances the pharmacokinetic properties of the resultant hybrid polypeptides.
7. EXAMPLE: HYBRID POLYPEPTIDES THAT FUNCTION

T1249, as depicted in FIG. 13, is a hybrid polypeptide comprising enhancer peptide sequences linked to an HIV core polypeptide. As demonstrated below, the T1249 hybrid polypeptide exhibits enhanced pharmacokinetic properties and potent in vitro activity against HIV-1, HIV-2, and SIV
isolates, with enhanced activity against HIV-1 clinical isolates in HuPBMC infectivity assays in vitro as well as in the HuPBMC SCID mouse model of HIV-1 infection .in vivo. In the biological assays described below, the activity of the T1249 is compared to the potent anti-viral T20 polypeptide.
The T2o polypeptide, also known as DP-178, is derived from HIV-1 gp41 protein sequence, and is disclosed and claimed in U.S. patent No. 5,464,933.
7.1. MATERIALS AND METHODS
7.1.1. PEPTIDE SYNTHESIS AND PURIFICATION
Peptides were synthesized using Fast Moc chemistry.
Generally, unless otherwise noted, the peptides contained amidated carboxyl termini and acetylated amino termini.
Purification was carried out by reverse phase HPLC.
T1249 (Ac-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF-NHZ) is a 39 amino acid peptide (MW = 5036.7) composed entirely of naturally occurring amino acids and is blocked at the amino terminus by an acetyl group and the carboxyl terminus is blocked by an amido group to enhance stability. T1387 is a 23 amino acid peptide lacking enhancer peptide sequences (Ac-TALLEQAQIQQEKNEYELQKLDK-NH2). Thus, T1387 represents the core polypeptide of the T1249 hybrid polypeptide. T1387 is blocked at its amino- and carboxy- termini in the same manner as T1249.
In particular, T1249 was synthesized using standard solid-phase synthesis techniques. The identity of the principal peak in the HPLC trace was confirmed by mass spectroscopy to be T1249.
T1249 was readily purified by reverse phase chromatography on a 6-inch column packed with a C18, 10 micron, 120A support.
7.1.2. VIRUS
The HIV-1~,1 virus (Popovic, M. et al., 1984, Science 224:497-508) was propagated in CEM cells cultured in RPMI
1640 containing 10°s fetal calf serum. Supernatant from the infected CEM cells was passed through a 0.2~m filter and the infectious titer estimated in a microinfectivity assay using the AA5 cell line to support virus replication. For this purpose, 201 of serially diluted virus was added to 20~c1 CEM

cells at a concentration of 6 x 105/ml in a 96-well microtitre plate. Each virus dilution was tested in triplicate. Cells were cultured for seven days by addition of fresh medium every other day. On day 7 post infection, supernatant samples were tested for virus replication as evidenced by reverse transcriptase activity released to the supernatant. The TCIDSO was calculated according to the Reed and Muench formula (Reed, L.J. et al., 1938, Am. J. Hyg.
27:493-497).
7.1.3. CELL FUSION ASSAY
Approximately 7 x 10" Molt-4 cells were incubated with 1 x 10' CEM cells chronically infected with the HIV-1,~,I virus in 96-well tissue culture plates in a final volume of 100,1 culture medium (RPM1 1640 containing 10% heat inactivated FBS, supplemented with 1% L-glutamine and 1% Pen-Strep) as previously described (Matthews, T.J. et al., 1987, Proc.
Natl. Acad. Sci. USA 84: 5424-5428). Peptide inhibitors were added in a volume of 10~C1 and the cell mixtures were incubated for 24 hr. at 37°C in 5% C02. At that time, multinucleated giant cells (syncytia, five cell widths or larger) were counted by microscopic examination at lOx and 40x magnification which allowed visualization of the entire '"cell in a single field. Treated cells were compared to infected, untreated controls and results expressed as percent inhibition of infected controls.
7.1.4. MAGI-CCR-5 INFECTIVITY ASSAYS
Approximately 1 x 106 Magi-CCR-5 cells (obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID; Chackerian, B. et al., 1997, J. Virol. 71:
3932-3939) were seeded into a 48-well tissue culture plate (approximately 2 x 104 cells/well in a volume of 300 ~1/well selective growth medium consisting of DMEM supplemented with 10% heat inactivated FBS, 1% L-glutamine, 1% Pen/Strep, -Hygromycin B, Geneticin, and Puromycin) and allowed to attach overnight at 37°C, 5% C02. Cell confluency was approximately 30% by the following day. Seeding medium was removed and diluted peptide inhibitor added in volumes of 50 ~1/well (media only in untreated controls), followed by 100 ~1/well of diluted virus (desired input virus titre of 100 - 200 pfu/well). Finally, 250 ~,1 of selective growth medium was added to each well and the plate incubated for 2 days at 37°C, 5% C02. Fixing and staining were done according to the protocol provided by NIAID with the MAGI-CCR5 cells.
Briefly, medium was removed from the plate and 500 ~1 of fixative added to each well. Plates were allowed to fix for 5 minutes at room temp. Fixative was removed, each well washed twice with DPBS, and 200 ~C1 of staining solution added to each well. The plate was then incubated at 37°C, 5% C02, for 50 minutes, staining solution removed, and each well washed twice with DPBS. The plate was allowed to air dry before blue cells were counted by microscopic, enumerating the entire well. Treated wells were compared to infected, untreated controls and results expressed as percent inhibition of infected controls.
7.1.5. REVERSE TRANSCRIPTASE ASSAY
The micro-reverse transcriptase (RT) assay was adapted from Goff et al. (Guff, S. et al., 1981, J. Virol. 38: 239-248) and Willey et al. (Willey, R. et al., 1988, J. Virol.
62: 139-147). Supernatants from virus/cell cultures were adjusted to 1% Triton-X100. 10 ~,1 of each supernatant/Triton X-100 sample were added to 50 ul of RT cocktail (75 mM KC1, 2 mM Clevelands reagent, 5 mM MgCl2, 5 ~g/ml poly A, 0.25 units/ml oligo dT, 0.05% NP40, 50 mM Tris-HC1, pH 7.8, 0.5 ~M
non-radioactive dTTP, and 10 cCi/ml 32P-dTTP) in a 96-well U-bottom microtitre plate and incubated at 37°C for 90 min.
After incubation, 40 ~1 of reaction mixture from each well was transferred to a Schleicher and Schuell (S+S) dot blot apparatus, under partial vacuum, containing a gridded 96-well filter-mat (Wallac catalog #1450-423) and filter backing w saturated with 2x SSC buffer (0.3M NaCl and 0.003M sodium citrate). Each well was washed 4 times with at least 200 ~l 2x SSC using full vacuum. Minifold was disassembled and gridded filter paper removed and washed 3 times with 2x SSC.
Finally, the filter membrane was drained on absorbent paper, allowed to air dry, and sealed in heat sealable bags.
Samples were placed in a phosphorscreen cassette and an erased (at least 8 min) phosphorscreen applied and closed.
Exposure was for 16 hr. Pixel Index Values (PIV), generated in volume reporting format retrieved from phosphorimaging (Molecular Dynamics Phosphorimager) blots, were used to determine the affected or inhibited fraction (Fa) for all doses of inhibitors) when compared to untreated, infected controls (analyzed by ImageQuant volume report, corrected for background).
7.1.6. HUMAN PBMC INFECTIVITY/NEUTRALIZATION
ASSAY
The prototypic assay used cell lines where the primary isolate assay utilizes PBMC, obtained through Interstate Blood Bank, activated for 2-3 days with a combination of OKT3 (0.5 ~g/ml) and CD28 antibodies (0.1 ~,g/ml). The target cells were banded on lymphocyte separation medium (LSM), washed, and frozen. Cells were thawed as required and activated as indicated above a minimum of 2-3 days prior to assay. In this 96-well format assay, cells were at a concentration of 2 x 106/ml in 5% IL-2 medium and a final volume of 100 ~l. Peptide stock solutions were made in DPBS
(1 mg/ml). Peptide dilutions were performed in 20% FBS RPM1 1640/5% IL-2 complete medium.
7~1~7~ IN VIVO HU-PBMC SCID MODEL

