CA2570324A1

CA2570324A1 - Rage fusion proteins and methods of use

Info

Publication number: CA2570324A1
Application number: CA002570324A
Authority: CA
Inventors: Adnan M. M. Mjalli; Ye Edward Tian; Jeffrey C. Webster; Robert Rothlein
Original assignee: Individual
Current assignee: vTv Therapeutics LLC
Priority date: 2004-08-03
Filing date: 2005-08-03
Publication date: 2006-02-16
Anticipated expiration: 2025-08-03
Also published as: US20110124102A1; IL180554A; UA93356C2; ZA200906459B; US7981423B2; KR20070039981A; US20080075733A1; WO2006017647A1; BRPI0514052A; JP2008512988A; EA200700404A1; IL180554A0; AU2005271452B2; NO20065949L; ES2473587T3; US20120171202A1; CA2570324C; ECSP077221A; US20110244516A1; KR101323411B1

Abstract

Disclosed are RAGE fusion proteins comprising RAGE polypeptide sequences linked to a second, non-RAGE polypeptide. The RAGE fusion protein may utilize a RAGE polypeptide domain comprising a RAGE ligand binding site and an interdomain linker directly linked to an immunoglobulin CH2 domain. Such fusion proteins may provide specific, high affinity binding to RAGE ligands.
Also disclosed is the use of the RAGE fusion proteins as therapeutics for RAGE-mediated pathologies.

Description

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RAGE FUSION PROTEINS AND METHODS OF USE
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims priority under 35 USC 119(e) from U.S.
Provisional Patent Application Serial No. 60/598,555 filed August 3, 2004. The disclosure of U.S.
Provisional Patent Application 60/ 598,555 is hereby incorporated by reference in its entirety herein.
FIELD OF THE INVENTION
The present invention relates to regulation of the Receptor for Advanced Glycated Endproducts (RAGE). More particularly, the present invention describes fitsion proteins comprising a RAGE polypeptide, methods of making such fusion proteins, and the use of such proteins for treatment of RAGE-based disorders.
BACKGROUND
Incubation of proteins or lipids with aldose sugars results in nonenzyniatic glycation and oxidation of amino groups on proteins to form Amadori adducts. Over time, the adducts undergo additional rearraalgements, dehydrations, and cross-linking with other proteins to form complexes known as Advanced Glycosylation End Products (AGEs). Factors which promote formation of AGEs include delayed protein turnover (e.g. as in amyloidoses), accumulation of macromolecules having high lysine content, and high blood glucose levels (e.g. as in diabetes) (Hori et al., J. Biol. Chem. 270: 25752-761, (1995)).
AGEs have been iinplicated in a variety of disorders including complications associated with diabetes and normal aging.
AGEs display specific and saturable binding to cell surface receptors on monocytes, macrophages, endothelial cells of the microvasculature, smooth muscle cells, mesengial cells, and neurons. The Receptor for Advanced Glycated Endproducts (RAGE) is a member of the irnmunoglobulin supergene family of molecules. The extracellular (N-terminal) domain of RAGE includes three immunoglobulin-type regions: one V (variable) type domain followed by two C-type (constant) domains (Neeper et al., J. Biol. Chen2., 267:14998-15004 (1992);
Schmidt et al., Circ. (Suppl.) 96#194 (1997)). A single transmembrane spanning domain and a short, highly charged cytosolic tail follow the extracellular domain. The N-terminal, extracellular domain can be isolated by proteolysis of RAGE or by molecular biological approaches to generate soluble RAGE (sRAGE) comprised of the V and C domains.
RAGE is expressed on multiple cell types including leulcocytes, neurons, microglial cells and vascular endothelium (e.g., Hori et al., J. Biol. Cliem., 270:25752-761 (1995)).

Increased levels of RAGE are also fow.id in aging tissues (Schleicher et al., J. Clin. Invest., 99 (3): 457-468 (1997)), and the diabetic retina, vasculature and lcidney (Schmidt et al., Nature Med., 1:1002-1004 (1995)).
In addition to AGEs, other compounds can bind to and modulate RAGE. RAGE
binds to multiple functionally and structurally diverse ligands including amyloid beta (A(3), serum amyloid A (SAA), Advanced Glycation End products (AGEs), S 100 (a proinflammatory member of the Calgranulin family), carboxymethyl lysine (CML), amphoterin and CDl lb/CD1 8 (Bucciarelli et al., Cell Mol. Life Sci., 59:1117-128 (2002);
Chavakis et al., Microbes Infect., 6:1219-1225 (2004); Kokkola et al., Scand.
J. Imrnunol., 61:1-9 (2005); Schmidt et al., J Clin. Invest., 108:949-955 (2001); Rocken et al., Am. J.
Patlaol., 162:1213-1220 (2003)).
Binding of ligands such as AGEs, S 1 00/calgranulin, P-amyloid, CML (Ne-Carboxymethyl lysine), and amphoterin to RAGE has been shown to modify expression of a variety of genes. These interactions may then initiate signal transduction mechanisms including p38 activation, p2lras, MAP kinases, Erk1-2 phosphorylation, and the activation of the transcriptional mediator of inflammatory signaling, NF-xB (Yeh et al., Diabetes, 50:1495-1504 (2001)). For example, in many cell types, interaction between RAGE and its ligands can generate oxidative stress, which thereby results in activation of the free radical sensitive transcription factor NF-xB, and the activation of NF-xB regulated genes, such as the cytokines IL-10 and TNF-a. Furthermore, RAGE expression is upregulated via NF-xB and shows increased expression at sites of inflammation or oxidative stress (Tanaka et al., J. Biol.
Chem., 275:25781-25790 (2000)). Thus, an ascending and often detrimental spiral may be fueled by a positive feedback loop initiated by ligand binding.
Activation of RAGE in different tissues and organs can lead to a number of pathophysiological consequences. RAGE has been ilnplicated in a variety of conditions including: acute and chronic inflammation (Hofinann et al., Cell 97:889-901 (1999)), the development of diabetic late complications such as increased vascular permeability (Wautier et al., J. Clin. Invest., 97:238-243 (1995)), nephropathy (Teillet et al., J.
Ain. Soc. Nephrol., 11:1488-1497 (2000)), arteriosclerosis (Vlassara et. al., The Finnish Medical Society DUODECIM, Ann. Med., 28:419-426 (1996)), and retinopathy (Hammes et al., Diabetologia, 42:603-607 (1999)). RAGE has also been implicated in Alzheimer's disease (Yan et al., Nature, 382:685-691 (1996)), and in tumor invasion and metastasis (Taguchi et al., Nature, 405:354-357 (2000)).

Despite the broad expression of RAGE and its apparent pleiotropic role in multiple diverse disease models, RAGE does not appear to be essential to normal development. For example, RAGE la7ockout mice are without an overt abnormal phenotype, suggesting that while RAGE can play a role in disease pathology when stimulated chronically, inhibition of RAGE does not appear to contribute to any unwanted acute phenotype (Liliensiek et al., J.
Clin. Invest., 113:1641-50 (2004)).

Antagoiuzing binding of physiological ligands to RAGE may down-regulate the pathophysiological changes brought about by excessive concentrations of AGEs and other RAGE ligands. By reducing binding of endogenous ligands to RAGE, symptoms associated with RAGE-mediated disorders may be reduced. Soluble RAGE (sRAGE) is able to effectively antagonize the binding of RAGE ligands to RAGE. However, sRAGE can have a half-life when administered in vivo that may be too short to be therapeutically useful for one or more disorders. Thus, there is a need to develop compounds that antagonize the binding of AGEs and other physiological ligands to the RAGE receptor where the compound has a desireable pharmacokinetic profile.
SUMMARY
Embodiments of the present invention comprise RAGE fusion proteins and methods of using such proteins. The present invention may be einbodied in a variety of ways.
Embodiments of the present invention may comprise a fusion protein comprising a RAGE
polypeptide linked to a second, non-RAGE polypeptide. In one embodiment, the fusion protein comprises a RAGE ligand binding site. The fusion protein may further comprise a RAGE polypeptide directly linked to a polypeptide coinprising CH2 domain of an immunoglobulin, or a portion of the CH2 domain.
The present invention also comprises a method to make a RAGE fusion protein.
In one embodiment the method comprises linking a RAGE polypeptide to a second, non-RAGE
polypeptide. In one embodiment, the RAGE polypeptide comprises a RAGE ligand binding site. The method may comprise linking a RAGE polypeptide directly to a polypeptide comprising the CH2 domain of an immunoglobulin or a portion of the CH2 domain.
In other embodiments, the present invention may comprise methods and compositions for treating a RAGE-mediated disorder in a subject. The method may comprise administering a fusion protein of the present invention to the subject. The composition may comprise a RAGE fusion protein of the present invention in a pharmaceutically acceptable carrier.

There are various advantages that may be associated with particular embodiments of the present invention. In one embodiment, the fusion proteins of the present invention may be metabolically stable when administered to a subject. Also, the fusion proteins of the present invention may exhibit high-affinity binding for RAGE ligands. In certain embodiments, the fusion proteins of the present invention bind to RAGE ligands with affinities in the high nanomolar to low micromolar range. By binding with high affinity to physiological RAGE ligands, the fusion proteins of the present invention may be used to inhibit binding of endogenoti.s ligands to RAGE, thereby providing a means to ameliorate RAGE-mediated diseases.
Also, the fusion proteins of the present invention may be provided in protein or nucleic acid form. In one example embodiment, the fusion protein may be administered systemically and remain in the vasculature to potentially treat vascular diseases mediated in part by RAGE. In another example einbodiment, the fusion protein may be administered locally to treat diseases where RAGE ligands contribute to the patllology of the disease.
Alternatively, a nucleic acid construct encoding the fusion protein may be delivered to a site by the use of an appropriate carrier such as a virus or nalced DNA where transient local expression may locally inhibit the interaction between RAGE ligands and receptors. Thus, administration may be transient (e.g., as where the fusion protein is administered) or more permanent in nature (e.g., as where the fusion protein is administered as a recombinant DNA).
There are additional features of the invention which will be described hereinafter. It is to be understood that the invention is not limited in its application to the details set forth in the following claims, description and figures. The invention is capable of other einbodiments and of being practiced or carried out in various ways.
BRIEF DESCRIPTION OF THE FIGURES
Various features, aspects and advantages of the present invention will become more apparent with reference to the following figures.
FIG. 1 shows various RAGE sequences in accordance with alternate embodiments of the present invention: Panel A, SEQ ID NO: 1, the amino acid sequence for human RAGE;
and SEQ ID NO: 2, the amino acid sequence for human RAGE without the signal sequence of amino acids 1-22; Panel B, SEQ ID NO: 3, the amino acid sequence for human RAGE
without the signal sequence of amino acids 1-23; Panel C, SEQ ID NO: 4, the amino acid sequence of human sRAGE; SEQ ID NO: 5, the amino acid sequence of human sRAGE
without the signal sequence of amino acids 1-22, and SEQ ID NO: 6, the amino acid sequence of human sRAGE without the signal sequence of amino acids 1-23; Panel D, SEQ
ID NO: 7, an amino acid sequence comprising the V-domain of 1luman RAGE; SEQ
ID NO:
8, an alternate amino acid sequence coinprising the V-domain of human RAGE;
SEQ ID NO:
9, an N-terminal fragment of the V-domain of human RAGE; SEQ ID NO: 10, an alternate N-terminal fraginent of the V-domain of human RAGE; SEQ ID NO: 11, the amino acid sequence for ainino acids 124-221 of human RAGE; SEQ ID NO: 12, the amino acid sequence for amino acids 227-317 of human RAGE; SEQ ID NO: 13, the amino acid sequence for amino acids 23-123 of human RAGE; Panel E, SEQ ID NO: 14, the amino acid sequence for amino acids 24-123 of human RAGE; SEQ ID NO: 15, the amino acid sequence for amino acids 23-136 of human RAGE; SEQ ID NO: 16, the amino acid sequence for amino acids 24-136 of human RAGE; SEQ ID NO: 17, the amino acid sequence for ainino acids 23-226 of human RAGE; SEQ ID NO: 18, the amino acid sequence for ainino acids 24-226 of human RAGE; Panel F, SEQ ID NO: 19, the amino acid sequence for amino acids 23-251 of human RAGE; SEQ ID NO: 20, the amino acid sequence for amino acids 24-251 of huma.n RAGE; SEQ ID NO: 21, a RAGE interdomain linker; SEQ ID NO: 22, a second RAGE interdomain linker; SEQ ID NO: 23, a third RAGE interdomain linker; SEQ
ID NO:
24, a fourth RAGE interdomain linker; Panel G, SEQ ID NO: 25, DNA encoding human RAGE amino acids 1-118; SEQ ID NO: 26, DNA encoding human RAGE amino acids 1-123; and SEQ ID NO: 27, DNA encoding human RAGE amino acids 1-136; Panel H, SEQ
ID NO: 28, DNA encoding human RAGE amino acids 1-230; and SEQ ID NO: 29, DNA
encoding human RAGE amino acids 1-25 1; Panel I, SEQ ID NO: 38, a partial amino acid sequence for the CH2 and CH3 domains of human IgG; SEQ ID NO:39, DNA encoding a portion of the human CH2 and CH3 domains of hunian IgG; SEQ ID NO: 40, an amino acid sequence for the CH2 and CH3 domains of human IgG; Panel J, SEQ ID NO: 41, a DNA
encoding the human CH2 and CH3 domains of human IgG; SEQ ID NO: 42, an amino acid sequence for the CH2 domain of human IgG; SEQ ID NO: 43, an amino acid sequence for the CH3 domain of human IgG; and SEQ ID NO: 44, a fifth RAGE interdomain linker.
FIG. 2 shows the DNA sequence (SEQ ID NO: 30) of a RAGE fusion protein (TTP-4000) coding region in accordance with an embodiment of the present invention.
Coding sequence 1-753 highlighted in bold encodes RAGE N-terminal protein sequence whereas sequence 754-1386 encodes human IgG Fc (yl) protein sequence.
FIG. 3 shows the DNA sequence (SEQ ID NO: 31) of an alternate RAGE fusion protein (TTP-3000) coding region in accordance with an embodiment of the present invention. Coding sequence 1-408 highlighted in bold encodes RAGE N-terminal protein sequence, whereas sequence 409-1041 codes human IgG Fc (yl) protein sequence.
FIG. 4 shows the amino acid sequences, SEQ ID NO: 32 (TTP-4000), SEQ ID NO:
33, and SEQ ID NO: 34, that each encode a four domain RAGE fusion protein in accordance with alternate embodiments of the present invention. RAGE sequence is highlighted with bold font.
FIG. 5 shows the amino acid sequences, SEQ ID NO: 35 (TTP-3 000), SEQ ID NO:
36, and SEQ ID NO: 37, that each encode a three domain RAGE fusion protein in accordance with altemate embodiments of the present invention. RAGE sequence is highlighted with bold font.
FIG. 6, Panel A, shows a comparison of the protein domains in human RAGE and human Ig gamina-1 Fc protein, and cleavage points used to make TTP-3000 (at position 136) and TTP-4000 (at position 251) in accordance with altenlate embodiments of the present invention; and Panel B shows the domain structure for TTP-3000 and TTP-4000 in accordance with alternate embodiments of the present invention.
FIG. 7 shows results of an in vitro binding assay for sRAGE, and RAGE fusion proteins TTP-4000 (TT4) and TTP-3000 (TT3), to the RAGE ligands amyloid-beta (A-beta), S 100b (S 100), and amphoterin (Ampho), in accordance with an embodiment of the present invention.
FIG. 8 shows results of an in vitro binding assay for RAGE fusion protein TTP-(TT4) ("Protein") to amyloid-beta as compared to a negative control only including the immunodetection reagents ("Complex Alone"), and antagonism of such binding by a RAGE
antagonist ("RAGE Ligand") in accordance with an embodiment of the present invention.
FIG. 9 shows results of an in vitro binding assay for RAGE fusion protein TTP-(TT3) ("Protein") to amyloid-beta as compared to a negative control only including the iimnunodetection reagents ("Complex Alone"), and antagonism of such binding by a RAGE
antagonist ("RAGE Ligand") in accordance with an embodiment of the present invention.
FIG. 10 shows results of a cell-based assay measuring the inhibition of S 100b-RAGE
induced production of TNF-a by RAGE fusion proteins TTP-3000 (TT3) and TTP-(TT4), and sRAGE in accordance with an embodiment of the present invention.
FIG. 11 shows a pharmacokinetic profile for RAGE fusion protein TTP-4000 in accordance with an embodiment of the present invention wherein each curve represents a different animal under the same experimental conditions.