Female SCID mice (5-7 weeks old) received 5-10x10' adult human PBMC injected intraperitoneally. Two weeks after reconstitution, mice were infected IP on day 0 with 103 TCIDSo HIV-1 9320 (AZT-sensitive isolate A018). Treatment with peptides was IP, bid, beginning day -1 and continuing through day 6. The extent of infection in blood cells, splenocytes, lymph nodes, and peritoneal cells was assayed by quantitative co-culture with human PBMC blasts weekly for three consecutive weeks following animal exsanguinations and tissue harvest (day 7, approximately 12-18 hours following the last drug treatment). Co-culture supernatants were evaluated for HIV-1 p24 antigen production as a measure of virus infection (Immunotek Coulter kits and protocol).
7.1.8. RAT PHARMACOKINETIC STUDIES
250-300 g male CD rats, double jugular catheter, obtained from Charles River Laboratories were used. Peptides were injected in one jugular catheter in a volume of 200 ~1 of peptide solution (approximately 3.75 mg/ml), dosing solution concentration was determined using the Edelhoch method, (Edelhoch, 1967, Biochemistry 6:1948-1954) method and adjusted based on animal weight such that each animal received a dose of 2.5 mg/kg). Approximately 250-300 ~1 of blood was removed at predetermined time intervals (0, 15, 30 min and 1, 2, 4, 6, and 8 hours) and added to EDTA capiject tubes. Plasma was removed from pelleted cells upon centrifugation and either frozen or immediately processed for fluorescence HPLC analysis.
7,1,9, FLUORESCENCE HPLC ANALYSIS OF
PLASMA SAMPLES
100 ~,1 of sample plasma was added to 900 ~C1 of precipitation buffer (acetonitrile, 1.0% TFA, detergent) resulting in precipitation of the majority of plasma proteins. Following centrifugation at 10,000 rpm for 10 min, 400 ~,1 of the supernatant was removed and added to 600 ~,1 of HPLC grade water. Serial dilutions were performed as dictated by concentration of peptide present in each sample in dilution buffer comprised of 40% precipitation buffer and 60% HPLC water. In addition to sample dilutions, serial dilutions of dosing solution were performed in buffer as well as in plasma and used to generate a standard curve relating peak area to known concentration of peptide. This curve was then used to calculate concentration of peptide in plasma taking into account all dilutions performed and quantity injected onto column.
7.1.10. XTT PROTOCOL
In order to measure cytotoxic/cytostatic effects of peptides, XTT assays (Weislow, O.S. et al., 1989, J. Natl.
Cancer Inst. 81:577-586) were performed in the presence of varying concentrations of peptide in order to effectively establish a selective index (SI). A TCSO was determined in this assay by incubating cells in the presence and absence of serially diluted peptide followed by the addition of XTT. In surviving/metabolizing cells XTT is reduced to a soluble brown dye, XTT-formazan. Absorbance is read and comparisons made between readings in the presence and absence of peptide to determine a TCSO utilizing the Karber method (see. e.a., Lennette, E.H. et al., eds., 1969, "Diagnostic Procedures for Viral and Rickettsial Infections," American Public Health Association, Inc., fourth ed., pp. 47-52). Molt 4, CEM
(80,000 cells/well) and a combination of the two cell types (70,000 and 10,000 respectively) were plated and incubated with serially diluted peptide for 24 hours in a total volume of 100 ~1. Following incubation, 25 ~,1 of XTT working stock (1 mg/ml XTT, 250 ~M PMS in complete medium containing 5%
DMSO) was added to each well and the plates incubated at 37°C. Color development was read and results used to express values generated from peptide containing wells as a percentage of the untreated control wells.
7.2. RESULTS
7.2.1. ANTIVIRAL ACTIVITY - FUSION ASSAYS
T1249 was directly compared to T20 in virus mediated cell-cell fusion assays conducted using chronically infected CEM cells mixed with uninfected Molt-4 cells, as shown in Table 3, below. T1249 fusion inhibition against lab isolates such as IIIb, MN, and RF is comparable to T20, and displays an approximately 2.5-5-fold improvement over T20. T1249 was also more active (3-28 fold improvement) than T20 against several syncytia-inducing clinical isolates, including an AZT
resistant isolate (G691-2), a pre-AZT treatment isolate (G762-3), and 9320 (isolate used in HuPBMC-SCID studies).
Most notably, T1249 was over 800-fold more potent than T20 against HIV-2 NIHZ.