FIG. 12 shows relative levels of TNF-a release from THP-1 cells due to stimulation by RAGE fusion protein TTP-4000 and human IgG stimulation as a measure of an inflanunatory response in accordance with an embodiment of the present invention FIG. 13 shows the use of RAGE fusion protein TTP-4000 to reduce restenosis in diabetic animals in accordance with alternate embodiments of the present invention, wherein panel A shows that TTP-4000 RAGE-fusion protein reduced the intima/media ratio as compared to a negative control (IgG), and panel B shows that TTP-4000 RAGE-fusion protein reduced vascular smooth muscle cell proliferation in a dose-responsive manner.
FIG. 14 shows use of RAGE fusion protein TTP-4000 to reduce amyloid formation and cognitive dysfunction in animals with Alzheimer's Disease (AD) in accordance with alternate embodiments of the present invention wherein panel A shows TTP-4000 RAGE-fusion protein reduced amyloid load in the brain, and panel B shows TTP-4000 RAGE-fusion protein improved cognitive function.
FIG. 15 shows saturation-binding curves with TTP-4000 to various immobilized klzown RAGE ligands in accordance with an embodiment of the present invention.
DETAILED DESCRIPTION
For the purposes of this specification, unless othem,ise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term "about."
Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification are approximations that can vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maxinzum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g. 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. Additionally, any reference referred to as being "incorporated herein" is to be understood as being incorporated in its entirety.
It is further noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless expressly and unequivocally limited to one referent. The term "or" is used interchangeably with the term "and/or" unless the context clearly indicates otherwise.
Also, the terms "portion" and "fragment" are used interchangeably to refer to parts of a polypeptide, nucleic acid, or other molecular construct.
As used herein, the term "upstream" refers to a residue that is N-terminal to a second residue where the molecule is a protein, or 5' to a second residue where the molecule is a nucleic acid. Also as used herein, the term "downstream" refers to a residue that is C-terminal to a second residue where the molecule is a protein, or 3' to a second residue where the molecule is a nucleic acid.
Unless defined otherwise, all tecluiical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Practitioners are particularly directed to Current Protocols in Molecular Biology (Ansubel) for definitions and terms of the art. Abbreviations for ainino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 coinmon L-ainino acids.
A "nucleic acid" is a polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term is used to include single-stranded nucleic acids, double-stranded nucleic acids, and RNA and DNA made from nucleotide or nucleoside analogues.
The term "vector" refers to a nucleic acid molecule that may be used to transport a second nucleic acid molecule into a cell. In one einbodiment, the vector allows for replication of DNA sequences inserted into the vector. The vector may comprise a promoter to enhance expression of the nucleic acid molecule in at least some host cells. Vectors may replicate autonomously (extrachromasomal) or may be integrated into a host cell chromosome. In one embodiment, the vector may comprise an expression vector capable of producing a protein derived from at least part of a nucleic acid sequence inserted into the vector.
As is known in the art, conditions for hybridizing nucleic acid sequences to each other can be described as ranging from low to high stringency. Generally, highly stringent hybridization conditions refer to washing hybrids in low salt buffer at high temperatures.
Hybridization may be to filter bound DNA using hybridization solutions standard in the art such as 0.5M NaHPOa, 7% sodium dodecyl sulfate (SDS), at 65 C, and washing in 0.25 M

NaHP04, 3.5% SDS followed by washing 0.1 x SSC/0.1% SDS at a teinperature ranging from room temperature to 68 C depending on the length of the probe (see e.g.
Ausubel, F.M.
et al., Short Protocols itz Molecular Biology, 4t" Ed., Chapter 2, John Wiley & Sons, N.Y).
For example, a high stringency wash comprises washing in 6x SSC/0.05% sodium pyrophosphate at 37 C for a 14 base oligonucleotide probe, or at 48 C for a 17 base oligonucleotide probe, or at 55 C for a 20 base oligonucleotide probe, or at 60 C for a 25 base oligonucleotide probe, or at 65 C for a nucleotide probe about 250 nucleotides in length.
Nucleic acid probes may be labeled with radionucleotides by end-labeling with, for example, [7-32P]ATP, or incorporation of radiolabeled nucleotides such as [a-32P]dCTP
by random primer labeling. Alternatively, probes may be labeled by incorporation of biotinylated or fluorescein labeled nucleotides, and the probe detected using Streptavidin or anti-fluorescein antibodies.
As used herein, "small organic molecules" are molecules of molecular weight less than 2,000 Daltons that contain at least one carbon atom.
"Polypeptide" and "protein" are used interchangeably herein to describe protein molecules that may comprise either partial or full-length proteins.
The term "fusion protein" refers to a protein or polypeptide that has an amino acid sequence derived from two or more proteins. The fusion protein may also include linking regions of amino acids between amino acid portions derived from separate proteins.
As used herein, a "non-RAGE polypeptide" is any polypeptide that is not derived from RAGE or a fragment thereof. Such non-RAGE polypeptides include iminunoglobulin peptides, dimerizing polypeptides, stabilizing polypeptides, amphiphilic peptides, or polypeptides comprising amino acid sequences that provide "tags" for targeting or purification of the protein.
As used herein, "immunoglobulin peptides" may comprise an iininunoglobulin heavy chain or a portion thereof. In one embodiment, the portion of the heavy chain may be the Fc fragment or a portion thereof. As used herein, the Fe fragment comprises the heavy chain hinge polypeptide, and the CH2 and CH3 domains of the heavy chain of an immunoglobulin, in either monomeric or dimeric form. Or, the CH1 and Fc fragment may be used as the immunoglobulin polypeptide. The heavy chain (or portion thereo fl may be derived from any one of the known heavy chain isotypes: IgG (y), IgM ( ), IgD (8), IgE (s), or IgA (a). In addition, the heavy chain (or portion thereof) may be derived from any one of the known heavy chain subtypes: IgGl (yl), IgG2 (y2), IgG3 (y3), IgG4 (y4), IgAl (al), IgA2 (a2), or mutations of these isotypes or subtypes that alter the biological activity. An example of biological activity that may be altered includes reduction of an isotype's ability to bind to some Fc receptors as for example, by modification of the hinge region.
The tenns "identity" or "percent identical" refers to sequence identity between two amino acid sequences or between two nucleic acid sequences. Percent identity can be determined by aligning two sequences and refers to the number of identical residues (i.e., amino acid or nucleotide) at positions shared by the compared sequences.
Sequence alignment and comparison may be conducted using the algorithms standard in the art (e.g.
Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J. Mol.
Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci., USA, 85:2444) or by computerized versions of these algorithms (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI) publicly available as BLAST and FASTA. Also, ENTREZ, available through the National Institutes of Health, Bethesda MD, may be used for sequence comparison. In one embodiment, the percent identity of two sequences may be determined using GCG with a gap weight of 1, such that each amino acid gap is weighted as if it were a single ainino acid mismatch between the two sequences.
As used herein, the term "conserved residues" refers to amino acids that are the same among a plurality of proteins having the same structure and/or function. A
region of conserved residues may be important for protein structure or function. T11us, contiguous conserved residues as identified in a three-dimensional protein may be important for protein structure or function. To find conserved residues, or conserved regions of 3-D
structure, a comparison of sequences for the saine or similar proteins from different species, or of individuals of the same species, may be made.
As used herein, the term "homologue" means a polypeptide having a degree of homology with the wild-type amino acid sequence. Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent homology between two or more sequences (e.g. Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl.
Acad. Sci. USA, 80:726-730). For example, homologous sequences may be taken to include an amino acid sequences which in alternate embodiments are at least 75%
identical, 85%
identical, 90% identical, 95% identical, or 98% identical to each other.
As used herein, a polypeptide or protein "domain" comprises a region along a polypeptide or protein that comprises an independent unit. Domains may be defined in terms of structure, sequence and/or biological activity. In one embodiment, a polypeptide domain may comprise a region of a protein that folds in a manner that is substantially independent from the rest of the protein. Domains may be identified using domain databases such as, but not limited to PFAM, PRODOM, PROSITE, BLOCKS, PRINTS, SBASE, ISREC
PROFILES, SAMRT, and PROCLASS.
As used herein, "immunoglobulin domain" is a sequence of amino acids that is structurally homologous, or identical to, a domain of an immunoglobulin. The length of the sequence of amino acids of an immunoglobulin domain may be any lengtll. In one embodiment, an immunoglobulin domain may be less than 250 amino acids. In an example embodiment, an immunoglobulin domain may be about 80-150 amino acids in length. For example, the variable region, and the CH1, CH2, and CH3 regions of an IgG are each immunoglobulin domains. In another example, the variable, the CH1, CH2, CH3 and CH4 regions of an IgM are each iininunoglobulin domains.
As used herein, a "RAGE immunoglobulin domain" is a sequence of amino acids from RAGE protein that is structurally homologous, or identical to, a domain of an immunoglobulin. For example, a RAGE immunoglobulin domain may comprise the RAGE
V-domain, the RAGE Ig-like C2-type 1 domain ("Cl domain"), or the RAGE Ig-like C2-type 2 doinain,("C2 domain").
As used herein, an "interdomain liiiker" comprises a polypeptide that joins two domains together. An Fc hinge region is an example of an interdomain liiiker in an IgG.
As used herein, "directly linlced" identifies a covalent linkage between two different groups (e.g., nucleic acid sequences, polypeptides, polypeptide domains) that does not have any intervening atoms between the two groups that are being linked.
As used herein, "ligand binding domain" refers to a domain of a protein responsible for binding a ligand. The term ligand binding domain includes homologues of a ligand binding domain or portions thereof. In this regard, deliberate amino acid substitutions may be made in the ligand binding site on the basis of similarity in polarity, charge, solubility, hydrophobicity, or hydrophilicity of the residues, as long as the binding specificity of the ligand binding domain is retained.
As used herein, a "ligand binding site" comprises residues in a protein that directly interact with a ligand, or residues involved in positioning the ligand in close proximity to those residues that directly interact with the ligaaid. The interaction of residues in the ligand binding site may be defined by the spatial proximity of the residues to a ligand in the model or structure. The term ligand binding site includes homologues of a ligand binding site, or portions thereof. In this regard, deliberate amino acid substitutions may be made in the ligand binding site on the basis of similarity in polarity, charge, solubility, hydrophobicity, or hydrophilicity of the residues, as long as the binding specificity of the ligand binding site is retained. A ligand binding site may exist in one or more ligand binding domains of a protein or polypeptide.
As used herein, the term "interact" refers to a condition of proximity between a ligand or compound, or portions or fragments thereof, and a portion of a second molecule of interest.
The interaction may be non-covalent, for example, as a result of hydrogen-bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or it may be covalent.
As used herein, a "ligand" refers to a molecule or coinpound or entity that interacts with a ligand binding site, including substrates or analogues or parts thereof. As described herein, the term "ligand" may refer to compounds that bind to the protein of interest. A
ligand may be an agonist, an antagonist, or a modulator. Or, a ligand may not have a biological effect. Or, a ligand may block the binding of other ligands thereby inhibiting a biological effect. Ligands may include, but are not limited to, sinall molecule inhibitors.
These small molecules may include peptides, peptidomimetics, organic compounds and the like. Ligands may also include polypeptides and/or proteins.
As used herein, a "modulator coinpound" refers to a molecule which changes or alters the biological activity of a molecule of interest. A modulator coinpound may increase or decrease activity, or change the physical or chemical characteristics, or fimctional or immunological properties, of the molecule of interest. For RAGE, a modulator compound may increase or decrease activity, or change the characteristics, or functional or immunological properties of the RAGE, or a portion threof A modulator compound may include natural and/or chemically synthesized or artificial peptides, modified peptides (e.g., phosphopeptides), antibodies, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, glycolipids, heterocyclic compounds, nucleosides or nucleotides or parts thereof, and small organic or inorganic molecules. A modulator compound may be an endogenous physiological compound or it may be a natural or synthetic compound. Or, the modulator compound may be a small organic molecule. The term "modulator compound"
also includes a chemically modified ligand or compotmd, and includes isomers and racemic forms.

An "agonist" comprises a compound that binds to a receptor to form a complex that elicits a pharmacological response specific to the receptor involved.

An "antagonist" comprises a compound that binds to an agonist or to a receptor to form a complex that does not give rise to a substantial pharmacological response and can inhibit the biological response induced by an agonist.
RAGE agonists may therefore bind to RAGE and stimulate RAGE-mediated cellular processes, and RAGE antagonists may inhibit RAGE-mediated processes froin being stimulated by a RAGE agonist. For example, in one embodiment, the cellular process stimulated by RAGE agonists comprises activation of TNF-a gene transcription.
The term "peptide mimetics" refers to structures that serve as substitutes for peptides in interactions between molecules (Morgan et al., 1989, Aizn. Reports Med.
Chena., 24:243-252). Peptide mimetics may include synthetic structures that may or may not contain amino acids and/or peptide bonds but that retain the structural and functional features of a peptide, or agonist, or antagonist. Peptide mimetics also include peptoids, oligopeptoids (Simon et al., 1972, Proc. Natl. Acad, Sci., USA, 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide, or agonist or antagonist of the invention.
The term "treating" refers to improving a symptom of a disease or disorder and may comprise curing the disorder, substantially preventing the onset of the disorder, or iinproving the subject's condition. The term "treatment" as used herein, refers to the full spectrum of treatments for a given disorder from which the patient is suffering, including alleviation of one symptom or most of the symptoms resulting from that disorder, a cure for the particular disorder, or prevention of the onset of the disorder.
, As used herein, the term "EC50" is defined as the concentration of an agent that results in 50% of a measured, biological effect. For example, the EC50 of a therapeutic agent having a measurable biological effect may comprise the value at which the agent displays 50% of the biological effect.
As used herein, the term "IC50" is defined as the concentration of an agent that results in 50% inhibition of a measured effect. For example, the IC50 of an antagonist of RAGE
binding may comprise the value at which the antagonist reduces ligand binding to the ligand binding site of RAGE by 50%.
As used herein, an "effective amount" means the amount of an agent that is effective for producing a desired effect in a subject. The term "therapeutically effective amount"
denotes that amount of a drug or pharmaceutical agent that will elicit therapeutic response of an animal or human that is being sought. The actual dose which comprises the effective amount may depend upon the route of administration, the size and health of the subject, the disorder being treated, and the like.
The term "pharmaceutically acceptable carrier" as used herein may refer to compounds and compositions that are suitable for use in human or animal subjects, as for example, for therapeutic compositions administered for the treatment of a RAGE-mediated disorder or disease.
The term "pharmaceutical composition" is used herein to denote a composition that may be administered to a mammalzan host, e.g., orally, parenterally, topically, by inhalation spray, intranasally, or rectally, in unit dosage formulations containing conventional non-toxic carriers, diluents, adjuvants, vehicles and the like.
The term "parenteral" as used herein, includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques.
RAGE Fusion Proteins Embodiments of the present invention comprise RAGE fasion proteins, methods of making such fusion proteins, and methods of use of such fusion proteins. The present invention may be einbodied in a variety of ways.
For example, embodiments of the present invention provide fusion proteins comprising a RAGE polypeptide linked to a second, non-RAGE polypeptide. In one embodiment, the fusion protein may comprise a RAGE ligand binding site. In an ?0 embodiment, the ligand binding site comprises the most N-terminal domain of the fusion protein. The RAGE ligand binding site may comprise the V domain of RAGE, or a portion thereof. Iu an einbodiment, the RAGE ligand binding site coinprises SEQ ID NO:
9 or a sequence 90% identical thereto, or SEQ IID NO: 10 or a sequence 90% identical thereto.
In an embodiment, the RAGE polypeptide may be linked to a polypeptide comprising 5 an immunoglobulin domain or a portion (e.g., a fragment thereof) of an immunoglobulin domain. In one embodiment, the polypeptide comprising an immunoglobulin domain comprises at least a portion of at least one of the CH2 or the CH3 domains of a human IgG.
A RAGE protein or polypeptide may comprise full-length human RAGE protein (e.g., SEQ ID NO: 1), or a fragment of human RAGE. As used herein, a fragment of a RAGE
) polypeptide is at least 5 amino acids in length, may be greater than 30 amino acids in length, but is less than the full amino acid sequence. In alternate embodiments, the RAGE
polypeptide may comprise a sequence that is 70%, or 80%, or 85%, or 90%
identical to human RAGE, or a fragment thereof. For example, in one embodiment, the RAGE
polypeptide may comprise human RAGE, or a fragment thereof, with Glycine as the first residue rather than a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGE may comprise full-length RAGE with the signal sequence removed (e.g., SEQ ID NO: 2 or SEQ
ID NO: 3) (FIGS. 1A and 1B) or a portion of that amino acid sequence.
The fusion proteins of the present invention may also comprise sRAGE (e.g., SEQ ID
NO: 4), a polypeptide 90% identical to sRAGE, or a fragment of sRAGE. As used herein, sRAGE is the RAGE protein that does not include the transmembrane region or the cytoplasmic tail (Park et al., Nature Med., 4:1025-1031 (1998)). For example, the RAGE
polypeptide may comprise human sRAGE, or a fragment thereof, with Glycine as the first residue rather than a Methionine (see e.g., Neeper et al., (1992)). Or, a RAGE
polypeptide may comprise human sRAGE with the signal sequence removed (e.g., SEQ ID NO: 5 or SEQ
ID NO: 6) (FIG. 1 C) or a portion of that amino acid sequence.
In other embodiments, the RAGE protein may comprise a RAGE V domain (e.g., SEQ ID NO: 7 or SEQ ID NO: 8; FIG. ID) (Neeper et al., (1992); Schmidt et al.
(1997)). Or, a sequence 90% identical to the RAGE V domain or a fragment thereof may be used.
Or, the RAGE protein may coinprise a fragment of the RAGE V domain (e.g., SEQ
ID NO: 9 or SEQ ID NO: 10, FIG. 1D). In one embodiment the RAGE protein may comprise a ligand binding site. In an einbodiment, the ligand binding site may comprise SEQ ID NO: 9, or a sequence 90% identical thereto, or SEQ ID NO: 10, or a sequence 90%
identical thereto. In yet anotller embodiment, the RAGE fragment is a synthetic peptide.
Thus, the RAGE polypeptide used in the fusion proteins of the present invention may comprise a fragment of full length RAGE. As is kilown in the art, RAGE
comprises three immunoglobulin-like polypeptide domains, the V domain, and the Cl and C2 domains each linked to each other by an interdomain linker. Full-length RAGE also includes a transmembra.ne polypeptide and a cytoplasmic tail downstream (C-terminal) of the C2 domain, and linked to the C2 domain.
In an embodiment, the RAGE polypeptide does not include any signal sequence residues. The signal sequence of RAGE may comprise either residues 1-22 or residues 1-23 of full length RAGE.
For example, the RAGE polypeptide may comprise amino acids 23-116 of human RAGE (SEQ ID NO: 7) or a sequence 90% identical tliereto, or amino acids 24-116 of human RAGE (SEQ ID NO: 8) or a sequence 90% identical thereto, corresponding to the V
domain of RAGE. Or, the RAGE polypeptide may comprise amino acids 124-221 of human RAGE (SEQ ID NO: 11) or a sequence 90% identical thereto, corresponding to the domain of RAGE. In another embodiment, the RAGE polypeptide may comprise amino acids 227-317 of hutnan RAGE (SEQ ID NO: 12) or a sequence 90% identical thereto, corresponding to the C2 domain of RAGE. Or, the RAGE polypeptide may comprise amino acids 23-123 of human RAGE (SEQ ID NO: 13) or a sequence 90% identical thereto, or ainino acids 24-123 of huinan RAGE (SEQ ID NO: 14) or a sequence 90% identical thereto, corresponding to the V domain of RAGE and a downstream interdomain linker. Or, the RAGE polypeptide may comprise amino acids 23-226 of human RAGE (SEQ ID NO: 17) or a sequence 90% identical thereto, or amino acids 24-226 of human RAGE (SEQ ID
NO: 18) or a sequence 90% identical thereto, corresponding to the V-domain, the C1 domain and the interdomain linker linking these two domains. Or, the RAGE polypeptide may comprise amino acids 23-339 of human RAGE (SEQ ID NO: 5) or a sequence 90% identical thereto, or 24-339 of human RAGE (SEQ ID NO: 6) or a sequence 90% identical thereto, corresponding to sRAGE (i.e.; encoding the V, Cl, and C2 domains and interdomain linkers).
Or, fragments of each of these sequences may be used.
The fusion protein may include several types of peptides that are not derived from RAGE or a fragment thereof. The second polypeptide of the fusion protein may comprise a polypeptide derived from an immunoglobulin. In one embodiment, the immunoglobulin polypeptide may comprise an immunoglobulin heavy chain or a portion (i.e., fragment) thereof. For example, the heavy chain fragment may comprise a polypeptide derived from the Fc fragment of an immunoglobulin, wherein the Fc fiagment comprises the heavy chain hinge polypeptide, and CH2 and CH3 domains of the imrnunoglobulin heavy chain as a monomer. The heavy chain (or portion tliereof) may be derived from any one of the known heavy chain isotypes: IgG (y), IgM ( ), IgD (b), IgE (s); or IgA (a). In addition, the heavy chain (or portion thereof) may be derived from any one of the known heavy chain subtypes:
IgGl (yl), IgG2 (y2), IgG3 (y3), IgG4 (y4), IgAl (al), IgA2 (a2), or mutations of these isotypes or subtypes that alter the biological activity. The second polypeptide may comprise the CH2 and CH3 domains of a human IgGl or portions of either, or both, of these domains.
As an example embodiments, the polypeptide comprising the CH2 and CH3 domains of a human IgGl or a portion thereof may comprise SEQ ID NO: 38 or SEQ ID NO: 40.
The Fe portion of the immunoglobulin chain may be proinflammatory in vivo.
Thus, in one embodiment, the RAGE fusion protein of the present invention comprises an interdomain linker derived from RAGE rather than an interdomain hinge polypeptide derived from an immunoglobulin.
Thus in one embodiment, the fitsion protein may further comprise a RAGE
polypeptide directly linked to a polypeptide comprising a CH2 domain of an immunoglobulin, or a fragment or portion of the CH2 domain of an iv.nmunoglobulin. In one embodiment, the CH2 domain, or a fragment thereof comprises SEQ ID NO: 42. In one embodiment, the RAGE polypeptide may comprise a ligand binding site. The RAGE ligand binding site may comprise the V domain of RAGE, or a portion thereof. In an embodiment, the RAGE ligand binding site comprises SEQ ID NO: 9 or a sequence 90% identical thereto, or SEQ ID NO:
or a sequence 90% identical thereto.
The RAGE polypeptide used in the fnsion proteins of the present invention may comprise a RAGE immunoglobulin domain. Additionally or alternatively, the fraginent of RAGE may comprise an interdomain linker. Or, the RAGE polypeptide may comprise a 10 RAGE immunoglobulin domain linked to an upstream (i.e., closerto the N-terminus) or downstream (i.e., closer to the C-terminus) interdomain linker. In yet.
another embodiment, the RAGE polypeptide may comprise two (or more) RAGE immunoglobulin domains each linked to each other by an interdomain linker. The RAGE polypeptide may further comprise multiple RAGE immunoglobulin domains linked to each other by one or more interdomain linkers and having a tenninal interdomain linker attached to the N-terminal RAGE
immunoglobulin domain and/or the C-terminal immunoglobulin domain. Additional combinations of RAGE immunoglobulin domains and interdomain linkers are within the scope of the present invention.
In one embodiment, the RAGE polypeptide comprises a RAGE interdoinain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE
immunoglobulin domain is linked to the N-tenninal amino acid of the interdomain linker, and the C-terminal amino acid of the RAGE interdomain linlcer is directly linked to the N-terminal amino acid of a polypeptide comprising a CH2 domain of an immunoglobulin, or a fragment thereof. The polypeptide comprising a CH2 domain of an immunoglobulin may comprise the CH2 and CH3 domains of a humaii IgGl or a portion of either, or both, of these domains. As an example einbodiment, the polypeptide comprising the CH2 and Cx3 domains, or a portion tllereof, of a human IgG1 may comprise SEQ ID NO: 38 or SEQ ID
NO: 40.
As described above, the fusion protein of the present invention may comprise a single or multiple domains from RAGE. Also, the RAGE polypeptide comprising an interdomain linker linlced to a RAGE polypeptide domain may comprise a fragment of full-length RAGE
protein. For example, the RAGE polypeptide may coinprise amino acids 23-136 of human RAGE (SEQ ID NO: 15) or a sequence 90% identical thereto or amino acids 24-136 of human RAGE (SEQ ID NO: 16) or a sequence 90% identical thereto corresponding to the V

domain of RAGE and a downstream interdomain linker. Or, the RAGE polypeptide may comprise amino acids 23-251 of human RAGE (SEQ ID NO: 19) or a sequence 90%
identical thereto, or amino acids 24-251 of human RAGE (SEQ ID NO: 20) or a sequence 90% identical thereto, corresponding to the V-domain, the C1 domain, the interdomain linlcer linking these two domains, and a second interdomain liillcer downstream of Cl.
For example, in one embodiment, the fusion protein may comprise two immunoglobulin domains derived from RAGE protein and two immunoglobulin domains derived from a huinan Fe polypeptide. The fusion protein may comprise a first RAGE
immunoglobulin domain and a first RAGE interdomain linker linked to a second RAGE
immunoglobulin doinain and a second RAGE interdoniain linker, such that the N-terminal amino acid of the first interdomain linker is linked to the C-tenninal amino acid of the first RAGE immunoglobulin domain, the N-tenninal amino acid of the second RAGE
immunoglobulin domain is linked to C-terminal amino acid of the first interdomain linker, the N-terminal amino acid of the second interdomain linker is linked to C-terminal ainino acid of the second RAGE immunoglobulin domain, and the C-terminal amino acid of the RAGE second interdomain linker is directly linked to the N-terininal amino acid of the CH2 immunoglobulin domain. In one embodiment, a four domain RAGE fusion protein may comprise SEQ ID NO: 32. In alternate embodiments, a four domain RAGE fusion protein comprises SEQ ID NO: 33 or SEQ ID NO: 34.
Alternatively, a three domain fusion protein may comprise one iinmunoglobulin domain derived from RAGE and two iinmunoglobulin domains derived from a human Fc polypeptide. For exainple, the fusion protein may comprise a single RAGE
immunoglobulin domain linked via a RAGE interdomain linker to the N-terminal amino acid of a immunoglobulin domain or a portion of a CH2 immunoglobulin domain. In one embodiment, a three domain RAGE fusion protein may comprise SEQ ID NO: 35. In alternate embodiments, a three domain RAGE fusion protein may comprise SEQ ID NO: 36 or SEQ
ID NO: 37.
A RAGE interdomain linker fragment may comprise a peptide sequence that is naturally downstream of, and thus, lii-dced to, a RAGE immunoglobulin domain.
For example, for the RAGE V domain, the interdomain linker may comprise amino acid sequences that are naturally downstream from the V domain. In an embodiment, the linlcer may comprise SEQ ID NO: 21, corresponding to amino acids 117-123 of full-length RAGE.
Or, the linker may comprise a peptide having additional portions of the natural RAGE
sequence. For example, a interdomain linker comprising several amino acids (e.g., 1-3, 1-5, or 1-10, or 1-15 amino acids) upstream and downstream of SEQ ID NO: 21 may be used.
Thus, in one embodiment, the interdomain linker comprises SEQ ID NO: 23 comprising amino acids 117-136 of full-length RAGE. Or, fragments of SEQ IID NO: 21 deleting, for example, 1, 2, or 3, amino acids from either end of the linlcer may be used.
In alternate embodiments, the linker may comprise a sequence that is 70% identical, or 80%
identical, or 90% identical to SEQ ID NO: 21 or SEQ ID NO: 23.
For the RAGE Cl domain, the linlcer may comprise peptide sequence that is naturally downstream of the C 1 domain. In an embodiment, the linker may comprise SEQ ID
NO: 22, corresponding to amino acids 222-251 of fitll-length RAGE. Or, the linker may comprise a peptide having additional portions of the natural RAGE sequence. For example, a linker coinprising several (1-3, 1-5, or 1-10, or 1-15 amino acids) amino acids upstream and downstream of SEQ ID NO: 22 may be used. Or, fragments of SEQ ID NO: 22 may be used, deleting for example, 1-3, 1-5, or 1-10, or 1-15 amino acids from either end of the linker. For exainple, in one embodiment, a RAGE interdomain linker may comprise SEQ ID NO:
24, corresponding to amino acids 222-226. Or an interdomain linker may comprise SEQ ID NO:
44, corresponding to RAGE amino acids 318-342.
Methods of Producing RAGE Fusion Proteins The present invention also comprises a method to malce a RAGE fusion protein.
Thus, in one embodiment, the present invention coinprises a metllod of making a RAGE
fusion protein comprising the step of covalently linking a RAGE polypeptide linked to a second, non-RAGE polypeptide wherein the RAGE polypeptide comprises a RAGE
ligand binding site. For example, the linked RAGE polypeptide and the second, non-RAGE
polypeptide may be encoded by a recombinant DNA construct. The method may further comprise the step of incorporating the DNA construct into an expression vector. Also, the method may comprise the step of inserting the expression vector into a host cell.
For example, embodiments of the present invention provide fusion proteins comprising a RAGE polypeptide linked to a second, non-RAGE polypeptide. In one embodiment, the fusion protein may comprise a RAGE ligand binding site. In an embodiment, the ligand binding site comprises the most N-terminal domain of the fusion protein. The RAGE ligand binding site may comprise the V domain of RAGE, or a portion thereof. In an embodiment, the RAGE ligand binding site comprises SEQ ID NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90% identical thereto.
In an embodiment, the RAGE polypeptide may be linked to a polypeptide comprising an immunoglobulin domain or a portion (e.g., a fragment thereof) of an immunoglobulin domain. In one embodiment, the polypeptide coinprising an immunoglobulin domain comprises at least a portion of at least one of the CH2 or the CH3 domains of a human IgG.
The fusion protein may be engineered by recombinant DNA techniques. For example, in one embodiinent, the present invention may comprise an isolated nucleic acid sequence encoding a RAGE polypeptide linked to a second, non-RAGE polypeptide.
In an embodiment, the RAGE polypeptide may comprise a RAGE ligand binding site.
The RAGE protein or polypeptide may comprise full-length human RAGE (e.g., SEQ
ID NO: 1), or a fragment of human RAGE. In an embodiment, the RAGE polypeptide does not include any signal sequence residues. The signal sequence of RAGE may comprise either residues 1-22 or residues 1-23 of full length RAGE (SEQ ID NO: 1). In alternate embodiments, the RAGE polypeptide may comprise a sequence 70%, or 80%, or 90%
identical to human RAGE, or a fragment thereof. For example, in one embodiment, the RAGE polypeptide may comprise human RAGE, or a fragment thereof, with Glycine as the first residue rather than a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGE
may comprise full-length RAGE with the signal sequence removed (e.g., SEQ ID
NO: 2 or SEQ ID NO: 3) (FIGS. lA and 1B) or a portion of that amino acid sequence. The fusion proteins of the present invention may also comprise sRAGE (e.g., SEQ ID NO:
4), a polypeptide 90% identical to sRAGE, or a fragment of sRAGE. For example, the RAGE
polypeptide may comprise human sRAGE, or a fragment thereof, witli Glycine as the first residue rather than a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGE may comprise sRAGE with the signal sequence removed (e.g., SEQ ID NO: 5 or SEQ ID
NO: 6) (FIG. 1 C) or a portion of that amino acid sequence. In other embodiments, the RAGE protein may comprise a V domain (e.g., SEQ ID NO: 7 or SEQ ID NO: 8; FIG. 1D). Or, a sequence 90% identical to the V domain or a fragment tllereof may be used. Or, the RAGE
protein may comprise a fragment of RAGE comprising a portion of the V domain (e.g., SEQ ID NO:
9 or SEQ IDNO: 10, FIG. 1D). In an embodiment, the ligaid binding site may comprise SEQ ID NO: 9, or a sequence 90% identical thereto, or SEQ ID NO: 10, or a sequence 90%
identical tliereto. In yet another embodiment, the RAGE fiaginent is a synthetic peptide.
In an embodiment, the nucleic acid sequence comprises SEQ ID NO: 25 to encode amino acids 1-118 of human RAGE or a fragment thereof. For example, a sequence comprising nucleotides 1- 348 of SEQ ID NO: 25 may be used to encode amino acids 1-116 of human RAGE. Or, the nucleic acid may coinprise SEQ ID NO: 26 to encode amino acids 1-123 of human RAGE. Or, the nucleic acid may comprise SEQ ID NO: 27 to encode amino acids 1-136 of human RAGE. Or, the nucleic acid may comprise SEQ ID NO: 28 to encode amino acids 1-230 of human RAGE. Or, the nucleic acid may comprise SEQ ID NO:
29 to encode amino acids 1-251 of human RAGE. Or fragments of these nucleic acid sequences may be used to encode RAGE polypeptide fragments.
The fusion protein may include several types of peptides that are not derived from RAGE or a fragment thereof. The second polypeptide of the fusion protein may comprise a polypeptide derived from an imrnunoglobulin. The heavy chain (or portion thereof) may be derived from any one of the lcnown heavy chain isotypes: IgG (y), IgM ( ), IgD
(8), IgE (s), or IgA (a). In addition, the heavy chain (or portion thereof) may be derived from any one of the lcnown heavy chain subtypes: IgGl (yl), IgG2 (y2), IgG3 (y3), IgG4 (y4), IgAI (al), IgA2 (a2), or mutations of these isotypes or subtypes that alter the biological activity. The second polypeptide may comprise the CH2 and CH3 domains of a human IgGl or a portion of either, or both, of these domains. As an example embodiments, the polypeptide comprising the CH2 and CH3 domains of a human IgGl or a portion thereof may comprise SEQ
ID NO:
38 or SEQ ID NO: 40. The iminunoglobulin peptide may be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 41.
The Fe portion of the immunoglobulin chain may be proinflammatory in vivo.
Thus, the RAGE fusion protein of the present invention may comprise an interdomain linker derived from RAGE rather than an interdomain hinge polypeptide derived from an immunoglobulin. For example, in one einbodiment, the fusion protein may be encoded by a recombinant DNA construct. Also, the method may comprise the step of incorporating the DNA construct into an expression vector. Also, the method may comprise transfecting the expression vector into a host cell.
Thus, in one embodiment, the present invention comprises a method of making a RAGE fusion protein comprising the step of covalently linking a RAGE
polypeptide to a polypeptide comprising a CH2 domain of an immunoglobulin or a portion of a CH2 domain of an immunoglobulin. In one embodiment, the fusion protein may comprise a RAGE
ligand binding site. The RAGE ligand binding site may comprise the V domain of RAGE, or a portion thereof. In an embodiment, the RAGE ligand binding site comprises SEQ
ID NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%
identical thereto For example, in one embodiment, the present invention comprises a nucleic acid encoding a RAGE polypeptide directly linked to a polypeptide comprising a CH2 domain of an immunoglobulin, or a fragment thereof. In one embodiment, the CH2 domain, or a fragment thereof, comprises SEQ ID NO: 42. The second polypeptide may comprise the CH2 and CH3 domains of a human IgGl. As an example embodiment, the polypeptide comprising the Cx2 and CH3 domains of a human IgGl may comprise SEQ ID NO: 38 or SEQ ID
NO:
40. The iminunoglobulin peptide may be encoded by the nucleic acid sequence of SEQ ID
NO: 39 or SEQ IID NO: 41.
In one embodiment, the RAGE polypeptide may comprise a RAGE interdomain linker linked to a RAGE immunoglobuliil domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-terminal amino acid of the interdomain linlcer, and the C-terminal amino acid of the RAGE interdomain linker is directly linlced to the N-terminal amino acid of a polypeptide comprising a CH2 domain of an iminunoglobulin, or a fragment thereof. The polypeptide comprising a CH2 domain of an immunoglobulin may comprise a polypeptide comprising the CH2 and CH3 domains of a human IgGl or a portion of both, or either, of these domains. As an example embodiment, the polypeptide comprising the CH2 and CH3 domains of a human IgGl, or a portion thereof, may comprise SEQ ID NO:
38 or SEQ ID NO: 40.
The fusion protein of the present invention may comprise a single or multiple domains from RAGE. Also, the RAGE polypeptide comprising an interdomain linker linked to a RAGE iinmunoglobulin domain may comprise a fragment of a full-lengtli RAGE protein.
For example, in one embodiment, the fusion protein may comprise two iminunoglobulin domains derived from RAGE protein and two iminunoglobulin domains derived from a human Fc polypeptide. The fusion protein may comprise a first RAGE
immunoglobulin domain and a first interdomain linker linlced to a second RAGE immunoglobulin domain and a second RAGE interdomain linker, such that the N-terminal amino acid of the first interdomain linker is linlced to the C-terminal amino acid of the first RAGE
immunoglobulin domain, the N-terminal amino acid of the second RAGE immunoglobulin domain is linked to C-terminal amino acid of the first interdomain linker, the N-terminal amino acid of the second interdomain linker is linked to C-terminal amino acid of the RAGE
second immunoglobulin domain, and the C-terminal ainino acid of the RAGE second interdomain liiiker is directly linlced to the N-terminal amino acid of the polypeptide comprising a CH2 immunoglobulin domain or fragment thereof. For example, the RAGE polypeptide may comprise amino acids 23-251 of human RAGE (SEQ ID NO: 19) or a sequence 90%
identical thereto, or amino acids 24-251 of human RAGE (SEQ ID NO: 20) or a sequence 90% identical thereto, corresponding to the V-domain, the Cl domain, the interdomain linlcer linking these two domains, and a second interdomain linker downstream of Cl.
In one embodiment, a nucleic acid construct comprising SEQ ID NO: 30 or a fragment thereof may encode for a four domain RAGE fusion protein.

Alternatively, a three domain fusion protein may comprise one inununoglobulin domain derived from RAGE and two immunoglobulin domains derived from a human Fc polypeptide. For example, the fusion protein may comprise a single RAGE
immunoglobulin domain linked via a RAGE interdomain linlcer to the N-terminal amino acid of the polypeptide comprising a CH2 immunoglobulin domain or a fragment thereof. For example, the RAGE polypeptide may comprise amino acids 23-136 of humalz RAGE (SEQ ID
NO: 15) or a sequence 90% identical thereto or amino acids 24-136 of huinan RAGE (SEQ
ID NO:
16) or a sequence 90% identical thereto corresponding to the V domain of RAGE
and a downstream interdomain linker. In one einbodiment, a nucleic acid construct comprising SEQ ID NO: 31 or a fragment thereof may encode for a three domain RAGE fusion proteiil.
A RAGE interdoinain linker fragment may comprise a peptide sequence that is naturally downstream of, and thus, linked to, a.RAGE immunoglobulin domain.
For example, for the RAGE V domain, the interdomain linlcer may comprise amino acid sequences that are naturally downstream from the V domain. In an embodiment, the linker may comprise SEQ ID NO: 21, corresponding to amino acids 117-123 of full-length RAGE.
Or, the linker may comprise a peptide having additional portions of the natural RAGE
sequence. For example, a interdomain linker comprising several amino acids (e.g., 1-3, 1-5, or 1-10, or 1-15 amino acids) upstream and downstream of SEQ ID NO: 21 may be used.
Thus, in one embodiment, the interdomain linker comprises SEQ ID NO: 23 comprising amino acids 117-136 of full-length RAGE. Or, fragments of SEQ ID NO: 21 deleting, for example, 1, 2, or 3, amino acids from either end of the linker may be used. In alternate embodiments, the linker may comprise a sequence that is 70% identical, or 80%
identical, or 90% identical to SEQ ID NO: 21 or SEQ ID NO: 23.
For the RAGE Cl domain, the linker may comprise peptide sequence that is naturally downstream of the Cl domain. In an embodiment, the linlcer may comprise SEQ ID
NO: 22, corresponding to amino acids 222-251 of full-length RAGE. Or, the linker may comprise a peptide having additional portions of the natural RAGE sequence. For example, a linker comprising several (1-3, 1-5, or 1-10, or 1-15 amino acids) amino acids upstreain and downstream of SEQ ID NO: 22 may be used. Or, fragments of SEQ ID NO: 22 may be used, deleting for example, 1-3, 1-5, or 1-10, or 1-15 amino acids from either end of the linker. For example, in one embodiment, a RAGE interdomain linker may comprise SEQ ID NO:
24, corresponding to amino acids 222-226. Or an interdomain linker may comprise SEQ ID NO:
44, corresponding to RAGE amino acids 318-342.