Virus Isolate T20 n T1249 n Fold (ng~~) (ng/ml) Different a HIV-1 IIIb 2.5 9 1.0 9 2.5 HIV-1 6691-2 (AZT-R)406.0 1 16.0 1 25 HIV-1 6762-3 (Pre- 340.1 1 12.2 1 28 AZT) HIV-1 NQ1 20.0 7 3.1 7 6 _ HIV-1 RF 6.1 7 2.1 7 3 i HIV-1 9320 118.4 1 34.5 1 3 HIV-2 NIHZ 3610.0 >10 4.3 2 840 7.2.2. ANTIVIRAL ACTIVITY - Magi-CCR-5 INFECTIVITY ASSAYS
Magi-CCR-5 infectivity assays allow direct comparisons to be made of syncytia and non-syncytia inducing virus isolates, as well as comparisons between laboratory and clinical isolates. The assay is also a direct measure of virus infection (TAT expression following infection, transactivating an LTR driven beta-galactosidase production), as opposed to commonly used indirect measures of infectivity such as p24 antigen or reverse transcriptase production.
Magi-CCR-5 infectivity assays (see Table 4 below) reveal that T1249 is consistently more effective than T20 against all isolates tested, in terms of both ECSo and Vn/Vo = 0.1 -inhibition calculations. T1249 shows considerable improvement in potency against the clinical isolate HIV-1 301714 (>25-fold), which is one of the least sensitive isolates to T20. In addition, T1249 is at least 100-fold more potent than T20 against the SIV isolate B670. These data, along with fusion data suggest that T1249 is a potent peptide inhibitor of HIV-1, HIV-2, and SIV.

i Virus EC-50 Vn/Vo=0.1 E Vn/Vo=0.18C-50 Vn/Vo=0.1 Isolate C- Fold Fold 50 DifferenceDifference IIIB

( subtype B, NSI) HIV-1 13 200 0. 20 43 10 (AZT-R) pNL4-3 SIV-B670 2313 >10000 21 100 110 >100 7.2.3. ANTIVIRAL ACTIVITY - HuPBMC INFECTIVITY ASSAYS
T1249 was directly compared to T20 in HuPBMC infectivity assays (Table 5, below), which represent a recognized surrogate in vitro system to predict plasma drug concentrations required for viral inhibition in vivo. These comparisons revealed that T1249 is more potent against all HIV-1 isolates tested to date, with all Vn/Vo = 0.1 (dose required to reduce virus titer by one log) values being reduced to sub-microgram concentrations. Many of the least sensitive clinical isolates to T20 exhibited 10-fold or greater sensitivity to T1249. It is noteworthy that HIV-1 9320, the isolate used in the HuPBMC SCID mouse model of infection, is 46-fold less sensitive to T20 than to T1249, indicating a very good correlation with the in vivo results.

Virus Isolate (HIV-1)Vn/Vo = Vn/Vo = Fold 0.1 0.1 Difference (ng/ml) (ng/ml) 9320 6000 ~ 130 46 301714 (subtype B, 8000 700 11 NSI) 302056 (subtype B, 800 90 9 NSI) 301593 (subtype B, 3500 200 18 SI) 302077 (subtype A) 3300 230 14 302143 (SI) 1600 220 7 6691-2 (AZT-R) 1300 400 3 7.2.4. ANTIVIRAL ACTIVTTY - T20 RESISTANT LAB
ISOLATES
T1249 was directly compared to T20 in virus mediated cell-cell fusion assays conducted using chronically infected CEM cells mixed with uninfected Molt-4 cells (Table 6, below). T1249 was nearly 200-fold more potent than T20 against a T20-resistant isolate.

Virus T20 n T1249 n Fold Isolate (ng/ml) (ng/ml) Difference HIV-1 pNL4-3 405.3 3 2.1 3 193 SM

3 (T20 Resistant) In Magi-CCR-5 assays (see Table 7, below), T1249 is as much as 50,000-fold more potent than T20 against T20-resistant isolates such as pNL4-3 SM and pNL4-3 STM (Rimsky, L. and Matthews, T., 1998, J. Virol. 72:986-993).

Virus EC- Vn/Vo EC-50 Vn/Vo=0.1 EC-50 Vn/Vo=0.1 Isolate 50 = 0.1 Fold Fold (HIV-1) DifferenceDifference pNL4-3 166 210 1 13 166 16 pNL4-3 SM 90 900 4 11 23 82 (T20-R) pNL4-3 SM 410 2600 4 11 103 236 (T20-R) Duke pNL4-3 STM >50 >5000 1 13 >50000 >3846 (T20/T649- 000 0 R) T1249 was directly compared to T20 in HuPBMC infectivity assays (see Table 8, below), evaluating differences in potency against a resistant isolate. T1249 is greater than 250-fold more potent than T20 against the resistant isolate pNL4-3 SM.

Virus Isolate (HIV-1)Vn/Vo ~ 0.1 Vn/Vo = Fold (ng/ml) 0.1 Difference (ng/ml) pNL4-3 3500 30 117 pNL4-3 SM (T20-R) >10000 40 >250 --7.2.5. ANTIVIRAL ACTIVITY - IN VIVO SCID-HuPBMC MODEL
In vivo antiviral activity of T1249 was directly compared to T20 activity in the HuPBMC-SCID mouse model of HIV-1 9320 infection (FIG. 3). Two weeks after reconstitution with HuPBMCs, mice were infected IP on day 0 with I03 TCIDSO HIV-1 9320 passed in PBMCs (AZT-sensitive isolate A018). Treatment with peptides was IP, bid, for total daily doses of 67 mg/kg (T20), 20 mg/kg (T1249), 6.7 mg/kg (T1249), 2.0 mg/kg (T1249), and 0.67 mg/kg (T1249), for 8 days beginning on day -1. The extent of infection in blood cells, splenocytes, lymph nodes, and peritoneal cells was assayed by quantitative co-culture with human PBMC blasts weekly for three consecutive weeks following animal exsanguinations and tissue harvest (day 7, approx. 12 to 18 hours following last drug treatment). Co-culture supernatants were evaluated for HIV-1 p24 antigen production as a measure of virus infection. Infectious virus was not detectable in the blood or lymph tissues of the T20-treated animals, although, virus was detected in the peritoneal washes and spleen preparation. All compartments were negative for infectious virus at the 6.7 mg/kg dose of T1249, indicating at least a 10-fold improvement over T20 treatment.
At the 2.0 mg/kg dose of T1249, both the lymph and the spleen were completely free of detectable infectious virus, with a 2 loglo reduction in virus titer in the peritoneal wash and a 1 loglo reduction in virus titer in the blood, compared to infected controls. At the lowest dose of T1249, 0.67 mg/kg, the peritoneal washes and blood were equivalent to infected control; however, at least a 1 loglo drop in infectious virus titer was observed in both the lymph and the spleen tissues.
Overall, the results indicate that T1249 is between 30 and 100-fold more potent against HIV-1 9320, in vivo, under these conditions.

7.2.6. PHARMACOKINETIC STUDIES - RAT
Cannulated rats were used to further define the pharmacokinetic profile of T1249. Male CD rats, 250-300 g, were dosed IV through a jugular catheter with T1249 and T20 (FIGS. 4A-5). The resulting plasma samples were evaluated using fluorescence HPLC to estimate peptide quantities in extracted plasma. The beta-phase half-life and total AUC of T1249 was nearly three times greater than T20 (FIG. 5).
7.2.7. CYTOTOXICITY
No overt evidence of T1249 cytotoxicity has been observed in vitro, as demonstrated in FIG. 6.
In addition, T1249 is not acutely toxic (death within 24 hours) at 167 mg/kg (highest dose tested) given IV through jugular cannula (0.3 ml over 2-3 min).
7.2.8. DIRECT BINDING TO gp41 CONSTRUCT

T1249 was radiolabelled with l2sI and HPLC- purified to maximum specific activity. T20 was iodinated in the same manner. Saturation binding of to M41~178 (a truncated gp41 ectodomain fusion protein lacking the T20 amino acid sequence) immobilized on microtitre plates at 0.5 mg/~C1 is shown in FIG.7. Nonspecific binding was defined as binding of the radioligand in the presence of 1 ACM unlabeled peptide.
Specific binding was the difference between total and nonspecific binding. The results demonstrate that l2sl-T1249 and lzsI-T20 have similar binding affinities of 1-2 nM.
Linear inverse Scatchard plots suggests that each ligand binds to a homogeneous class of sites.
The kinetics of l2sl-T1249 and l2sl-T20 binding was determined on scintillating microtitre plates coated with 0.5 ~,g/ml M41~178. The time course for association and dissociation is shown in FIG.8. Dissociation of bound radioligand was measured following the addition of unlabeled peptide to a final concentration of to ~cM in one-tenth of the total assay volume. initial on- and off-rates for l2sl_T1249 were significantly slower than those of l2sl-T20.
Dissociation patterns for both radioligands were unchanged when dissociation was initiated with the other unlabeled peptide (i.e., l2sl-T1249 with T20) .
To further demonstrate that both ligands compete for the same target site, unlabeled T1249 and T20 were titrated in the presence of a single concentration of either lzsl-T1249 or 1251-T20. Ligand was added just after the unlabeled peptide to start the incubation. The competition curves shown in FIG.9 suggest that although both ligands have similar affinities, a higher concentration of either unlabeled T20 or T1249 is required to fully compete for bound l2sl_T1249.
7.2.9. DIRECT BINDING TO THE HR1 Circular dichroism (CD) spectroscopy was used to measure the secondary structure of T1249 in solution (phosphate-buffered saline, pH 7) alone and in combination with a 45-residue peptide (T1346) from the HR1 (heptad repeat 1) binding region of gp 41. FIG. 14A illustrates the CD
spectrum of T1249 alone in solution (10 ~,M, 1QC). The spectrum is typical of peptides which adopt an alpha-helical structure. In particular, deconvolution of this spectrum using single value decomposition with a basis set of 33 protein spectra predicts the helix content of T1249 (alone in solution) to be 50%. FIG. 14B illustrates a representative CD spectrum of T1249 mixed with T1346. The closed squares (~) represent a theoretical CD spectrum predicted for a "non-interaction model" wherein the peptides are hypothesized ~5 to not interact in solution. The actual experimental spectrum (~) differs markedly from this theoretical "non-interaction model" spectrum, demonstrating that the two peptides do, indeed, interact, producing a measurable structural change which is observed in the CD spectrum.

WO 99/59615 PCT/LlS99/11219 7.2.10. PROTEASE PROTECTION OF THE T1249 The susceptibility of the chimeric protein M41~178, described in Section 7.2.8 above, to proteinase-K digestion was determined and analyzed by polyacrylamide gel electrophoresis. The results are illustrated in FIG. 15.
When either M41~178 (untreated; FIG 15, lane 2) or T1249 (untreated; FIG. 15, lane 4) are incubated individually with proteinase K (FIG. 15, lanes 3 and 5, respectively), both are digested. However, when T1249 is incubated with M41o178 prior to addition of proteinase-K
(FIG. 15, lane 7), a protected HR-1 fragment of approximately 6500 Daltons results. Sequencing of the protected fragment demonstrates that it corresponds to a region of primary sequence located within the ectodomain of gp4l. The protected fragment encompasses the soluble HR1 peptide (T1346) used in the CD studies described in Section 7.2.9 above, and further contains an additional seven amino acid residues located on the amino terminus. This protection can be attributed to the binding of T1249 to a specific sequence of gp41 which is contained in the M41~178 construct.
8. EXAMPLE: RESPIRATORY SYNCYTIAL
VIRUS HYBRID POLYPEPTIDES
The following example describes respiratory syncytial virus (RSV) hybrid polypeptides with enhanced pharmacokinetic properties. In addition, results are presented, below, which demonstrate that the RSV hybrid polypeptides represent potent inhibitors of RSV infection.
8.1. MATERIALS AND METHODS
8.1.1. PEPTIDE-SYNTHESIS AND PURIFICATION
RSV polypeptides were synthesized using standard Fast Moc chemistry. Generally, unless otherwise noted, the peptides contained amidated carboxyl termini and acetylated amino termini. Purification was carried out by reverse phase HPLC.