The metllod may further comprise the step of incorporating the DNA construct into an expression vector. Thus, in a embodiment, the present invention coinprises an expression vector that encodes for a fusion protein comprising a RAGE polypeptide directly linlced to a polypeptide comprising a CH2 domain of an iminunoglobulin or a portion of a CH2 domain of an immunoglobulin. In an embodiment, the RAGE polypeptide comprise constructs, such as those described herein, having a RAGE interdomain linlcer linked to a RAGE
immunoglobulin domain such that the C-terminal amino acid of the RAGE
imtnunoglobulin domain is linked to the N-terminal amino acid of the interdomain linlcer, and the C-terminal a.inino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising a CH2 domain of an immunoglobulin, or a portion thereof. For example, the expression vector used to transfect the cells may comprise the nucleic acid sequence SEQ ID NO:30, or a fragment thereof, or SEQ ID NO: 31, or a fragment thereof.
The method may further comprise the step of transfecting a cell with the expression vector of the present invention. Thus, in an embodiment, the present invention comprises a cell transfected with the expression vector that expressed the RAGE fusion protein of the present invention, such that the cell expresses a fusion protein comprising a RAGE
polypeptide directly linked to a polypeptide comprising a CH2 domain of an im.inunoglobulin or a portion of a CH2 domain of an immunoglobulin. In an embodiment, the RAGE
polypeptide comprise constructs, such as those described herein, having a RAGE
interdomain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-tenninal amino acid of the interdomain linlcer, and the C-terminal amino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising a CH2 domain of an immunoglobulin, or a portion thereof. For example, the expression vector may comprise the nucleic acid sequence SEQ ID NO:30, or a fragment thereof, or SEQ ID NO: 31, or a fragment thereof.
For example, plasmids may be constructed to express RAGE-IgG Fc fusion proteins by fusing different lengths of a 5' cDNA sequence of human RAGE with a 3' cDNA
sequence of human IgGl Fc (yl). The expression cassette sequences may be inserted into an expression vector such as pcDNA3.1 expression vector (Invitrogen, CA) using standard recombinant techniques.
Also, the method may comprise transfecting the expression vector into a host cell. In one embodiment, the recombinant may be transfected into Chinese Hamster Ovary cells and expression optimized. In alternate embodiments, the cells may produce 0.1 to 20 grams/liter, or 0.5 to 10 grams/liter, or about 1-2 grams/liter.

As is lmown in the art, such nucleic acid constructs may be modified by mutation, as for example, by PCR amplification of a nucleic acid template with primers comprising the mutation of interest. In this way, polypeptides comprising varying affinity for RAGE ligands may be designed. In one embodiment, the mutated sequences may be 90% or more identical to the starting DNA. As such, variants may include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27 C below the melting temperature (TM) of the DNA duplex in 1 molar salt).

The coding sequence may be expressed by transfecting the expression vector into an appropriate host. For example, the recombinant vectors may be stably transfected into Chinese Hamster Ovary (CHO) cells, and cells expressing the fusion protein selected and cloned. In an embodiment, cells expressing the recombinant construct are selected for plasmid-encoded neomycin resistance by applying antibiotic G418. Individual clones may be selected and clones expressing high levels of recombinant protein as detected by Western Blot analysis of the cell supernatant may be expanded, and the gene product purified by affinity cllromatography using Protein A columns.
Sample embodiments of recombinant nucleic acids that encode the fusion proteins of the present invention are shown in FIGS. 2-5. For exainple, as described above, the fusion protein produced by the recombinant DNA construct may comprise a RAGE
polypeptide linked to a second, non-RAGE polypeptide. The fusion protein may comprise two domains derived from RAGE protein and two domains derived from an immuno globulin.. An example nucleic acid construct encoding a fusion protein, TTP-4000 (TT4), having this type of structure is shown as FIG. 2 (SEQ ID NO: 30). As shown in FIG. 2, coding sequence 1-753 (highlighted in bold) encodes the RAGE N-temiinal protein sequence whereas the sequence from 754-1386 encodes the IgG Fe protein sequence.
When derived from SEQ ID NO: 30, or a sequence 90% identical thereto, the fusion protein may comprise the four domain amino acid sequence of SEQ ID NO: 32, or the polypeptide with the signal sequence removed (e.g., SEQ ID NO: 33 or SEQ ID
NO: 34) (FIG. 4). In FIG. 4, the RAGE amino acid sequence is highlighted with bold font. The immunoglobulin sequence is the CH2 and CH3 immunoglobulin domains of IgG. As sliown in FIG. 6B, the first 251 amino acids of the full-length TTP-4000 RAGE fusion protein contains as the RAGE polypeptide sequence a signal sequence comprising amino acids 1-22/23, the V immunoglobulin domain (including the ligand binding site) comprising amino acids 23/24-116, an interdoinain linker comprising amino acids 117 to 123, a second immunoglobulin domain (Cl) comprising amino acids 124-221, and a downstream interdomain linker comprising amino acids 222-251.
In an embodiment, the fusion protein may not necessarily comprise the second RAGE
immunoglobulin domain. For example, the f-usion protein may comprise one immunoglobulin domain derived from RAGE and two iinmunoglobulin domains derived from a human Fc polypeptide. An example nucleic acid construct encoding this type of fusion protein is shown as FIG. 3 (SEQ ID NO: 31). As shown in FIG. 3, the coding sequence from nucleotides 1 to 408 (highlighted in bold) encodes the RAGE N-terminal protein sequence, whereas the sequence from 409-1041 codes the IgGl Fc (yl) protein sequence.
When derived from SEQ ID NO: 31, or a sequence 90% identical thereto, the fusion protein may comprise the three domain amino acid sequence of SEQ ID NO: 35, or the polypeptide with the signal sequence removed (e.g., SEQ ID NO: 36 or SEQ ID
NO: 37) (FIG. 5). hi FIG. 5, the RAGE amino acid sequence is higlilighted with bold font. As shown in FIG. 6B, the first 136 ainino acids of the full-length TTP-3000 RAGE fusion protein contains as the RAGE polypeptide a signal sequence comprising amino acids 1-22/23, the V
irmnunoglobulin domain (including the ligand binding site) comprising amino acids 23/24-116, and an interdomain linlcer comprising amino acids 117 to 136. The sequence from 137 to 346 includes the CH2 and CH3 immunoglobulin domains of IgG.
The fusion proteins of the present invention may comprise improved in vivo stability over RAGE polypeptides not comprising a second polypeptide. The fusion protein may be further modified to increase stability, efficacy, potency and bioavailability.
Thus, the fusion proteins of the present invention may be modified by post-translational processing or by chemical modification. For example, the fusion protein may be synthetically prepared to include L-, D-, or umlatural amino acids, alpha-disubstituted amino acids, or N-alkyl ainino acids. Additionally, proteins may be modified by acetylation, acylation, ADP-ribosylation, amidation, attachment of lipids such as phosphatidyinositol, formation of disulfide bonds, and the like. Furthermore, polyethylene glycol can be added to increase the biological stability of the fusion protein.
Bindin2 of RAGE Antagonists to RAGE fusion proteins The fusion proteins of the present invention may comprise a number of applications.
For example, the fusion protein of the present invention may be used in a binding assay to identify RAGE ligands, such as RAGE agonists, antagonists, or modulators.

For example, in one embodinient, the present invention provides a method for detection of RAGE modulators cornprising: (a) providing a fusion protein comprising a RAGE polypeptide linked to a second, non-RAGE polypeptide, where the RAGE
polypeptide comprises a ligand binding site; (b) mixing a compound of interest and a ligand having a known binding affinity for RAGE with the fusion protein; and (c) measuring binding of the known RAGE ligand to the RAGE fusion protein in the presence of the compound of interest.
In an embodiment, the ligand binding site comprises the most N-tenninal domain of the fusion protein.

The RAGE fusion proteins may also provide kits for the detection of RAGE
modulators. For example, in one embodiment, a kit of the present invention may comprise (a) a compound having known binding affinity to RAGE as a positive control;
(b) a RAGE
fusion protein comprising a RAGE polypeptide linked to a second, non-RAGE
polypeptide, wherein the RAGE polypeptide comprises a RAGE ligand binding site; and (c) instructions for use. In an embodiment, the ligand binding site comprises the most N-terminal domain of the fiision protein.
The RAGE protein or polypeptide may comprise full-length huinan RAGE (e.g., SEQ
ID NO: 1), or a fragment of human RAGE. In an embodiment, the RAGE polypeptide does not include any signal sequence residues. The signal sequence of RAGE may comprise either residues 1-22 or residues 1-23 of full length RAGE (SEQ ID NO: 1). In alternate embodiments, the RAGE polypeptide may coinprise a sequence 70%, 80%, or 90%
identical to huinan RAGE, or a fragment thereof. For example, in one embodiment, the RAGE
polypeptide may comprise human RAGE, or a fragment thereof, with Glycine as the first residue rather than a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGE may comprise full-length RAGE with the signal sequence removed (e.g., SEQ ID NO: 2 or SEQ
ID NO: 3) (FIGS. 1A and 1B) or a portion of that amino acid sequence. The fusion proteins of the present invention may also comprise sRAGE (e.g., SEQ ID NO: 4), a polypeptide 90%
identical to sRAGE, or a fragment of sRAGE. For example, the RAGE polypeptide may comprise human sRAGE, or a fragment thereof, with Glycine as the first residue rather than a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGE may comprise sRAGE
with the signal sequence removed (e.g., SEQ ID NO: 5 or SEQ ID NO: 6) (FIG.
1C) or a portion of that amino acid sequence. In other embodiments, the RAGE protein may coinprise a V domain (e.g., SEQ ID NO: 7 or SEQ ID NO: 8; FIG. 1D). Or, a sequence 90%
identical to the V domain or a fragment thereof may be used. Or, the RAGE protein may comprise a fragment of RAGE comprising a portion of the V domain (e.g., SEQ ID NO: 9 or SEQ ID

NO: 10, FIG. 1D). In an embodiment, the ligand binding site may comprise SEQ
ID NO: 9, or a sequence 90% identical thereto, or SEQ ID NO: 10, or a sequence 90%
identical thereto.
In yet another embodiment, the RAGE fragment is a synthetic peptide.
The fusion protein may include several types of peptides that are not derived from RAGE or a fraginent tliereof. The second polypeptide of the fusion protein may comprise a polypeptide derived from an immunoglobulin. The heavy chain (or portion thereof) may be derived from any one of the laiown heavy chain isotypes: IgG (y), IgM ( ), IgD
(8), IgE (E), or IgA (a). In addition, the heavy chain (or portion thereof) may be derived from any one of the known heavy chain subtypes: IgGl (yl), IgG2 (y2), IgG3 (y3), IgG4 (y4), IgAl (al), IgA2 (a2), or mutations of these isotypes or subtypes that alter the biological activity. The second polypeptide may comprise the CH2 and CH3 domains of a human IgGI or a portion of either, or bot11, of these domains. As an example embodiments, the polypeptide coinprising the CH2 and CH3 domains of a human IgGI or a portion thereof may comprise SEQ
ID NO:
38 or SEQ ID NO: 40. The immunoglobulin peptide may be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 41.
The Fc portion of the immunoglobulin chain may be proinflammatory in vivo.
Thus, the RAGE fusion protein of the present invention may comprise an Fc sequence derived from RAGE rather than an immunoglobulin chain. In an embodiment, the fusion protein may comprise a RAGE immunoglobulin domain linked to a polypeptide comprising a CH2 immunoglobulin domain or a fragment thereof. In one embodiment, the RAGE
polypeptide may comprise a RAGE interdomain linker linked to a RAGE
immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-terminal amino acid of the interdomain linker, and the C-terminal amino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising a CH2 domain of an iminunoglobulin, or a fragment thereof. The polypeptide comprising a CH2 domain of an immunoglobulin may comprise a polypeptide comprising the CH2 and CH3 domains of a human IgGl or a portion of both, or either, of these domains. As an example embodiment, the polypeptide comprising the CH2 and CH3 domains of a human IgG1, or a portion thereof, may comprise SEQ ID NO: 38 or SEQ ID NO: 40.
The fusion protein of the present invention may coinprise a single or multiple domains from RAGE. Also, the RAGE polypeptide comprising an interdomain linker linked to a RAGE inununoglobulin domain may comprise a fragment of a full-length RAGE
protein.
For example, in one embodiment, the fusion protein may comprise two immunoglobulin domains derived from RAGE protein and two immunoglobulin domains derived from a human Fc polypeptide. The fusion protein may comprise a first RAGE
immunoglobulin domain and a first interdomain linker linked to a second RAGE immunoglobulin domain and a second RAGE interdomain linker, such that the N-terminal amino acid of the first interdomain linker is linked to the C-terminal amino acid of the first RAGE
iminunoglobulin domain, the N-terminal ainino acid of the second RAGE immunoglobulin domain is linked to C-terminal amino acid of the first interdomain linker, the N-terminal amino acid of the second interdomain linker is linked to C-terminal amino acid of the RAGE
second immunoglobulin domain, and the C-terininal amino acid of the RAGE second interdomain linker is directly linl,-.ed to the N-terminal amino acid of the polypeptide comprising a CH2 immunoglobulin domain or fragment thereof. For example, the RAGE polypeptide may comprise amino acids 23-251 of human RAGE (SEQ ID NO: 19) or a sequence 90%
identical thereto, or amino acids 24-251 of human RAGE (SEQ ID NO: 20) or a sequence 90% identical tliereto, corresponding to the V-domain, the Cl domain, the interdomain linker linking these two domains, and a second interdomain linker downstream of Cl.
In one embodiment, a nucleic acid construct comprising SEQ ID NO: 30 or a fraginent thereof may encode for a four domain RAGE fusion protein.
Alternatively, a three domain fusion protein may comprise one immunoglobulin domain derived from RAGE and two immunoglobulin domains derived from a human Fc polypeptide. For example, the fusion protein may coinprise a single RAGE
immunoglobulin domain linked via a RAGE interdomain linker to the N-terminal amino acid of the polypeptide comprising a CH2 immunoglobulin domain or a fragment thereof. For example, the RAGE polypeptide may comprise amino acids 23-136 of human RAGE (SEQ ID NO:
15) or a sequence 90% identical thereto or amino acids 24-136 of human RAGE (SEQ
ID NO:
16) or a sequence 90% identical thereto corresponding to the V domain of RAGE
and a downstream interdomain linlcer. In one embodiment, a nucleic acid construct comprising SEQ ID NO: 31 or a fragment thereof may encode for a three domain RAGE fusion protein.
As described herein, RAGE interdomain linker fragment may comprise a peptide sequence that is naturally downstream of, and thus, linked to, a RAGE
immunoglobulin domain. For example, for the RAGE V domain, the interdomain linker may comprise amino acid sequences that are naturally downstream from the V domain. In an embodiment, the linker may comprise SEQ ID NO: 21, corresponding to amino acids 117-123 of full-length RAGE. Or, the linker may comprise a peptide having additional portions of the natural RAGE sequence. For example, a interdomain linker comprising several amino acids (e.g., 1-3, 1-5, or 1-10, or 1-15 amino acids) upstream and downstream of SEQ ID NO: 21 may be used. Thus, in one embodiment, the interdomain linker coinprises SEQ ID NO: 23 comprising amino acids 117-136 of full-length RAGE. Or, fragments of SEQ ID
NO: 21 deleting, for example, 1, 2, or 3, amino acids from either end of the linker may be used. In alternate embodiments, the linker may comprise a sequence that is 70%
identical, or 80%
identical, or 90% identical to SEQ ID NO: 21 or SEQ ID NO: 23.
For the RAGE Cl domain, the linker may comprise peptide sequence that is naturally downstream of the Cl domain. In an embodiment, the linker may comprise SEQ ID
NO: 22, corresponding to amino acids 222-251 of full-length RAGE. Or, the linker may comprise a peptide haviiig additional portions of the natural RAGE sequence. For example, a linker comprising several (1-3, 1-5, or 1-10, or 1-15 amino acids) ainino acids upstream and downstream of SEQ ID NO: 22 may be used. Or, fragments of SEQ ID NO: 22 may be used, deleting for example, 1-3, 1-5, or 1-10, or 1-15 ainino acids from either end of the linker. For example, in one embodiment, a RAGE interdomain linker may comprise SEQ ID NO:
24, corresponding to ainino acids 222-226. Or an interdomain linker may comprise SEQ ID NO:
44, corresponding to RAGE amino acids 318-342.
For example, the RAGE fusion protein may be used in a binding assay to identify potential RAGE ligands. In one example embodiment of sucli a binding assay, a known RAGE ligand may coated onto a solid substrate (e.g., Maxisorb plates) at a concentration of about 5 micrograms per well, where each well contains a total voluine of about microliters ( L). The plates may be incubated at 4 C overnight to allow the ligand to absorb.
Alternatively, shorter incubation periods at higher temperature (e.g., room temperature) may be used. After a period of time to allow for the ligand to bind to the substrate, the assay wells may be aspirated and a blocking buffer (e.g., 1% BSA in 50 mM imidizole buffer, pH
7.2) may be added to block nonspecific binding. For exalnple, blocking buffer may be added to the plates for 1 hour at room teinperature. The plates may then be aspirated and/or washed with a wash buffer. In one embodiment, a buffer comprising 20 mM Iinidizole, 150 mM
NaCI, 0.05% Tween-20, 5 mM CaC12 and 5mM MgC12, pH 7.2 may be used as a wash buffer.
The fusion protein may then added at increasing dilutions to the assay wells.
The RAGE
fusion protein may then be allowed to incubate with the immobilized ligand in the assay well such that binding can attain equilibrium. In one embodiment, the RAGE fusion protein is allowed to incubate with the immobilized ligand for about one hour at 37 C. In alternate embodiments, longer incubation periods at lower temperatures may be used.
After the fusion protein and immobilized ligand have been incubated, the plate may be washed to remove any unbound fusion protein. The fusion protein bound to the immobilized ligand may be detected in a variety of ways. In one embodiment, detection employs an ELISA. Thus, in one embodiment, an immunodetection complex containing a monoclonal mouse anti-human IgGl, biotinylated goat anti-mouse IgG, and an avidin linlced alkaline phosphatase may be added to the fusion protein immobilized in the assay well. The immunodetection complex may be allowed to bind to the immobilized fusion protein such that binding between the fusion protein and the immunodetection complex attains equilibrium. For example, the coinplex may be allowed to bind to the fusion protein for one hour at room temperature. At that point, any unbound complex may be removed by washing the assay well with wash buffer. The bound complex may be detected by adding the allcaline phosphatase substrate, paJ a-nitrophenylphosphate (PNPP), and measuring conversion of PNPP to para-nitrophenol (PNP) as an increase in absorbance at 405 nm.
In an embodiment, RAGE ligand bind to the RAGE fusion protein with nanomolar (nM) or micromolar ( M) affinity. An experiment illustrating binding of RAGE
ligands to RAGE fusion proteins of the present invention is shown in FIG. 7. Solutions of (TT3 ) and TTP-4000 (TT4) having initial concentrations of 1.082 mg/mL, and 370 g/mL, respectively, were prepared. As shown FIG. 7, at various dilutions, the fusion proteins TTP-3000 and TTP-4000 are able to bind to immobilized RAGE ligands Amyloid-beta (Abeta) (Amyloid Beta (1-40) from Biosource), S I OOb (S 100), and amphoterin (Ampho), resulting in an increase in absorbance. In the absence of ligand (i.e., coating with only BSA) there was no increase in absorbance.
The binding assay of the present invention may be used to quantify ligand binding to RAGE. In alternate embodiments, RAGE ligands may bind to the fusion protein of the present invention with binding affinities ranging from 0.1 to 1000 nanomolar (nM), or from 1 to 500 nM, or from 10 to 80 nM.
The fusion protein of the present invention may also be used to identify compounds having the ability to bind to RAGE. As shown in FIGS. 8 and 9, respectively, a RAGE
ligand may be assayed for its ability to compete with immobilized amyloid beta for binding to TTP-4000 (TT4) or TTP-3000 (TT3) fusion proteins. Thus, it may be seen that a RAGE
ligand at a final assay concentration (FAC) of 10 M can displace binding of RAGE fusion protein to amyloid-beta at concentrations of 1:3, 1:10, 1:30, and 1:100 of the initial TTP-4000 solution (FIG. 8) or TTP-3000 (FIG. 9).