8.1.2. RESPIRATORY SYNCYTIAL VIRUS
PLAQUE REDUCTION ASSAY
All necessary dilutions of peptides were performed in clean, sterile 96-well TC plate. A total of eleven dilutions for each peptide and one control well containing no peptide were assembled. The final concentration range of peptide started at 50~,g/ml or 10o~,g/ml, with a total of eleven two-fold dilutions. The RSV was prepared at a concentration of 100PFU/well in 1001 3% EMEM, as determined by a known titer of RSV. The virus is then added to all of the wells.
The media was removed from one sub-confluent 96-well plate of Hep2 cells. The material from the dilution plate was transferred onto the cell plates starting with row 1 and then transferring row 12, row 11, etc. until all rows were transferred. Plates were placed back into the incubator for 48 hours.
The cells were checked to ensure that syncytia were present in the control wells. Media was removed and approximately 50 ~Cls of 0.25% Crystal Violet in methanol was added to each well. The wells were rinsed immediately in water to remove excess stain and allowed to dry. Using a dissecting microscope, the number of syncytia in each well was counted.
8.2. RESULTS
Pharmacokinetic studies with the RSV hybrid peptides T1301 (Ac-WQEWDEYDASISQVNEKINQALAYIREADELWA WF-NHZ) and T1302 (Ac-WQAWDEYDASISQVNEKINQALAYIREADELW AWF-NH2) containing enhancer peptide sequences demonstrated a greatly enhanced half-life relative to core peptide T786 (Ac-VYPSDEYDASISQVNEEINQALAYIRKADELLENV-NH2), as demonstrated in FIG. l0A-lOB. Hybrid polypeptides T1301, T1302 and T1303 (Ac-WQAWDEYDASISDVNEKINQALAYIREADELWEWF-NH2) also showed a greatly enhanced half-size relative to core peptide T1476 (Ac-DEYDASISQVNEKINQALAYIREADEL-NH2).
RSV hybrid polypeptides T1301, T1302 and T1303, as well as polypeptide T786 and T1293, were tested for their ability to inhibit RSV plaque formation of HEp2 cells. As indicated in FIGS. 11A and 11B, both the tested hybrid RSV
polypeptides, as well as the T786 core polypeptide were able to inhibit RSV infection. Surprisingly, the T1293 hybrid polypeptide was also revealed to be a potent anti-RSV
compound (FIG. 13).

9. EXAMPLE: LUTEINIZING HORMONE
HYBRID POLYPEPTIDES
The example presented herein describes luteinizing hormone (LH) hybrid proteins with enhanced pharmacokinetic properties. The following LH hybrid peptides were synthesized and purified using the methods described above:
core peptide T1323 (Ac-QHWSYGLRPG-NH2) and hybrid polypeptide T1324 (Ac-WQEWEQKIQHWSYGLRPGWASLWEWF-NHz) which comprises the core polypeptide T1323 amino acid sequence coupled with enhancer peptides at its amino- and carboxy-termini. As demonstrated in FIG. 12A and 12B, the T1324 hybrid peptide exhibited a significantly increased half-life when compared to the T1323 core peptide which lacks the enhancer peptide sequences.

10. EXAMPLE: PHARMACOLOGY OF HYBRID

T1249, depicted in FIG. 13, is a hybrid polypeptide comprising enhancer peptide sequences linked to a core polypeptide derived from a mix of viral sequences. As demonstrated in the Example presented in Section 7 above, the T1249 hybrid polypeptide exhibits enhanced pharmacokinetic properties and potent in vitro as well as in vivo activity against HIV-1. In the example presented below, the pharmacological properties of T1249 in both rodent and primate animal models are further described.

10.1. MATERIALS AND METHODS
10.1.1. SINGLE-DOSE ADMINISTRATION TO RODENTS
T1249 was administered to Sprague-Dawley albino rats in a single dose administered by continuous subcutaneous infusion (SCI), subcutaneous (SC) injection or intravenous (IV) injection. Each treatment group consisted of nine rats per sex per group. The groups received sterile preparations of T1249 bulk drug substance at a dose of 0.5, 2.0, or 6.5 mg/kg by CSI. One group received 50mM carbonate-bicarbonate, pH 8.5, administered as a control. The peptides were given for 12 hours via a polyvinyl chloride/polyethylene catheter surgically implanted subcutaneously in the nape of the neck. Two groups received a single dose of T1249 at a dose of 1.2 or 1.5 mg/kg by subcutaneous injection into the intrascapular region. Two groups received a single dose of T1249 at a dose of 1.5 or 5 mg/kg via intravenous injection.
The actual milligram amount of T1249 was calculated using the peptide content that was determined for the batch administrated.
Endpoints for analysis included cageside observations (twice daily for mortality and moribundity), clinical observations, clinical laboratory parameters, body weight and necropsy. Blood samples were obtained by a sparse sampling technique over a 12 hour time period from three rats per sex per group at each of the following times: 0.5, 1, 2, 4, 6, 8, 19, and 12 hours after dose administration. Sample analysis was performed using a PcAb ECLIA assay (Blackburn, G. et al., 1991, Clin. Chem. 37:1534-1539; Deaver, D., 1995, Nature 377:758).
For plasma and lymphatic pharmacokinetic analysis of T1249 in rats, T1249 was prepared as a sterile solution in bicarbonate buffer and administered as a single dose, bolus intravenous injection into the lateral tail vain at a dose of 20 mg/kg. Blood was collected from the animal from an in-dwelling jugular catheter. Samples were collected i~ediately after dosing and at 5, 15, and 30 minutes, and 1, 2, 4, and 6 hours after drug administration. For the analysis of lymphatic fluids, samples were taken immediately before dosing and every 20 minutes for the first six hours after dosing. Lymphatic fluid was collected from a catheter placed directly into the thoracic lymphacic duct as previously described (Kirkpatrick and Silver, 1970, The Journal of Surgical Research 10:147-158). The concentrations of T1249 in plasma and lymphatic fluid were determined using a standard T1249 Competitive ELISA assay (Hamilton, G. 1991, p. 139, in "Immunochemistry of Solid-Phase Immunoassay,", Butler, J., ed., CRC Press, Boston).
10.1.2. SINGLE-DOSE ADMINISTRATION TO PRIMATES
Sterile preparations of T1249 bulk drug substance were administered to cynomolgus monkeys in single doses administered by subcutaneous (SC), intramuscular (IM) or intravenous (IV) injection. In a sequential crossover design, one group of animals consisting of two per sex received a single bolus dose of T1249 by IV (0.8 mg/kg), IM
(0.8 mg/kg) or SC (0.4, 0.8, and 1.6 mg/kg) injection. A
washout period of at least three days separated each dosing day. Lyophilized T1249 was reconstituted in sterile phosphate buffered saline pH 7.4 immediately prior to dosing.
The actual milligram amount of test article was calculated using the peptide content that was determined for the batch administered.
Endpoints for analysis included cageside observations, physical examinations and body weight. For the IV phase of the study, blood samples were collected into heparinized tubes at the following time points: immediately after dosing, 0.25, 0.5, 1.5, 3, 6, 12, and 24 hours after dosing.
For the IM and SC phases of the study blood samples were collected in heparinized tubes from each animal at the following time points: 0.5, 1, 2, 3, 6, 12, and 24 hours after dosing. Plasma samples were prepared within one hour of collection and flash frozen in liquid nitrogen. Samples analysis was performed using a PcAb ECLIA assay (Blackburn, G. et al., 1991, Clin. Chem. 37:1534-1539; Deaver, D., 195;
Nature 377:758).
10.1.3. BRIDGING PHARMACOKINETIC STUDY
Six male cynomolgus monkeys were randomly assigned to three groups consisting of two animals per group. All doses of T1249 were given by bolus subcutaneous injection. The study was divided into two sessions. In Session 1, animals in groups 1, 2 and 3 received a sterile preparation of T1249 bulk drug substance (i.e., bulk +1249 dissolved in carbonate-bicarbonate, pH 8.5) twice daily for four consecutive days (Study Days 1-4) at doses of 0.2, 0.6 and 2.0 mg/kg/dose, respectively. A ten day washout period separated Session 1 and Session 2. In Session 2, animals in groups 1, 2, and 3 received a sterile preparation of T1249 drug product (i.e., in aqueous solution, pH 6.5, plus mannitol) twice daily for four consecutive days (Study Days 15-18) at doses of 0.2, 0.6 and 2.0 mg/kg/dose, respectively.
Blood samples for pharmacokinetic analyses were collected on Study Days 1 and 15 to assess single-dose pharmacokinetic parameters, and on Study Days 4 and 18 to assess steady-state plasma pharmacokinetic parameters.
Samples were collected at the following times: immediately pre-dose, and 0.5, 1.5, 3.0, 4.0, 6.0, 8.0 and 12.0 hours post-dose. Animals were monitored during Sessions 1 and 2 for clinical signs and changes in body weight.
10.2. RESULTS
10.2.1. PHARMACOKINETICS OF T1249 ADMINISTERED TO RATS
Rat models were used to perform an initial assessment of plasma pharmacokinetics and distribution of T1249. For animals in all dose groups, there were no changes in body weight, physical observations, hematology and clinical chemistry parameters or macroscopic pathology observations, related to the administration of T1249.

Rats that received T1249 by CSI achieved steady-state plasma peptide concentrations approximately four hours after administration. Both the steady-state concentration in plasma (Cpss) and calculated area under the plasma concentration versus time curve (AUC) were directly proportional to the administered dose, indicating that T1249 displays linear pharmacokinetics within the tested dose range of 0.5 to 6.5 mg/kg. Both the calculated pharmacokinetic parameters and the plasma concentration versus time curves for the CSI route of administration are presented in Table 9 and in FIG. 16A, respectively.

Dose Groups Parameter 0.5 mg/kg 2.o mg/kg 6.5 mg/kg Cpss (~,g/ml) 0.80 2.80 10.9 AUC~o_,2,,~ (~g'h/ml) 7.99 25.9 120 Administration of T1249 by bolus IV injection resulted in linear dose-dependent pharmacokinetics within the doses tested. In contrast, exposure to T1249 by SC injection was not dose-dependent within the dose range studied. The calculated pharmacokinetic parameters and plasma concentration versus time curves for both SC and TV
administration of T1249 are shown in Table 10 and FIG. 16B
respectively.

Dose Groups/Administration (SC) (IV) Parameter 1.2 mg/kg 15 mg/kg 1.5 mg/kg 5.0 mg/kg tl/2, terminal 2.02 2.00 2.46 1.86 (hours) _ 87 _ tmaX (hours) 1.09 1.88 - -Cmax (l~g/ml) 6.37 21.5 15.7 46.3 AUC~o_12n~ 27.0 107 45.6 118 (~Cgh/ml) AUCio_.., 27.6 110 47.1 120 (~gh/ml) The bioavailability of T1249 administered to rats by subcutaneously was determined relative to IV administration.
The results are shown in Table 11 below. At low dose (1.2 mg/kg) T1249 exhibited a relative bioavailability (FR) of 73o for subcutaneous administration. Relative bioavailability was 30% when high-dose (15 mg/kg) administration of T1249 concentration was greater than the concentration that inhibits 90% (IC9o) of HIV infectivity for the full 12 hours of the study at all doses examined.
TAB?~E 11 Route Dose AOC~o_m~ Normalized AUC~a_ FR
(m9/kg) (ug~h/ml) ~~>
(E,tg~h/ml) Low Dose SC 1.2 27.6 34.5~a~ 73 IV 1.5 47.1 - -High Dose SC 15 110 36.5~b~ 30 2 5 "' Normalized from a 1.2 mg/kg dose to a 1.5 mg/kg dose by multiplying AUC,o__, by 1.25.
'°' Normalized from a 15 mg/kg dose to a 5 mg/kg dose by dividing AUC,o_., by 3.
The kinetic data for both plasma and lymph concentrations of T1249 are illustrated in FIG. 16C and tabulated below in Table 12. T1249 rapidly penetrated into the lymphatic system and equilibrated with the plasma reservoir of drug within approximately one hour after _ 88 _ administration. Following equilibration between the two compartments, plasma and lymph levels of drug were comparable out to three hours post-dosing in four out of five animals.
One animal had consistently lower concentrations of T1249 in the lymph than the other animals, however this animal's lymph elimination profile was indistinguishable from other members of the group. Comparison of the elimination phase half-life (t1/2) for plasma and lymph suggest that the transit of T1249 between these two compartments is a diffusion-controlled process. After three hours, there appeared to be a second, more rapid elimination phase from the lymphatic system. This difference could be mechanism-based (e.g., due to redistribution or accelerated peptide degradation in the lymph) or due to other factors. The concentration of T1249 in lymphatic fluid six hours post-injection is greater than the IC9o for viral infectivity for common laboratory strains and for primary clinical isolates of HIV-1.
The extent of penetration of T1249 into cerebrospinal fluid (CSF) was also assessed. T1249 concentrations were below the limit of detection (LOD; 2.0 ng T1249/ml CSF) at all measurable time points, indicating that T1249 does not penetrate the central nervous system after a single dose administration.