Modulation of Cellular Effectors Embodiments of the fusion proteins of the present invention may be used to modulate a biological response mediated by RAGE. For example, the fusion proteins may be designed to modulate RAGE-induced increases in gene expression. Thus, in an embodiment, fusion proteins of the present invention may be used to modulate the function of biological enzymes.
For example, the interaction between RAGE and its ligands may generate oxidative stress and activation ofNF-xB, a.nd NF-xB regulated genes, such as the cytokines IL-1p, TNF-a, and the like. In addition, several other regulatory pathways, such as those involving p2lras, MAP
kinases, ERK1, and ERK2, have been shown to be activated by binding of AGEs and other ligands to RAGE.
Use of the fusion proteins of the present invention to modulate expression of the cellular effector TNF-a is shown in FIG. 10. THP-1 myeloid cells may be cultured in RPMI-1640 media supplemented with 10% FBS and induced to secrete TNF-a via stimulation of RAGE with S 100b. When such stimulation occurs in the presence of a RAGE
fusion protein, induction of TNF-a by S 100b binding to RAGE may be inhibited. Thus, as shown in FIG.
10, addition of 10 g TTP-3000 (TT3) or TTP-4000 (TT4) RAGE fusion protein reduces S I00b induction of TNF-a by about 50% to 75%. Fusion protein TTP-4000 may be at least as effective in blocking S I OOb induction of TNF-a as is sRAGE (FIG. 10).
Specificity of the inhibition for the RAGE sequences of TTP-4000 and TTP-3000 is shown by the experiment in which IgG alone was added to S 100b stimulated cells. Addition of IgG and S
l 00b to the assay shows the same levels of TNF-a as S I OOb alone.
Physiolowical Characteristics of RAGE Fusion Proteins While sRAGE can have a therapeutic benefit in the modulation of RAGE-mediated diseases, human sRAGE may have limitations as a stand-alone therapeutic based on the relatively short half-life of sRAGE in plasma. For example, whereas rodent sRAGE has a half-life in normal and diabetic rats of approximately 20 hours, human sRAGE
has a half-life of less than 2 hours when assessed by retention of immunoreactivity sRAGE
(Renard et al., J.
Pharinacol. Exp. Ther., 290:1458-1466 (1999)).
To generate a RAGE therapeutic that has similar binding characteristics as sRAGE, but a more stable pharmacokinetic profile, a RAGE fusion protein comprising a RAGE ligand binding site linked to one or more human immunoglobulin domains may be used.
As is lrnown in the art, the immunoglobulin domains may include the Fc portion of the immunoglobulin heavy chain.

The immunoglobulin Fc portion may confer several attributes to a fusion protein. For example, the Fc fusion protein may increase the serum half-life of such fusion proteins, often from hours to several days. The increase in pharmacokinetic stability is generally a result of the interaction of the linker between CH2 and CH3 regions of the Fc fragment witlz the FcRn receptor (Wines et al., J. bnmunol., 164:5313-5318 (2000)).
Although fusion proteins comprising an iinmunoglobulin Fc polypeptide may provide the advantage of increased stability, immunoglobulin fusion proteins may elicit an inflaminatory response when introduced into a host. The inflammatory response may be due, in large part, to the Fc portion of the immunoglobulin of the fusion protein.
The proinflammatory response may be a desirable feature if the target is expressed on a diseased cell type that needs to be eliminated (e.g., a cancer cell, an or a population of lymphocytes causing an autoimmune disease). The proinflammatory response may be a neutral feature if the target is a soluble protein, as most soluble proteins do not activate immunoglobulins.
However, the proinflammatory response may be a negative feature if the target is expressed on cell types whose destruction would lead to untoward side-effects. Also, the proinflammatory response may be a negative feature if an inflaminatory cascade is established at the site of a fusion protein binding to a tissue target, since many mediators of inflammation may be detrimental to surrounding tissue, and/or may cause systemic effects.
The primary proinflammatory site on immunoglobulin Fc fragments resides on the hinge region between the CH1 and CH2. This hinge region interacts with the FcRl-3 on various leukocytes and trigger these cells to attack the target. (Wines et aL, J. Ifnnaun Z., 164:5313-5318 (2000)).
As therapeutics for RAGE-mediated diseases, RAGE fusion proteins may not require the generation of an inflammatory response. Thus, embodiments of the RAGE
fusion proteins of the present invention may comprise a fusion protein comprising a.R.AGE
polypeptide linked to an immunoglobulin domain(s) where the Fc hinge region from the immunoglobulin is removed and replaced with a RAGE polypeptide. In this way, interaction between the RAGE fusion protein and Fc receptors on inflanunatory cells may be minimized. It may be important, however, to maintain proper stacking and other three-dimensional structural interactions between the various immunoglobulin domains of the fusion protein. Thus, embodiments of the fusion proteins of the present invention may substitute the biologically inert, but structurally similar RAGE interdomain linker that separates the V and C1 domains of RAGE, or the linker that separates the C1 and C2 domains of RAGE, in lieu of the normal hinge region of the immunoglobulin heavy chain.
Thus, the RAGE polypeptide of the fusion protein may comprise an interdoinain linker sequence that is naturally found downstream of a RAGE immunoglobulin domain to form a RAGE
immunglobulin domain/linker fragment. In this way, the tliree dimensional interactions between the immunoglobulin domains contributed by either RAGE or the immunoglobulin may be maintained.

In an embodiment, a RAGE fusion protein of the present invention may comprise a substantial increase in pharmacokinetic stability as compared to sRAGE. For example, FIG.

11 shows that once the RAGE fusion protein TTP-4000 has saturated its ligands, it may retain a half-life of greater than 300 hours. This may be contrasted with the half-life for sRAGE of only a few hours in liuman plasma.
Thus, in an embodiment, the RAGE fusion proteins of the present invention may be used to antagonize binding of physiological ligands to RAGE as a means to treat RAGE-mediated diseases without generating an unacceptable amount of inflammation.
The fusion proteins of the present invention may exhibit a substantial decrease in generating a proinflanunatory response as coinpared to IgG. For example, as shown in FIG.

12, the RAGE fusion protein TTP-4000 does not stimulate TNF-a release from cells under conditions where human IgG stimulation of TNF-ca release is detected.

Treatment of Disease with RAGE Fusion Proteins The present invention may also comprise methods for the treatment of RAGE-mediated disorder in a human subject. In an embodiment, the method may comprise administering to a subject a fusion protein coinprising a RAGE polypeptide comprising a RAGE ligand binding site linlced to a second, non-RAGE polypeptide. In one embodiment, the fusion protein may comprise a RAGE ligand binding site. In an embodiment, the ligand binding site comprises the most N-terminal domain of the fusion protein. The RAGE ligand binding site may comprise the V domain of RAGE, or a portion thereof. In an embodiment, the RAGE ligand binding site comprises SEQ ID NO: 9 or a sequence 90%
identical thereto, or SEQ ID NO: 10 or a sequence 90% identical thereto.
In an embodiment, the RAGE polypeptide may be linked to a polypeptide comprising an immunoglobulin domain or a portion (e.g., a fragment tllereof) of an immunoglobulin domain. In one embodiment, the polypeptide comprising an immunoglobulin domain comprises at least a portion of at least one of the CH2 or the CH3 domains of a human IgG.
The RAGE protein or polypeptide may comprise full-length human RAGE (e.g., SEQ
ID NO: 1), or a fragment of human RAGE. In an embodiment, the RAGE polypeptide does not include any signal sequence residues. The signal sequence of RAGE may comprise either residues 1-22 or residues 1-23 of full length.RAGE (SEQ ID NO: 1). In alternate embodiments, the RAGE polypeptide may comprise a sequence that is 70%, 80% or 90%
identical to human RAGE, or a fragment thereof. For example, in one embodiment, the RAGE polypeptide may comprise human RAGE, or a fragment thereof, with Glycine as the first residue rather than a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGE
may comprise full-length RAGE with the signal sequence removed (e.g., SEQ ID
NO: 2 or SEQ ID NO: 3) (FIGS. lA and 1B) or a portion of that amino acid sequence. The fusion proteins of the present invention may also comprise sRAGE (e.g., SEQ ID NO:
4), a polypeptide 90% identical to sRAGE, or a fraginent of sRAGE. For example, the RAGE
polypeptide may comprise human sRAGE, or a fragment thereof, with Glycine as the first residue rather than a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGE may comprise sRAGE with the signal sequence removed (e.g., SEQ ID NO: 5 or SEQ ID
NO: 6) (FIG. 1 C) or a portion of that amino acid sequence. In other embodiments, the RAGE protein may comprise a V domain (e.g., SEQ ID NO: 7 or SEQ ID NO: 8; FIG. 1D). Or, a sequence 90% identical to the V domain or a fragment thereof may be used. Or, the RAGE
protein may comprise a fragment of RAGE comprising a portion of the V domain (e.g., SEQ ID NO:
9 or SEQ IDNO: 10, FIG. 1D). In an embodiment, the ligand binding site may comprise SEQ ID NO: 9, or a sequence 90% identical thereto, or SEQ ID NO: 10, or a sequence 90%
identical thereto. In yet another einbodiment, the RAGE fragment is a synthetic peptide.
The fusion proteiv.l may include several types of peptides that are not derived from RAGE or a fragment thereof. The second polypeptide of the fusion protein may comprise a polypeptide derived from an immunoglobulin. The heavy chain (or portion thereof) may be derived from any one of the lcnown heavy chain isotypes: IgG (7), IgM (g), IgD
(S), IgE (s), or IgA (a). In addition, the heavy chain (or portion thereof) may be derived from any one of the lrnown heavy chain subtypes: IgGl (yl), IgG2 (y2), IgG3 (y3), IgG4 (y4), IgAl (al), IgA2 (a2), or mutations of these isotypes or subtypes that alter the biological activity. The second polypeptide may comprise the CH2 and CH3 domains of a human IgGl or a portion of either, or both, of these domains. As an example embodiments, the polypeptide comprising the CH2 and CH3 domains of a human IgGl or a portion thereof may comprise SEQ
ID NO:
38 or SEQ ID NO: 40. The immunoglobulin peptide may be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 41.
For example, the RAGE polypeptide may comprise amino acids 23-116 of human RAGE (SEQ ID NO: 7) or a sequence 90% identical thereto, or ainino acids 24-116 of human RAGE (SEQ ID NO: 8) or a sequence 90% identical thereto, corresponding to the V
domain of RAGE. Or, the RAGE polypeptide inay comprise amino acids 124-221 of human RAGE (SEQ ID NO: 11) or a sequence 90% identical thereto, corresponding to the Cl domain of RAGE. In another embodiment, the RAGE polypeptide may comprise amino acids 227-317 of human RAGE (SEQ ID NO: 12) or a sequence 90% identical thereto, corresponding to the C2 domain of RAGE. Or, the RAGE polypeptide may comprise amino acids 23-123 of human RAGE (SEQ ID NO: 13) or a sequence 90% identical thereto, or amino acids 24-123 of human RAGE (SEQ ID NO: 14) or a sequence 90% identical thereto, corresponding to the V domain of RAGE and a dowiistream interdomain linker.
Or, the RAGE polypeptide may comprise amino acids 23-226 of human RAGE (SEQ ID NO: 17) or a sequence 90% identical thereto, or amino acids 24-226 of human RAGE (SEQ ID
NO: 18) or a sequence 90% identical thereto, corresponding to the V-domain, the Cl domain and the interdomain linker linking these two domains. Or, the RAGE polypeptide may comprise amino acids 23-339 of human RAGE (SEQ ID NO: 5) or a sequence 90% identical thereto, or 24-339 of human RAGE (SEQ ID NO: 6) or a sequence 90% identical thereto, corresponding to sRAGE (i.e., encoding the V, C1, and C2 domains and interdomain linkers).
Or, fragments of each of these sequences may be used.
The Fc portion of the immunoglobulin chain may be proinflaminatory in vivo.
Thus, in one embodiment, the RAGE fusion protein of the present invention comprises an interdomain linker derived from RAGE rather than an interdomain hinge polypeptide derived from an ivnmunoglobulin.

Thus in one embodiment, the fusion protein may further comprise a RAGE
polypeptide directly linlced to a polypeptide comprising a CH2 domain of an immun.oglobulin, or a fragment thereof. In one einbodiment, the CH2 domain, or a fragment thereof comprises SEQ ID NO: 42.
In one embodiment, the RAGE polypeptide comprises a RAGE interdomain linker linked to a RAGE inununoglobulin domain such that the C-terminal amino acid of the RAGE
immunoglobulin domain is linked to the N-terminal amino acid of the interdomain linker, and the C-terminal ainino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising a CH2 domain of an immunoglobulin, or a fragment thereof. The polypeptide comprising a CH2 domain of an immunoglobulin may comprise the CH2 and CH3 domains of a human IgGl. As an example embodiment, the polypeptide comprising the CH2 and CH3 domains of a human IgGl may comprise NO: 38 or SEQ ID NO: 40.

The fusion protein of the present invention may comprise a single or multiple domains from RAGE. Also, the RAGE polypeptide comprising an interdomain linker linked to a RAGE immunoglobulin domain may comprise a fragment of a full-length RAGE
protein.
For example, in one embodiment, the fusion protein may comprise two immunoglobulin domains derived from RAGE protein and two immunoglobulin domains derived from a human Fc polypeptide. The fusion protein may comprise a first RAGE
inlxnunoglobulin domain and a first interdomain linker linked to a second RAGE immunoglobulin domain and a second RAGE interdomain linker, such that the N-terminal amino acid of the first interdomain linker is linked to the C-terminal amino acid of the first RAGE
iminunoglobulin domain, the N-terminal amino acid of the second RAGE immunoglobulin domain is linked to C-terminal amino acid of the first interdomain linker, the N-terminal atnino acid of the second interdomain liiiker is linked to C-terminal ainino acid of the RAGE
second immunoglobulin domain, and the C-terminal amino acid of the RAGE second interdomain linker is directly linked to the N-terminal amino acid of the polypeptide comprising a CH2 immunoglobulin domain or fragment thereof. For example, the RAGE polypeptide may comprise amino acids 23-251 of human RAGE (SEQ ID NO: 19) or a sequence 90%
identical thereto, or amino acids 24-251 of human RAGE (SEQ ID NO: 20) or a sequence 90% identical thereto, corresponding to the V-domain, the Cl domain, the interdomain linker linking these two domains, and a second interdomain linker downstream of Cl.
In one embodiment, a nucleic acid construct comprising SEQ ID NO: 30 or a fragment thereof may encode for a four domain RAGE fusion protein.
Alternatively, a three domain fusion protein may comprise one iininunoglobulin domain derived from RAGE and two immunoglobulin domains derived from a human Fc polypeptide. For example, the fusion protein may comprise a single RAGE
iunmunoglobulin domain linlced via a RAGE interdomain linker to the N-terminal amino acid of the polypeptide comprising a CH2 immunoglobulin domain or a fragment thereof. For example, the RAGE polypeptide may comprise amino acids 23-136 of liuman RAGE (SEQ ID
NO: 15) or a sequence 90% identical thereto or amino acids 24-136 of human RAGE (SEQ
ID NO:
16) or a sequence 90% identical thereto corresponding to the V domain of RAGE
and a downstreain interdomain linker. In one embodiinent, a nucleic acid construct comprising SEQ ID NO: 31 or a fragment thereof may encode for a three domain RAGE fusion protein.
A RAGE interdomain linlcer fragment may comprise a peptide sequence that is naturally downstream of, and thus, linked to, a RAGE immunoglobulin domain.
For example, for the RAGE V domain, the interdoinain linlcer may comprise amino acid sequences that are naturally downstream from the V domain. In an embodiment, the linker may comprise SEQ ID NO: 21, corresponding to amino acids 117-123 of full-length RAGE.
Or, the linker may comprise a peptide having additional portions of the natural RAGE
sequence. For example, a interdomain linker comprising several amino acids (e.g., 1-3, 1-5, or 1-10, or 1-15 ainino acids) upstream and downstream of SEQ ID NO: 21 maybe used.
Thus, in one embodiment, the interdomain linker comprises SEQ ID NO: 23 comprising amino acids 117-136 of full-length RAGE. Or, fragments of SEQ ID NO: 21 deleting, for example, 1, 2, or 3, amino acids fiom either end of the linlcer may be used.
In alternate embodiments, the linlcer may comprise a sequence that is 70% identical, or 80%
identical, or 90% identical to SEQ ID NO: 21 or SEQ ID NO: 23.