Parameter Plasma Lymph t1~2, 2. 60.41 1. 30.27 elimination(hours) Cmex (hg/ml) 291 133~a~/155~b~

AUC~p_6n) (/.tgh/ml) 506 348~a~/411~b~

AUC,o_m~ (~gh/ml) 598 390~a~/449~b~

C1 (ml/h) 7.8 11.5 _ 89 -"' Calculated averages include one animal (Rat #1) that exhibited significantly lower lymph concentrations but a similar kinetic profile by comparison to the other animals in the group.
'"' Calculated averages that exclude Rat #1.
10.2.2. P13ARMACOKINETICS OF T1249 ADMINISTERED TO PRIMATES
Primate models were used to evaluate the relationship between dose level and various pharmacokinetic parameters associated with the parenteral administration of T1249.
Plasma concentrations greater than 6.0 ~,g/ml of T1249 were achieved by all routes of administration and quantifiable :0 levels (i.e., levels greater than 0.5 ~g/ml) were detected at 24 hours after SC and IV administration. The elimination t"2 was comparable for all routes of administration (5.4 hours, 4.8 hours and 5.6 hours for IV, SC and IM administration, respectively). Plasma concentrations of T1249 that exceed the IC9o values for laboratory strains and clinical isolates of HIV-1 were observed at all measured time points throughout the 24 hour sampling period.
A comparison of the data obtained for the parenteral administration of 0.8 mg/kg T1249 via all routes of administration (SC, IV, and IM) is presented in FIG. 17A.
FIG. 15B illustrates a comparison of the data obtained from SC injection at three different dose levels of T1249 (0.4 mg/kg, 0.8 mg/kg, and 1.6 mg/kg). The insert in FIG.
178 contains a plot of the calculated AUC versus administered dose.
T1249 displays linear pharmacokinetics in cynomolgus monkeys following SC administration within the range of administered doses, indicating that saturation of the clearance mechanism or mechanisms has not occurred within this range. A summary of the pharmacokinetic data following parenteral administration of T1249 to cynomolgus monkeys is provided in Table 13, below. A comparison of the plasma AUC
values indicates that, relative to intravenous administration, the bioavailability of T1249 is approximately 64% when given by intramuscular injection and 92% when given by subcutaneous injection.
Table 13 Parameter Admini strationRoute (Dose mg/kg) Level, SC (0.4) SC (0.8)SC (1.6) IM (0.8) IV (0.8) ti/z, cermiaal 6.230.52 4.8310.485.550.92 5.570.24 5.350.95 (h) t",ax (h) 3.971.18 4.581.454.7211.81 2.320.43 -C"~x (~g/ml) 3.170.09 6.8511.0113.312.55 6.3711.6926.70.25 IO

AUC,o_z4~ 37.516.6 8.12111.4168134.0 56.412.3 87.4125.0 (~gh/ml) AUC~o__, 40.98.2 85.313.618144.0 59.5I3.1 92.525.0 (~gh/ml) - 92.3 - 64.4 -10.2.3. BRIDGING PHARMACOKINETIC STUDY
Bridging pharmacokinetic studies were performed in order to compare the plasma pharmacokinetic profiles of the T1249 bulk drug substances used in the nonclinical trials described above to the formulated T1249 drug product which would be administered to an actual subject or patient, e.g., to treat HIV infection. The study was designed as a parallel group, one-way, cross-over comparison of three dose levels of T1249 bulk drug substance and three dose levels of formulated drug product. Plasma pharmacokinetics were assessed after single-dose administration and after steady state was achieved.
Administration of T1249 by subcutaneous injection resulted in measurable levels of peptide in all dose groups.
The plasma concentration-time curves were roughly parallel within all dose groups following the initial dose (Days 1 and 15) and at steady state (Days 4 and 18) for both T1249 bulk, drug substance and formulated T1249 drug product.
Furthermore AUC,o-l2nr, values varied in direct proportion to the dose level for both drug formulations. Calculated AUC,o-lz,,r, values for the drug product ranged from 43 % to 80 of the AUC,o_lzn=, values calculated for drug substance following single dose administration, and from 36o to 71o at steady state.
T1249 bulk drug substance and drug product demonstrated similar pharmacokinetic profiles in cynomolgus monkeys following bolus subcutaneous administration at the dose levels and dose volume tested. A direct comparison of the shapes of the plasma concentration-time curves in the present study and the shapes of curves from a previous study in cynomolgus monkeys suggests that there is a depot effect when T1249 is administered by subcutaneous injection. This is suggested by the increases in time at which maximal plasma concentration ~tmax) is achieved and t1,2~
These results indicate that the formulation of bulk drug substance used in the pharmacology program yields comparable AUC values and other kinetic parameters to those observed following the administration of the formulated drug product.
These observations indicate that clinical administration of T1249 will result in total patient exposure to T1249.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims (20)

WHAT IS CLAIMED IS:

1. A hybrid polypeptide comprising an enhancer peptide sequence linked to a core polypeptide.

2. The hybrid polypeptide of Claim 1, wherein the enhancer peptide sequence comprises: WXXWXXXI, WXXWXXX, WXXWXX, WXXWX, WXXW, WXXXWXWX, XXXWXWX, XXWXWX, XWXWX, WXWX, WXXXWXW, WXXXW, IXXXWXXW, XXXWXXW, XXWXXW, XWXXW, XWXXXW, XWXWXXX, XWXWXX, XWXWX, XWXW, WXWXXXW or XWXXXW.

3. The hybrid polypeptide of Claim 1 wherein the enhancer peptide sequence comprises WQEWEQKI or WASLWEWF.

4. The hybrid polypeptide of Claim 1, wherein the enhancer peptide sequence is linked to the amino-terminal end of the core polypeptide.

5. The hybrid polypeptide of Claim 4, further comprising an enhancer peptide sequence linked to the carboxy-terminal end of the core polypeptide.

6. The hybrid polypeptide of Claim 1, wherein the enhancer peptide sequence is linked to the carboxy-terminal end of the core polypeptide.

7. The hybrid polypeptide of Claim 1 wherein the core polypeptide is a therapeutic reagent.

8. The hybrid polypeptide of Claim 1 wherein the core polypeptide is a bioactive peptide, a growth factor, cytokine, differentiation factor, interleukin, interferon, colony stimulating factor, hormone or angiogenic factor amino acid sequence.

9. The hybrid polypeptide of Claim 1, wherein the core polypeptide comprises the following amino acid sequence:
YTSLIHSLIEESQNQQEKNEQELLELDK; LEENITALLEEAQIQQEKNMYELQKLNS;

LEANISQSLEQAQIQQEKNMYELQKLNS; NNYTSLIHSLIEESQNQQEKNEQELLEL;
DFLEENITALLEEAQIQQEKNMYELQKL; RYLEANISQSLEQAQIQQEKNMYELQKL;
RYLEANITALLEQAQIQQEKNEYELQKL; NNYTSLIHSLIEESQNQQEKNEQELLELDK;
TALLEQAQIQQEKNEYELQKLDK;
TALLEQAQIQQEKNEYELQKLDE;
TALLEQAQIQQEKNEYELQKLIE;
TALLEQAQIQQEKIEYELQKLDK;
TALLEQAQIQQEKIEYELQKLDE;
TALLEQAQIQQEKIEYELQKLIE;
TALLEQAQIQQEKIEYELQKLE;
TALLEQAQIQQEKIEYELQKLAK;
TALLEQAQIQQEKIEYELQKLAE;
TALLEQAQIQQEKAEYELQKLE;
TALLEQAQIQQEKNEYELQKLE;
TALLEQAQIQQEKGEYELQKLE;
TALLEQAQIQQEKAEYELQKLAK;
TALLEQAQIQQEKNEYELQKLAK;
TALLEQAQIQQEKGEYELQKLAK;
TALLEQAQIQQEKAEYELQKLAE;
TALLEQAQIQQEKNEYELQKLAE;
TALLEQAQIQQEKGEYELQKLAE;
DEFDASISQVNEKINQSLAFIRKSDELL;
DEYDASISQVNEKINQALAYIREADEL;
DEYDASISQVNEEINQALAYIRKADEL; DEFDESISQVNEKIEESLAFIRKSDELL;
DEFDESISQVNEKIEESLAFIRKSDEL; or QHWSYGLRPG.

10. The hybrid polypeptide of Claim 9, wherein the enhancer peptide sequence is linked to the amino-terminal end of the core polypeptide.

11. The hybrid polypeptide of Claim 10, further comprising an enhancer peptide sequence linked to the carboxy-terminal end of the core polypeptide.

12. The hybrid polypeptide of Claim 9, wherein the enhancer peptide sequence is linked to the carboxy-terminal end of the core polypeptide.

13. The hybrid polypeptide of Claim 9, wherein the enhancer peptide sequence comprises WQEWEQKI or WASLWEWF.

14. The hybrid polypeptide of Claim 9, wherein the hybrid polypeptide comprises the amino acid sequence:
WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF, WQEWEQKITALLEQAQIQQEKIEYELQKLIEWEWF or VYPSDEYDASISQVNEEINQALAYIRKADELLENV.

15. The hybrid polypeptide of Claim 14, further comprising an amino terminal acetyl group and a carboxy terminal amido group.

16. A core polypeptide comprising:
YTSLIHSLIEESQNQQEKNEQELLELDK; LEENITALLEEAQIQQEKNMYELQKLNS;
LEANISQSLEQAQIQQEKNMYELQKLNS; NNYTSLIHSLIEESQNQQEKNEQELLEL;
DFLEENITALLEEAQIQQEKNMYELQKL; RYLEANISQSLEQAQIQQEKNMYELQKL;
RYLEANITALLEQAQIQQEKNEYELQKL; NNYTSLIHSLIEESQNQQEKNEQELLELDK;
TALLEQAQIQQEKNEYELQKLDK;
TALLEQAQIQQERNEYELQKLDE;
TALLEQAQIQQEKNEYELQKLIE;
TALLEQAQIQQEKIEYELQKLDK;
TALLEQAQIQQEKIEYELQKLDE;
TALLEQAQIQQEKIEYELQKLIE;
TALLEQAQIQQEKIEYELQKLE;
TALLEQAQIQQEKIEYELQKLAK;
TALLEQAQIQQEKIEYELQKLAE;
TALLEQAQIQQEKAEYELQKLE;
TALLEQAQIQQEKNEYELQKLE;
TALLEQAQIQQEKGEYELQKLE;
TALLEQAQIQQEKAEYELQKLAK;
TALLEQAQIQQEKNEYELQKLAK;
TALLEQAQIQQEKGEYELQKLAK;

TALLEQAQIQQEKAEYELQKLAE;
TALLEQAQIQQEKNEYELQKLAE;
TALLEQAQIQQEKGEYELQKLAE;
DEFDASISQVNEKINQSLAFIRKSDELL;
DEYDASISQVNEKINQALAYIREADEL;
DEYDASISQVNEEINQALAYIRKADEL; DEFDESISQVNEKIEESLAFIRKSDELL;
DEFDESISQVNEKIEESLAFIRKSDEL; or QHWSYGLRPG.

17. The core polypeptide of Claim 16, further comprising an amino terminal acetyl group and a carboxy terminal amido group.

18. A method for enhancing the pharmacokinetic properties of a core polypeptide comprising linking a consensus enhancer peptide sequence to a core polypeptide to form a hybrid polypeptide, such that, when introduced into a living system, the hybrid polypeptide exhibits enhanced pharmacokinetic properties relative those exhibited by the core polypeptide.

19. The method of Claim 18 wherein the core polypeptide is a therapeutic reagent.

20. The method of Claim 18 wherein the core polypeptide is a bioactive peptide, growth factor, cytokine, differentiation factor, interleukin, interferon, colony stimulating factor, hormone or angiogenic factor.