For the RAGE Cl domain, the linker may comprise peptide sequence that is naturally downstream of the C1 domain. In an embodiment, the linker may comprise SEQ ID
NO: 22, corresponding to amino acids 222-251 of fitll-length RAGE. Or, the linker inay comprise a peptide having additional portions of the natural RAGE sequence. For example, a linlcer comprising several (1-3, 1-5, or 1-10, or 1-15 amino acids) amino acids upstrea.in and downstream of SEQ ID NO: 22 may be used. Or, fragments of SEQ ID NO: 22 may be used, deleting for example, 1-3, 1-5, or 1-10, or 1-15 amino acids from either end of the linker. For example, in one embodiment, a RAGE interdomain linker may comprise SEQ ID NO:
24, corresponding to amino acids 222-226. Or an interdomain linker may comprise SEQ ID NO:
44, corresponding to RAGE amino acids 318-342.

In an embodiment, a fusion protein of the present invention may be adininistered by various routes. Adininistration of the RAGE fusion protein of the present invention may employ intraperitoneal (IP) injection. Alternatively, the RAGE fusion protein may be administered orally, intranasally, or as an aerosol. In another embodiment, administration is intravenous (N). The RAGE fusion protein may also be injected subcutaneously.
In another embodiment, administration of the fusion protein is intra-arterial. In another embodiment, administration is sublingual. Also, administration may employ a time-release capsule. In yet another embodiment, administration may be transrectal, as by a suppository or the like. For example, subcutaneous administration may be useful to treat chronic disorders when the self-administration is desireable.
A variety of animal models have been used to validate the use of compounds that modulate RAGE as therapeutics. Examples of these models are as follows:

a) sRAGE inhibited neointimal formation in a rat model of restenosis following arterial injury in both diabetic and normal rats by inhibiting endothelial, smooth inuscle and macrophage activation via RAGE (Zhou et al., Ci3 culation 107:2238-2243 (2003));
b) Ii-Aubition of RAGE/ligand interactions, using either sRAGE or an anti-RAGE
antibody, reduced amyloid plaque formation in a mouse model of systemic amyloidosis (Yan et al., Nat. Med., 6:643-651 (2000)). Accompanying the reduction in amyloid plaques was a reduction in the iulflammatory cytokines, interleulcin-6 (IL-6) and macrophage colony stimulating factor (M-CSF) as well as reduced activation of NF-kB in the treated animals;
c) RAGE transgenic mice (RAGE overexpressers and RAGE dominant negative expressers) exhibit plaque formation and cognitive deficits in a mouse model of AD
(Arancio et al., EMBO J., 23:4096-4105 (2004));
d) Treatment of diabetic rats with sRAGE reduced vascular permeability (Bonnardel-Phu et al., Diabetes, 48:2052-2058 (1999));
e) Treatment with sRAGE reduced atherosclerotic lesions in diabetic apolipoprotein E-null mice and prevented the functional and morphological indices of diabetic nepllropathy in db/db mice (Hudson et al., Arch. Biochern.. Biophys., 419:80-(2003)); and f) sRAGE attenuated the severity of inflammation in a mouse model of collagen-induced artbritis (Hofinann et al., Genes Immunol., 3:123-135 (2002)), a mouse model of experimental allergic encephalomyelitis (Yan et al., Nat. Med. 9:28-293 (2003)) and a mouse model of inflammatory bowel disease (Hofinann et al., Cell, 97:889-(1999)). Thus, in an embodiment, the fiision proteins of the present invention may be used to treat a symptom ofdiabetes and/or complications resulting from diabetes mediated by RAGE.
In alternate embodiments, the syinptom of diabetes or diabetic late complications may comprise diabetic nephropathy, diabetic retinopathy, a diabetic foot ulcer, a cardiovascular complication of diabetes, or diabetic neuropathy.
Originally identified as a receptor for molecules whose expression is associated with the pathology of diabetes, RAGE itself is essential to the pathophysiology of diabetic complications. In vivo, inhibition of RAGE interaction with its ligand(s) has been shown to be therapeutic in multiple models of diabetic complications and inflammation (Hudson et al., Arch. Biochem. Biophys., 419:80-88 (2003)). For example, a two-month treatment with anti-RAGE antibodies normalized kidney function and reduced abnormal kidney histopathology in diabetic mice (Flyvbjerg et al., Diabetes 53:166-172 (2004)). Furthermore, treatment with a soluble fon.n of RAGE (sRAGE) which binds to RAGE ligands and inhibits RAGE/ligand interactions, reduced atherosclerotic lesions in diabetic apolipoprotein E-null mice and attenuated the fiuictional and morphological pathology of diabetic nephropathy in db/db mice (Bucciarelli et al., Circulation 106:2827-2835 (2002)).
Also, it has been shown that nonenzymatic glycoxidation of macromolecules ultiinately resulting in the formation of advanced glycation endproducts (AGEs) is enhanced at sites of inflammation, in renal failure, in the presence of h.yperglycemia and other conditions associated with systemic or local oxidant stress (Dyer et al., .I.
Clin. Invest., 91:2463-2469 (1993); Reddy et al., Biochem., 34:10872-10878 (1995); Dyer et al., J. Biol.
Chem., 266:11654-11660 (1991); Degenhardt et al., Cell Mol. Biol., 44:1139-1145 (1998)).
Accumulation of AGEs in the vasculature can occur focally, as in the joint amyloid composed of AGE-f32-microglobulin found in patients with dialysis-related amyloidosis (Miyata et al., J. Clin. h2vest., 92:1243-1252 (1993); Miyata et al., J Clin.
Invest., 98:1088-1094 (1996)), or generally, as exemplified by the vasculature and tissues of patients with diabetes (Schmidt et al., Nature Med., 1:1002-1004 (1995)). The progressive accuinulation of AGEs over time in patients with diabetes suggests that endogenous clearance mechanisms are not able to function effectively at sites of AGE deposition. Such accumulated AGEs have the capacity to alter cellular properties by a nuinber of mechanisms. Although RAGE is expressed at low levels in normal tissues and vasculature, in an enviromnent where the receptor's ligan.ds accumulate, it has been shown that RAGE becomes upregulated (Li et al., J. Biol. Chem., 272:16498-16506 (1997); Li et al., J. Biol. Chem., 273:30870-30878 (1998);
Tanaka et al., J Biol. Chem., 275:25781-25790 (2000)). RAGE expression is increased in endothelium, smooth muscle cells and infiltrating mononuclear phagocytes in diabetic vasculature. Also, studies in cell culture have demonstrated that AGE-RAGE
interaction causes changes in cellular properties important in vascular homeostasis.
Use of the RAGE fusion proteins in the treatment of diabetes related pathology is illustrated in FIG. 13. The RAGE fusion protein TTP-4000 was evah.iated in a diabetic rat model of restenosis which involved measuring smooth muscle proliferation and intimal expansion following vascular injury. As illustrated in FIG. 13, TTP-4000 treatment may significantly reduce the intima/media (UM) ratio (FIG. 13A; Table 1) in diabetes-associated restenosis in a dose-responsive manner. Also, TTP-4000 treatment may significantly reduce restenosis-associated vascular smooth muscle cell proliferation in a dose-responsive manner.

Table 1 Effect of TTP-4000 in Rat Model of Restenosis IgG (n=9) TTP-4000 (n=9) TTP-4000 (n=9) Low dose** High dose**
(0.3 mg/animal qod x 4) (1.0 mg/animal qod x 4) Luminal area (mm ) 0.2 0.03 0.18 ~ 0.04 0.16 ~ 0.02 Medialarea(min) 0.12~0.01 0.11~0.02 0.11~0.01 I/Mratio 1.71~0.27 1.61~0.26 1.44* 0.15 *p<0.05 ** For both high and low dose, a loading dose of 3 mg/animal was used.
In other embodiments, the fusion proteins of the present invention may also be used to treat or reverse ainyloidoses and Alzheimer's disease. RAGE is a receptor for amyloid beta (A(3) as well as other ainyloidogenic proteins including SAA and amylin (Yan et al., Nature, 382:685-691 (1996); Yan et al., Proc. Natl. Acad. Sci., USA, 94:5296-5301 (1997); Yan et al., Nat. Med., 6:643-651 (2000); Sousa et al., Lab Invest., 80:1101-1110 (2000)). Also, the RAGE ligands, including AGEs, S 100b and Ap proteins; are found in tissue surrounding the senile plaque in man (Luth et al., Cereb. Cortex 15:211-220 (2005); Petzold et al, Neurosci.
Lett.., 336:167-170 (2003); Sasaki et al., Brain Res., 12:256-262 (2001; Yan et al., Restor.
Neurol Nes uosci., 12:167-173 (1998)). It has been shown that RAGE binds B-sheet fibrillar material regardless of the composition of the subunits (amyloid-B peptide, amylin, serum amyloid A, prion-derived peptide) (Yan et al., Nature, 382:685-691 (1996); Yan et al., Nat.
Med., 6:643-651 (2000)). In addition, deposition of amyloid has been shown to result in enhanced expression of RAGE. For example, in the brains of patients with Alzheimer's disease (AD), RAGE expression increases in neurons and glia (Yan, et al., Nature 382:685-691 (1996)). Concurrent with expression of RAGE ligands, RAGE is upregulated in astrocytes and microglial cells in the hippocampus of individuals with AD but is not upregulated in individuals that do not have AD (Lue et al., Exp. Neurol., 171:29-45 (2001)).
These findings suggest that cells expressing RAGE are activated via RAGE/RAGE
ligand interactions in the vicinity of the senile plaque. Also, in vitro, A(3-mediated activation of microglial cells can be blocked with antibodies directed against the ligand-binding domain of RAGE (Yan et al., Proc. Natl. Acad. Sci., USA, 94:5296-5301 (1997)). It has also been demonstrated that RAGE can serve as a focal point for fibril assembly (Deane et al., Nat.
Med. 9:907-913 (2003)).
Also, in vivo inhibition of RAGE/ligand interactions using either sRAGE or an anti-RAGE antibody can reduce amyloid plaque formation in a mouse model of systemic amyloidosis (Yan et al., Nat. Med., 6:643-651 (2000)). Double transgenic mice that over-express human RAGE and human amyloid precursor protein (APP) with the Swedish and London inutations (mutant hAPP) in neurons develop learning defects and neuropathological abnormalities earlier than their single mutant hAPP transgenic counterparts.
In contrast, double transgenic mice with diminished Ap signaling capacity due to neurons expressing a dominant negative fonn of RAGE on the same mutant hAPP background, show a delayed onset of neuropathological and learning abnormalities compared to their single APP
transgenic counterpart (Arancio et al., EMBO J., 23:4096-4105 (2004)).
In addition, inhibition of RAGE-amyloid interaction has been shown to decrease expression of cellular RAGE and cell stress markers (as well as NF-xB
activation), and diminish amyloid deposition (Yan et al., Nat. Med., 6:643-651 (2000)) suggesting a role for RAGE-amyloid interaction in both perturbation of cellular properties in an environment enriched for ainyloid (even at early stages) as well as in amyloid accumulation.
Thus, the RAGE fusion proteins of the present invention may also be used to treat reduce amyloidosis and to reduce amyloid plaques and cognitive dysfunction associated witll Alzheimer's Disease (AD). As described above, sRAGE has been shown to reduce both amyloid plaque formation in the brain and subsequent increase in inflammatory markers in an animal model of AD. FIGS. 14A and 14B show that mice that have AD, and are treated for 3 months with either TTP-4000 or mouse sRAGE had fewer amyloid beta (A(3) plaques and less cognitive dysfunction than animals that received a vehicle or a human IgG
negative control (IgGl). Like sRAGE, TTP-4000 may also reduce the inflammatory cytokines IL-1 and TNF-oc (data not shown) associated with AD.
Also, fusion proteins of the present invention may be used to treat atherosclerosis and other cardiovascular disorders. Thus, it has been shown that ischemic heart disease is particularly high in patients with. diabetes (Robertson, et al., Lab Invest., 18:538-551 (1968);
Kannel et al, J. Am. Med. Assoc., 241:2035-2038 (1979); Kannel et al., Diab.
Care, 2:120-126 (1979)). In addition, studies have shown that atherosclerosis in patients with diabetes is more accelerated and extensive than in patients not suffering from diabetes (see e.g. Waller et al., Am. J. Med., 69:498-506 (1980); Crall et al, An. J. Med. 64:221-230 (1978); Hamby et al., Chest, 2:251-257 (1976); and Pyorala et al., Diab. Metab. Rev., 3:463-524 (1978)).
Although the reasons for accelerated atherosclerosis in the setting of diabetes are many, it has been shown that reduction of AGEs can reduce plaque formation.
For example, the RAGE fusion proteins of the present invention may also be used to treat stroke. When TTP-4000 was compared to sRAGE in a disease relevant animal model of strolce, TTP-4000 was found to provide a significantly greater reduction in infarct volume.
In this model, the middle carotid artery of a mouse is ligated and then reperfused to form an infart. To assess the efficacy of RAGE fusion proteins to treat or prevent stroke, mice were treated with sRAGE or TTP-4000 or control immunoglobulin just prior to reperfusion. As can be seen in Table 2, TTP-4000 was more efficacious than sRAGE in limiting the area of infarct in these animals suggesting that TTP-4000, because of its better half-life in plasma, was able to maintain greater protection than sR.AGE.
Table 2 Reduction of Infarct in Stroke % Reduction of Infarct**
sRAGE 15%*
TTP-4000 (300 gg) 38%*
TTP-4000 (300 g) 21%*
TTP-4000 (300 g) 10%*
IgG Isotype control 4%
(300 g) *Significant to p<0.001; **Compared to saline In another embodiment, the fusion proteins of the present invention may be used to treat cancer. In one embodiment, the cancer treated using the fusion proteins of the present invention comprises cancer cells that express RAGE. For example, cancers that may be treated with the RAGE fusion protein of the present invention include some lung cancers, some gliomas, some papillomas, and the like. Amphoterin is a high mobility group I
nonhistone chromosomal DNA binding protein (Rauvala et al., J. Biol. Claem., 262:16625-16635 (1987); Parkikinen et al., J. Biol. Clzena. 268:19726-19738 (1993)) which has been shown to interact with RAGE. It has been shown that amphoterin promotes neurite outgrowth, as well as serving as a surface for assembly of protease complexes in the fibrinolytic system (also known to contribute to cell mobility). In addition, a local tumor growth inhibitory effect of bloclcing RAGE has been observed in a primary tumor model (C6 glioma), the Lewis lung metastasis model (Taguchi et al., Nature 405:354-360 (2000)), and spontaneously arising papillomas in mice expressing the v-Ha-ras transgene (Leder et al., PNoc. Natl. Acad. Sci., 87:9178 ;9182 (1990)).
In yet another embodiment, fusion proteins of the present invention may be used to treat inflammation. For exainple, a\in alternate einbodiments, the fusion protein of the present invention is used to treat inflammation associated with autoimmunity, inflammation associated with inflammatory bowel disease, inflammation associated with rizeumatoid arthritis, inflammation associated with psoriasis, inflammation associated with multiple sclerosis, inflammation associated with hypoxia, inflammation associated with stroke, inflammation associated with heart attack, inflammation associated with hemorraghic shock, inflatnmation associated with sepsis, inflammation associated with organ transplantation, or inflammation associated with impaired wound healing.
For example, following thrombolytic treatment, inflaminatory cells such as granulocytes infiltrate the ischemic tissue and produce oxygen radicals that can destroy more cells than were killed by the hypoxia. Inhibiting the receptor on the neutrophil responsible for the neutrophils being able to infiltrate the tissue with antibodies or other protein antagonists has been shown to ameliorate the response. Since RAGE is a ligand for this neutrophil receptor, a fusion protein containing a fragment of RAGE may act as a decoy and prevent the neutrophil from trafficking to the reperfused site and thus prevent further tissue destruction. The role of RAGE in prevention of infla.inmation may be indicated by studies showing that sRAGE inhibited neointimal expansion in a rat model of restenosis following arterial injury in both diabetic and norinal rats, presumably by inhibiting endothelial, smooth muscle cell proliferation and macrophage activation via RAGE (Zhou et al., Circulation, 107:2238-2243 (2003)). In addition, sRAGE inhibited models of inflammation including delayed-type hypersensitivity, experimental autoimmune encephalitis and inflammatory bowel disease (Hofinan et al., Cell, 97:889-901 (1999)).
Also, in an embodiment, the fusion proteins of the present invention may be used to treat auto-immune based disorders. For example, the fusion proteins of the present invention may be used to treat kidney failure. Thus, the fusion proteins of the present invention may be used to treat systemic lupus nephritis or inflammatory lupus nephritis. For example, the S 100/calgranulins have been shown to comprise a family of closely related calcium-binding polypeptides characterized by two EF-hand regions linked by a connecting peptide (Schafer et al., TIBS, 21:134-140 (1996); Zimmer et al., Brain Res. Bull., 37:417-429 (1995);
Rammes et al., J. Biol. Chena., 272:9496-9502 (1997); Lugering et al., Euf-.
J. Clira. Invest., 25:659-664 (1995)). Although they laclc signal peptides, it has long been known that S 1 00/calgranulins gain access to the extracellular space, especially at sites of chronic iunmune/ivlflammatory responses, as in cystic fibrosis and rheumatoid arthritis. RAGE is a receptor for mauy members of the S 100/calgranulin family, mediating their proinflammatory effects on cells such as lymphocytes and mononuclear phagocytes. Also, studies on delayed-type hypersensitivity response, colitis in IL- 10 null mice, collagen-induced arthritis, and experimental autoimmune encephalitis models suggest that RAGE-ligand interaction (presumably wit11 S-100/calgranulins) has a proximal role in the inflammatory cascade.
Thus, in various selected embodiments, the present invention may provide a method for inhibiting the interaction of an AGE with RAGE in a subject by administering to the subject a therapeutically effective ainount of a fusion protein of the present invention. The subject treated using the RAGE fusion proteins of the present invention may be an animal. In an embodiment, the subject is a human. The subject may be suffering from an AGE-related disease such as diabetes, diabetic complications such as nephropathy, neuropathy, retinopathy, foot ulcer, amyloidoses, or renal failure, and inflammation. Or, the subject may be an individual with Alzheimer's disease. In an alternative embodiment, the subject may be an individual with cancer. In yet other embodiments, the subject may be suffering from systemic lupus erythmetosis or inflammatory lupus nephritis. Other diseases may be mediated by RAGE and thus, may be treated using the fusion prbteins of the present invention. Thus, in additional alternative embodiments of the present invention, the fusion proteins may be used for treatment of Crohn's disease, arthritis, vasculitis, nephropathies, retinopathies, and neuropathies in human or animal subjects.
A therapeutically effective amount may comprise an amount which is capable of preventing the interaction of RAGE with an AGE or other types of endogenous RAGE
ligands in a subject. Accordingly, the amount will vary with the subject being treated.
Administration of the compound may be hourly, daily, weekly, monthly, yearly, or as a single event. hl various alternative embodiments, the effective amount of the fusion protein may range from about 1 ng/kg body weight to about 100 mg/kg body weight, or from about 10 g/kg body weight to about 50 mg/kg body weight, or from about 100 gg/kg body weight to about 10 mg/kg body weight. The actual effective amount may be established by dose/response assays using methods standard in the art (Johnson et al., Diabetes. 42: 1179, (1993)). Thus, as is lcnown to those in the art, the effective amount may depend on bioavailability, bioactivity, and biodegradability of the compound.

Compositions The present invention may comprise a coinposition comprising a fusion protein of the present invention mixed with a pharmaceutically acceptable carrier. The fusion protein may comprise a RAGE polypeptide linked to a second, non-RAGE polypeptide. In one embodiment, the fusion protein may comprise a RAGE ligand binding site. In an embodiment, the ligand binding site comprises the most N-terminal domain of the fusion protein. The RAGE ligand binding site may comprise the V domain of RAGE, or a portion thereof. In an embodiment, the RAGE ligand binding site comprises SEQ ID NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90% identical thereto.
In an embodiment, the RAGE polypeptide may be lii-Aced to a polypeptide comprising an immunoglobulin domain or a portion (e.g., a fraginent thereof) of an inn.nunoglobulin domain. In one embodiment, the the polypeptide comprising an immunoglobulin domain comprises at least a portion of at least one of the CH2 or the CH3 domains of a huinan IgG.
The RAGE protein or polypeptide may comprise full-lengtli human RAGE (e.g., SEQ
ID NO: 1), or a fragment of human RAGE. In an embodiment, the RAGE polypeptide does not include any signal sequence residues. The signal sequence of RAGE may comprise either residues 1-22 or residues 1-23 of full length RAGE (SEQ ID NO: 1). In alternate embodiments, the RAGE polypeptide may comprise a sequence that is 70%, 80% or 90%
identical to human RAGE, or a fragment thereof. For example, in one embodiment, the RAGE polypeptide may comprise human RAGE, or a fragment thereof, with Glycine as the first residue rather than a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGE
may comprise full-length RAGE with the signal sequence removed (e.g., SEQ ID
NO: 2 or SEQ ID NO: 3) (FIGS. 1A and 1B) or a portion of that amino acid sequence. The fusion proteins of the present invention may also comprise sRAGE (e.g., SEQ ID NO:
4), a polypeptide 90% identical to sRAGE, or a fragment of sRAGE. For example, the RAGE
polypeptide may comprise human sRAGE, or a fragment thereof, with Glycine as the first residue rather than a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGE may comprise sRAGE with the signal sequence removed (e.g., SEQ ID NO: 5 or SEQ ID
NO: 6) (FIG. 1C) or a portion of that amino acid sequence. In otlier embodiments, the RAGE protein may comprise a V domain (e.g., SEQ ID NO: 7 or SEQ ID NO: 8; FIG. ID). Or, a sequence 90% identical to the V domain or a fragment thereof may be used. Or, the RAGE
protein may comprise a fragment of RAGE comprising a portion of the V domain (e.g., SEQ ID NO:
9 or SEQ ID NO: 10, FIG. 1D). In an einbodiment, the ligand binding site may comprise SEQ ID NO: 9, or a sequence 90% identical thereto, or SEQ ID NO: 10, or a sequence 90%
identical thereto. In yet another einbodiment, the RAGE fragment is a synthetic peptide.
For example, the RAGE polypeptide may comprise amino acids 23-116 of human RAGE (SEQ ID NO: 7) or a sequence 90% identical thereto, or amino acids 24-116 of human RAGE (SEQ ID NO: 8) or a sequence 90% identical thereto, corresponding to the V
domain of RAGE. Or, the RAGE polypeptide may comprise amino acids 124-221 of human RAGE (SEQ ID NO: 11) or a sequence 90% identical thereto, corresponding to the domain of RAGE. In another embodiment, the RAGE polypeptide may comprise amino acids 227-317 of human RAGE.(SEQ ID NO: 12) or a sequence 90% identical thereto, corresponding to the C2 doinain of RAGE. Or, the RAGE polypeptide may comprise amino acids 23-123 of human RAGE (SEQ ID NO: 13) or a sequence 90% identical thereto, or amino acids 24-123 of human RAGE (SEQ ID NO: 14) or a sequence 90% identical thereto, corresponding to the V domain of RAGE and a downstream interdoinain linker.
Or, the RAGE polypeptide may comprise amino acids 23-226 of human RAGE (SEQ ID NO: 17) or a sequence 90% identical thereto, or amino acids 24-226 of human RAGE (SEQ ID
NO: 18) or a sequence 90% identical thereto, corresponding to the V-domain, the C 1 domain and the interdomain linker linking these two domains. Or, the RAGE polypeptide may comprise amino acids 23-339 of human RAGE (SEQ ID NO: 5) or a sequence 90% identical thereto, or 24-339 of human RAGE (SEQ ID NO: 6) or a sequence 90% identical thereto, corresponding to sRAGE (i.e., encoding the V, C1, and C2 domains and interdomain linlcers).
Or, fragments of each of these sequences may be used.
The fusion protein may include several types of peptides that are not derived from RAGE or a fragment thereof. The second polypeptide of the fusion protein may comprise a polypeptide derived from an immunoglobulin. The heavy chain (or portion thereof) may be derived from any one of the Icnown heavy chain isotypes: IgG (y), IgM ( ), IgD
(8), IgE (s), or IgA (a). In addition, the heavy chain (or portion thereof) may be derived from any one of the 1{nown heavy chain subtypes: IgGI (yl), IgG2 (y2), IgG3 (y3), IgG4 ('y4), IgAl (al), IgA2 (a2), or mutations of these isotypes or subtypes that alter the biological activity. The second polypeptide may comprise the CH2 and CH3 domains of a human IgGl or a portion of either, or both, of these domains. As an example embodiments, the polypeptide comprising the CH2 and CH3 domains of a human IgGl or a portion thereof may comprise SEQ
ID NO:
38 or SEQ ID NO: 40. The immunoglobulin peptide may be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 41.

The Fc portion of the inimunoglobulin chain may be proinflaminatory in vivo.
Thus, in one embodiment, the RAGE fusion protein of the present invention comprises an interdomain linker derived from RAGE rather than an interdomain hinge polypeptide derived from an immunoglobulin.
Thus in one embodiment, the fusion protein may further comprise a RAGE
polypeptide directly linked to a polypeptide comprising a CH2 domain of an immunoglobulin, or a fragment thereof. In one einbodiment, the CH2 domain, or a fragment thereof com.prises SEQID NO:42.
In one embodiment, the RAGE polypeptide comprises a RAGE interdoinain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE
immunoglobulin doinain is linked to the N-terminal ainino acid of the interdomain linker, and the C-terminal amino acid of the RAGE interdomain linker is directly linlced to the N-terminal amino acid of a polypeptide comprising a CH2 domain of an immunoglobulin, or a fragment thereof. The polypeptide comprising a CH2 domain of an imi.nunoglobulin, or a portion thereof, may comprise the CH2 and CH3 domains of a human IgGI. As an exainple einbodiment, the polypeptide comprising the CH2 and CH3 domains of a human IgGl may comprise SEQ ID NO: 38 or SEQ ID NO: 40.
The fusion protein of the present invention may comprise a single or multiple domains from RAGE. Also, the RAGE polypeptide comprising an interdomain linker linked to a RAGE immunoglobulin domain may comprise a fragment of a full-length RAGE
protein.
For example, in one embodiment, the fusion protein may comprise two immunoglobulin domains derived from RAGE protein and two immunoglobulin domains derived from a human Fc polypeptide. The fusion protein may comprise a first RAGE
immunoglobulin domain and a first interdomain linker linked to a second RAGE immunoglobulin domain and a second RAGE interdomain linker, such that the N-tenninal amino acid of the first interdomain linlcer is linked to the C-terminal amino acid of the first RAGE
immunoglobulin domain, the N-terminal amino acid of the second RAGE immunoglobulin domain is linlwd to C-terminal amino acid of the first interdomain lifflwr, the N-tenninal a.inino acid of the second interdomain linlcer is linked to C-terminal amino acid of the RAGE
second immunoglobulin domain, and the C-terminal amino acid of the RAGE second interdomain linker is directly linked to the N-terminal amino acid of the polypeptide comprising a Cx2 immunoglobulin domain or fragment thereof. For example, the RAGE polypeptide may comprise amino acids 23-251 of human RAGE (SEQ ID NO: 19) or a sequence 90%
identical thereto, or amino acids 24-251 of human RAGE (SEQ ID NO: 20) or a sequence 90% identical thereto, corresponding to the V-domain, the Cl domain, the interdomain linker linking these two domains, and a second interdomain livilcer downstream of C1.
In one embodiment, a nucleic acid construct comprising SEQ ID NO: 30 or a fragment thereof may encode for a four domain RAGE fusion protein.

Alternatively, a three domain fusion protein may comprise one immunoglobulin domain derived from RAGE and two immunoglobulin domains derived from a human Fc polypeptide. For example, the fusion protein may comprise a single RAGE
immunoglobulin domain linked via a.RAGE interdomain linker to the N-tenninal amino acid of the polypeptide comprising a CH2 immunoglobulin domain or a fragment thereof. For example, the RAGE polypeptide may comprise amino acids 23-136 of human RAGE (SEQ ID NO:
15) or a sequence 90% identical thereto or amino acids 24-136 of human RAGE (SEQ
ID NO:
16) or a sequence 90% identical thereto corresponding to the V domain of RAGE
and a downstream interdomain linker. In one einbodiment, a nucleic acid construct comprising SEQ ID NO: 31 or a fragment thereof may encode for a three domain RAGE fusion protein.
A RAGE interdomain linker fragment may comprise a peptide sequence that is naturally downstream of, and thus, linlcedto, a RAGE immunoglobulin domain.
For example, for the RAGE V domain, the interdomain linker may comprise amino acid sequences that are naturally downstream from the V domain. In an embodiment, the linker may comprise SEQ ID NO: 21, corresponding to ainino acids 117-123 of full-lengtli RAGE. -Or, the linlcer may coinprise a peptide having additional portions of the natural RAGE
sequence. For example, a interdomain linker comprising several amino acids (e.g., 1-3, 1-5, or 1-10, or 1-15 amino acids) upstream and downstream of SEQ ID NO: 21 may be used.
Thus, in one embodiment, the interdomain linker comprises SEQ ID NO: 23 comprising amino acids 117-136 of full-length RAGE. Or, fraginents of SEQ ID NO: 21 deleting, for example, 1, 2, or 3, amino acids from either end of the linker may be used. In alternate embodiments, the linlcer may comprise a sequence that is 70% identical, or 80%
identical, or 90% identical to SEQ ID NO: 21 or SEQ ID NO: 23.
For the RAGE C 1 domain, the linker may comprise peptide sequence that is naturally downstream of the Cl domain. In an einbodiment, the linlcer may comprise SEQ
ID NO: 22, corresponding to amino acids 222-251 of full-length RAGE. Or, the linker may comprise a peptide having additional portions of the natural RAGE sequence. For example, a linker comprising several (1-3, 1-5, or 1-10, or 1-15 amino acids) amino acids upstream and downstream of SEQ ID NO: 22 may be used. Or, fragments of SEQ ID NO: 22 may be used, deleting for example, 1-3, 1-5, or 1-10, or 1-15 amino acids from either end of the linker. For example, in one embodiment, a RAGE interdomain linker may comprise SEQ II) NO:
24, corresponding to amino acids 222-226. Or an interdomain linker may comprise SEQ ID NO:
44, corresponding to RAGE amino acids 318-342.
Pharmaceutically acceptable carriers may comprise any of the standard pharmaceutically accepted carriers known in the art. The cairier may comprise a diluent. hi one embodiment, the pharmaceutical carrier may be a liquid and the fusion protein or nucleic acid construct may be in the form of a solution. In another einbodiment, the pharmaceutically acceptable carrier may be a solid in the form of a powder, a lyophilized powder, or a tablet. Or, the pharmaceutical carrier may be a gel, suppository, or cream. In altexnate embodiments, the carrier may comprise a liposome, a microcapsule, a polymer encapsulated cell, or a virus. Thus, the term pharmaceutically acceptable carrier encompasses, but is not limited to, any of the standard pharmaceutically accepted carriers, such as water, alcohols, phosphate buffered saline solution, sugars (e.g., sucrose or mannitol), oils or emulsions such as oil/water emulsions or a trigyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules.
Administration of the RAGE fusion proteins of the present invention may employ various routes. Thus, administration of the RAGE fusion protein of the present invention may employ intraperitoneal (IP) injection. Alternatively, the RAGE fusion protein may be administered orally, intranasally, or as an aerosol. In another embodiment, administration is intravenous (IV). The RAGE fusion protein may also be injected subcutaneously.
In another embodiment, administration of the fusion protein is intra-arterial. In another embodiment, adininistration is sublingual. Also, administration may employ a time-release capsule. In yet another embodiment, administration may be transrectal, as by a suppository or the like. For example, subcutaneous administration may be useful to treat chronic disorders when the self-administration is desireable.
The pharmaceutical coinpositions may be in the form of a sterile injectable solution in a non-toxic parenterally acceptable solvent or vehicle. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, 3-butanediol, isotonic sodium chloride solution, or aqueous buffers, as for example, physiologically acceptable citrate, acetate, glycine, histidine, phosphate, tris or succinate buffers. The injectable solution may contain stabilizers to protect against chemical degradation and aggregate formation.
Stabilizers may include antioxidants such as butylated hydroxy anisole (BHA), and butylated hydroxy toluene (BHT), buffers (citrates, glycine, histidine) or surfactants (polysorbate 80, poloxamers). The solution may also contain antimicrobial preservatives, such as benzyl alcohol and parabens. The solution may also contain surfactants to reduce aggregation, such as Polysorbate 80, poloxomer, or otlier surfactants known in the art. The solution may also contain other additives, such as a sugar(s) or saline, to adjust the osmotic pressure of the composition to be similar to human blood.
The pharmaceutical compositions may be in the form of a sterile lyophilized powder for injection upon reconstitution with a diluent. The diluent can be water for injection, bacteriostatic water for injection, or sterile saline. The lyophilized powder may be produced by freeze drying a solution of the fusion protein to produce the protein in dry form. As is lcnown in the art, the lyophilized protein generally has increased stability and a longer shelf life than a liquid solution of the protein. The lyophilized powder (cake) many contain a buffer to adjust the pH, as for example physiologically acceptable citrate, acetate, glycine, histidine, phosphate, tris or succinate buffer. The lyophilized powder may also contain lyoprotectants to maintain its physical and chemical stability. The commonly used lyoprotectants are non-reducing sugars and disaccharides such as sucrose, mannitol, or trehalose. The lyophilized powder may contain stabilizers to protect against chemical degradation and aggregate formation. Stabilizers may include, but are not limited to antioxidants (BHA, BHT), buffers (citrates, glycine, histidine), or surfactants (polysorbate 80, poloxamers).
The lyophilized powder may also contain antimicrobial preservatives, such as benzyl alcohol and parabens.
The lyophilized powder may also contain surfactants to reduce aggregation, such as, but not limited to, Polysorbate 80 and poloxomer. The lyophilized powder may also contain additives (e.g., sugars or saline) to adjust the osmotic pressure to be similar to human blood upon reconstitution of the powder. The lyophilized powder may also contain bullcing agents, such as sugars and disaccharides.
The phannaceutical compositions for injection may also be in the form of a oleaginous suspension. This suspension may be formulated according to the lrnown methods using suitable dispersing or wetting agents and suspending agents described above. In addition, sterile, fixed oils are conveniently employed as solvent or suspending medium. For this purpose, any bland fixed oil may be employed using synthetic mono- or diglycerides.
Also, oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as a liquid paraffin. For example, fatty acids such as oleic acid find use in the preparation of injectables. The oily suspensions may contain a thiclcening agent, for exainple beeswax, hard paraffin or cetyl alchol. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the present invention may also be in the form of oil-in-water einulsions or aqueous suspensions. The oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example a liquid paraffin, or a mixture thereof. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan.
Aqueous suspensions may also contain the active coinpounds in admixture with excipients. Such excipients may include suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, such as a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water may provide the active compound in admixture with a dispersing agent, suspending agent, and one or more preservatives. Suitable preservatives, dispersing agents, and suspending agents are described above.
The compositions may also be in the foirn of suppositories for rectal administration of the compounds of the invention. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will thus melt in the rectum to release the drug.
Such materials include cocoa butter atid polyethylene glycols, for example.
For topical use, creams, ointments, jellies, solutions or suspensions containing the compounds of the invention may be used. Topical applications may also include mouth washes and gargles. Suitable preservatives, antioxidants such as BHA and BHT, dispersants, surfactants, or buffers may be used.
The compounds of the present invention may also be administered in the form of liposome delivery systems, such as small tuzilainellar vesicles, large unilamellar vesicles, and inultilamellar vesicles. Liposomes may be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
In certain embodiments, the compounds of the present invention may be modified to further retard clearance from the circulation by metabolic enzymes. In one embodiment, the compounds may be modified by the covalent attachment of water-soluble polymers such as polyethylene glycol (PEG), copolymers of PEG and polypropylene glycol, polyvinylpyrrolidone or polyproline, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. Such modifications also may increase the compound's solubility in aqueous solution. Polymers such as PEG may be covalently attached to one or more reactive amino residues, sulfydryl residues or carboxyl residues. Numerous activated forms of PEG have been described, including active esters of carboxylic acid or carbonate derivatives, particularly those in which the leaving groups are N-hydroxsuccinimide, p-nitrophenol, imdazole or 1-hydroxy-2-nitrobenzene-3 sulfone for reaction with amino groups, multimode or halo acetyl derivatives for reaction with sulfliydryl groups, and ainino hydrazine or hydrazide derivatives for reaction with carbohydrate groups.
Additional methods for preparation of protein formulations which may be used with the fusion proteins of the present invention are described in U.S. Patents No.
6,267,958, and 5,567,677.
In a further aspect of the present invention, the RAGE modulators of the invention are utilized in adjuvant therapeutic or combination therapeutic treatments with other lcnown therapeutic agents. The following is a non-exhaustive listing of adjuvants and additional therapeutic agents which may be utilized in combination with the RAGE fusion protein modulators of the present invention:
Pharmacologic classifications of anticancer a g i1ts:
1. Alkylating agents: Cyclophosphamide, nitrosoureas, carboplatin, cisplatin, procarbazine 2. Antibiotics: Bleomycin, Daunorubicin, Doxorubicin 3. Antimetabolites: Methotrexate, Cytarabine, Fluorouracil 4. Plant alkaloids: Vinblastine, Vincristine, Etoposide, Paclitaxel, 5. Hormones: Tamoxifen, Octreotide acetate, Finasteride, Flutamide 6. Biologic response modifiers: Interferons, Interleukins, Pharmacologic classifications of treatment for Rheumatoid Arthritis 1. Analgesics: Aspirin 2. NSAIDs (Nonsteroidal anti-inflammatory drugs): Ibuprofen, Naproxen, Diclofenac 3. DIVIARDs (Disease-Modifying Antirheumatic drugs): Methotrexate, gold preparations, hydroxychloroquine, sulfasalazine 4. Biologic Response Modifiers, DMARDs: Etanercept, Infliximab Glucocorticoids Pharmacologic classifications of treatment for Diabetes Mellitus 1. Sulfonylureas: Tolbutamide, Tolazamide, Glyburide, Glipizide 2. Biguanides: Metformin 3. Miscellaneous oral agents: Acarbose, Troglitazone 4. Insulin Pharmacologic classifications of treatment for Alzheimer's Disease 1. Cholinesterase Inhibitor: Tacrine, Donepezil 2. Antipsychotics: Haloperidol, Thioridazine 3. Antidepressants: Desiprainine, Fluoxetine, Trazodone, Paroxetine 4. Anticonvulsants: Carbainazepine, Valproic acid In one embodiment, the present invention may therefore provide a method of treating RAGE mediated diseases, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a RAGE fusion protein in combination with therapeutic agents selected from the group consisting of allcylating agents, antimetabolites, plant alkaloids, antibiotics, hormones, biologic response modifiers, analgesics, NSAIDs, DMARDs, glucocorticoids, sulfonylureas, biguanides, insulin, cholinesterase inhibitors, antipsychotics, antidepressants, and anticonvulsants. In a further embodiment, the present invention provides the pharmaceutical composition of the invention as described above, further comprising one or more therapeutic agents selected from the group consisting of alkylating agents, antimetabolites, plant alkaloids, antibiotics, hormones, biologic response modifiers, analgesics, NSAIDs, DMARDs, glucocorticoids, sulfonylureas, biguanides, insulin, cholinesterase inhibitors, antipsychotics, antidepressants, and anticonvulsants.
EXAMPLES
Features and advantages of the inventive concept covered by the present invention are further illustrated in the examples which follow.
Example 1: Production of RAGE-ILYG Fc Fusion Proteins Two plasmids were constructed to express RAGE-IgG Fc fusion proteins. Both plasmids were constructed by ligating different lengths of a 5' eDNA sequence froin human RAGE with the same 3' eDNA sequence fiom human IgG Fc (yl). These expression sequences (i.e., ligation products) were then inserted in pcDNA3.l expression vector (Invitrogen, CA). The nucleic acid sequences that encode the fusion protein coding region are shown in FIGS. 2 and 3. For TTP-4000 fusion protein, the nucleic acid sequence from 1 to 753 (higl-Aighted in bold) encodes the RAGE N-tenninal protein sequence, whereas the nucleic acid sequence from 754 to 1386 encodes the IgG Fc protein sequence (FIG. 2). For TTP-3000, the nucleic acid sequence from 1 to 408 (highlighted in bold) encodes the RAGE
N-terminal protein sequence, whereas the nucleic acid sequence from 409 to 1041 encodes the IgG Fc protein sequence (FIG. 3).
To produce the RAGE fusion proteins, the expression vectors comprising the nucleic acid sequences of either SEQ ID NO: 30 or SEQ ID NO: 31 were stably transfected into CHO cells. Positive transfonnants were selected for neomycin resistance conferred by the plasmid and cloned. High producing clones as detected by Western Blot analysis of supernatant were expanded and the gene product was purified by affinity chromatography using Protein A columns. Expression was optimized so that cells were producing recombinant TTP-4000 at levels of about 1.3 grams per liter.
The expressed polypeptides encoding the two fusion proteins are illustrated in FIGS.
4-6. For the four domain structure of TTP-4000, the first 251 amino acids (shown in bold in FIG. 4) contain a signal sequence (1-22/23), the V immunoglobulin (and ligand binding) domain (23/24-116), a second interdomain linker (117-123), a second iinmunoglobulin domain (CHI) (124-221), and a second linlcer (222-251) of the human RAGE
protein (FIGS.
4, 6B). The sequence from 252 to 461 includes the CH2 and CH3 immunoglobulin domains of IgG.
For the three domain structure of TTP-3000, the first 136 amino acids (shown in bold) contain a signal sequence (1-22/23), the V inununoglobulin (and ligand binding) domain (23/24-116) and an interdomain linker sequence (117-136) of the human RAGE
protein (FIGS. 5, 6B). In addition, for TT3, the sequence from 137 to 346 includes the CH2 and CH3 immunoglobulin domains of IgG.

Example 2: Method for testing activity of a RAGE-IgGl fusion protein A. Iia vitro liLrand bindin~:
Known RAGE ligands were coated onto the surface of Maxisorb plates at a concentration of 5 micrograms per well. Plates were incubated at 4 C
overnight. Following ligand incubation, plates were aspirated and a blocking buffer of 1% BSA in 50 mM
imidizole buffer (pH 7.2) was added to the plates for 1 hour at room temperature. The plates were then aspirated and/or washed with wash buffer (20 mM Imidizole, 150 mM
NaCl, 0.05% Tween-20, 5 mM CaC12 and 5mM MgClz, pH 7.2). A solution of TTP-3000 (TT3) at an initial concentration of 1.082 mg/mL and a solution of TTP-4000 (TT4) at an initial concentration of 370 gghnL were prepared. The fusion protein was added at increasing dilutions of the initial sample. The RAGE fusion protein was allowed to incubate with the immobilized ligand at 37 C for one hour after which the plate was washed and assayed for binding of the fusion protein. Binding was detected by the addition of an immunodetection complex containing a monoclonal mouse anti-human IgGl diluted 1:11,000 to a fmal assay concentration (FAC) of 21 ng/100 L, a biotinylated goat anti-mouse IgG
diluted 1:500, to a FAC of 500 ng/ L, and an avidin-linked allcaline phosphatase. The complex was incubated with the immobilized fusion protein for one hour at room temperature after which the plate was washed and the alkaline phosphatase substrate para-nitrophenylphosphate (PNPP) was added. Binding of the complex to the immobilized fusion protein was quantified by measuring conversion of PNPP to para-nitrophenol (PNP) which was measured spectrophotoinetrically at 405 nm.
As illustrated in FIG. 7, the fusion proteins TTP-4000 (TT4) and TTP-3000 (TT3) specifically interact with known RAGE ligands amyloid-beta (Abeta), S 100b (S
100), and amphoterin (Ampho). In the absence of ligand, i.e., BSA coating alone (BSA or BSA +
wash) there was no increase in absorbance over levels attributable to non-specific binding of the immunodetection complex. Where amyloid beta is used as the labeled ligand it may be necessary to preincubate the amyloid beta before the assay. Preincubation may allow the amyloid beta to self-aggregate into pleated sheet form, as amyloid beta may preferentially bind to RAGE in the fonn of a pleated sheet.
Additional evidence for a specific interaction between RAGE fusion proteins TTP-4000 and TTP-3000 witli RAGE ligands is exemplified in studies showing that a RAGE
ligand is able to effectively compete with a lcnown RAGE ligand for binding to the fusion proteins. In these studies, amyloid-beta (A-beta) was irnmobilized on a Maxisorb plate and fusion protein added as described above. In addition, a RAGE ligand was added to some of the wells at the same time as the fusion protein.
It was found that the RAGE ligand could block binding of TTP-4000 (TT4) by about 25% to 30% where TTP-4000 was present at 123 gg/mL (1:3 dilution, FIG. 8).
When the initial solution of TTP-4000 was diluted by a factor of 10 or 30 (1:10 or 1:30), binding of the fusion protein to the immobilized ligand was completely inhibited by the RAGE
ligand.
Similarly, the RAGE ligand blocked binding of TTP-3000 (TT3) by about 50%
where TTP-3000 was present at 360 g/mL (1:3 dilution, FIG. 9). When the initial solution of TTP-3000 was diluted by a factor of 10 (1:10), binding of the fusion protein to the immobilized ligand was completely inhibited by the RAGE ligand. Thus, specificity of binding of the RAGE
fusion protein to the RAGE ligand was dose dependent. Also, as shown in FIGS.
8 and 9, there was essentially no binding detected in the absence of fusion protein, i.e., using only the immunodetection complex ("Complex alone").
B. Effect of RAGE fusion proteins in a cell based assay Previous worlc has shown that the myeloid THP-1 cells may secrete TNF-a in response to RAGE ligands. In this assay, THP-1 cells were cultured in RPMI-1640 media supplemented with 10% FBS using a protocol provided by ATCC. The cells were induced to secrete TNF-a via stiinulation of RAGE with 0.1 mg/ml S 100b both in the absence and the presence of the fusion proteins TTP-3000 (TT3) or TTP-4000 (TT4) (10 g), sRAGE (10 g), and a human IgG (10 g) (i.e., as a negative control). The amount of TNF-a secreted by the THP-1 cells was measured 24 hours after the addition of the proteins to the cell culture using a cominercially available ELISA kit for TNF-a (R&D Systems, Minneapolis, MN).
The results in FIG. 10 demonstrate that the fusion proteins inhibit the S
100b/RAGE-induced production of TNF-a in these cells. As shown in FIG. 10, upon addition of 10 g TTP-3000 or TTP-4000 RAGE fusion protein, induction of TNF-a by S l 00b (0.1 mg/ml FAC) was reduced by about 45% to 70%, respectively. Fusion protein TTP-4000 may be at least as effective in bloclcing S100b induction of TNF-a as is sRAGE (FIG. 10).
Specificity of the iiihibition for the RAGE sequences of TTP-4000 and TTP-3000 is shown by the experiment in which IgG alone was added to S l 00b stimulated cells. Addition of IgG and S l 00b to the assay shows the same levels of TNF-a as S 1 OOb alone. Specificity of the inhibition of TNF-a induction by TTP-4000 and TTP-3000 for RAGE sequences of the fusion protein is shown by an experiment in which IgG alone was added to S 100b stimulated cells. It can be seen that the addition of IgG, i.e., human IgG without the RAGE sequence (Sigma human IgG added at 10 g/well), and S 100b to the assay shows the same levels of TNF-a as S l 00b alone.
Example 3: Pharmacokinetic Profile of TTP-4000 To determine whether TTP-4000 would have a superior pharmacokinetic profile as compared to human sRAGE, rats and nonhuman primates were given an intravenous (IV) injection of TTP-4000 (5mg/kg) and then plasma was assessed for the presence of TTP-4000.
In these experiments, two naYve male monkeys received a single IV bolus dose of TTP-4000 (5mg/ml/kg) in a peripheral vein followed by an approximate 1.0 milliliter (mL) saline flush.
Blood samples (approximately 1.0 mL) were collected at pre-dose (i.e., prior to injection of the TTP-4000), or at 0.083, 0.25, 0.5, 2, 4, 8, 12, 24, 48, 72, 96, 120, 168, 240, 288, and 336 hours post dose into tubes containing (lithium heparin). Following collection, the tubes were placed on wet ice (maximum 30 minutes) until centrifugation under refrigeration (at 2 to 8 C) at 1500 x g for 15 minutes. Each harvested plasma sample was then stored frozen (-70 C
+10 C) until assayed for RAGE polypeptide using an ELISA at various time-points following the injection, as described in Example 6.
The kinetic profile shown in FIG. 11 reveals that once TTP-4000 has saturated its ligands as evidenced by the fairly steep slope of the alpha phase in 2 animals, it retains a terminal half-life of greater than 300 hours. This half-life is significantly greater than the half-life of human sRAGE in plasma (generally about 2 hours) and provides an opportunity for single iuijections for acute and semi-chronic indications. In FIG. 11 each curve represents a different animal under the same experimental conditions.
Example 4: TTP-4000 Fe Activation Experiments were performed to measure the activation of the Fc receptor by RAGE
fusion protein TTP-4000 as compared to human IgG. Fc receptor activation was measured by measuring TNF-a secretion from THP-1 cells that express the Fc receptor. In these experiments, a 96 well plate was coated with 10 g/well TTP-4000 or huinan IgG. Fc stimulation results in TNF-a secretion. The amount of TNF-a was measured by an Enzyine Linked Immunoabsorbent Assay (ELISA).
Thus, in this assay, the myeloid cell line, THP-1 (ATTC # TIB-202) was maintained in RPMI-1640 media supplemented with 10% fetal bovine serum per ATCC
instructions.
Typically, 40,000-80,000 cells per well were induced to secrete TNF-alpha via Fc receptor stimulation by precoating the well with 10 ug/well of eitlier heat aggregated (63 C for 30 min) TTP-4000 or human IgGl. The amount of TNF-alpha secreted by the THP-1 cells was measured in supernatants collected from 24 hours cultures of cells in the treated wells using a commercially available TNF ELISA. kit (R&D Systems, Minneapolis, MN # DTAOOC) per instructions.
Results are shown in FIG. 12 where it can be seen that TTP-4000 generates less than 2 ng/well TNF and IgG generated greater than 40 ng/well.
Example 5: In vivo activity of TTP-4000 The activity of TTP-4000 was compared to sRAGE in several ira vivo models of human disease.

A. TTP-4000 in an animal model of restenosis The RAGE fusion protein TTP-4000 was evaluated in a diabetic rat model of restenosis which involved measuring smooth muscle proliferation and intimal expansion 21 days following vascular injury. In these experiments, balloon injury of left common carotid artery was performed in Zucker diabetic and nondiabetic rats using standard procedure. A loading dose (3mg/rat) of IgG, TTP-4000 or phosphate buffered saline (PBS) was administered intraperitoneally (IP) oiie day prior injury. A maintenance dose was delivered every other day until day 7 after injury (i.e., at day 1, 3, 5 and 7 after injury). The inaintenance dose was high = 1 mg/animal for one group, or low = 0.3 mg/animal for the second group.
To measure vascular smooth muscle cell (VSMC) proliferation, animals were sacrificed at 4 days and 21 days after injury.
For the measurement of cell proliferation, 4 day animals received intraperitoneal injection of bromodeoxyuridine (BrDdU) 50 mg/lcg at 18, 12, and 2 hours before euthanasia. After sacrifice, the entire left and right carotid arteries were harvested.
Specimens were stored in Histochoice for at least 24 hours before embedding. Assessment of VSMC
proliferation was performed using mouse anti-BrdU monoclonal antibody. A fluorescence labeled goat anti-mouse secondary antibody was applied. The number of BrdU-positive nuclei per section were counted by two observers blinded to the treatment regimens.
The remaining rats were sacrificed at 21 days for morphometric analysis.
Morphometric analyses were performed by an observer blinded to the study groups, using computerized digital microscopic planimetry software Image-Pro Plus on serial sections, (5 imn apart) carotid arteries stained by Van Gieson staining. All data were expressed as mean ~: SD.
Statistical analysis was performed with use of SPSS software. Continuous variables were compared using unpaired t tests. A values of P< 0.05 was considered to be statistically significant.

As seen in FIGS. 13A and 13B, TTP-4000 treatment significantly reduced the intima/media ratio and vascular smooth muscle cell proliferation in a dose-responsive fashion. In FIG. 13 B, the y-axis represents the number of BrdU proliferating cells.
B. TTP4000 in an animal model of AD
Experiments were performed to evaluate whether TTP-4000 could affect amyloid formation and cognitive dysfunction in a mouse model of AD. The experiments utilized transgenic mice expressing the human Swedish mutant amyloid precursor protein (APP) under the control of the PDGF-B chain promoter. Over time, these mice generate high levels of the RAGE ligand, amyloid beta (AJ3). Previously, sRAGE treatment for 3 months has been shown to reduce both amyloid plaque formation in the brain and the associated increase in inflammatory markers in this model.
The APP mice (male) used in this experiment were designed by microinjection of the human APP gene (with the Swedish and London mutations) into mouse eggs under the control of the platelet-derived growth factor B (PDGF-B) chain gene promoter.
The mice were generated on a C57BL/6 background and were developed by Molecular Therapeutics Inc. Animals were fed ad libituin and maintained by brother sister mating. The mice generated from this construct develop amyloid deposits starting at 6 months of age. Animals were aged for 6 months and then maintained for 90 days and sacrificed for amyloid quantification.

APP transgenic mice were administered vehicle or TTP4000 every other day [qod (i.p.)]
for 90 days starting at 6 months of age. At the end of the experiment, animals were sacrificed and examined for A(3 plaque burden in the brain (i.e., plaque number). A 6-month control APP
group was used to determine the baseline of amyloid deposits. In addition, at the end of the study, the animals were subjected to behavioral (Morris water maze) analysis.
The investigators were blinded to the study coinpounds. Samples were given to the mice at 0.25 ml/mouse/every other day. In addition, one group of mice were given 200 ug/day of human sRAGE.
1. Amyloid Beta Deposition For histological examination, the animals were anesthetized with an intraperitoneal injection (IP) of sodium pentobarbital (50 mg/1{g). The animals were transcardially perfused with 4 C, phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. The brains were removed and placed in 4% paraformaldehyde over night. The brains were processed to paraffin and embedded. Ten serial 30- m thick sections through the brain were obtained.
Sections were subjected to primary antibody overnight at 4 C (A(3 peptide antibody) in order to detect the amyloid deposits in the brain of the transgenic animals (Guo et al., J. Neurosci., 22:5900-5909 (2002)). Sections were washed in Tris-buffered saline (TBS) and secondary antibody was added and incubated for 1 hour at room teinperature. After washing, the sections were incubated as instructed in the Vector ABC Elite kit (Vector Laboratories) and stained with diaminobenzoic acid (DAB). The reactions were stopped in water and cover-slipped after treatment with xylene. The amyloid area in each section was determined with a computer-assisted image analysis system, consisting of a Power Macintosh computer equipped with a Quick Capture frame grabber card, Hitachi CCD camera mounted on an Olympus microscope and camera stand. NIH Image Analysis Software, v. 1.55 was used.
The images were captured and the total area of amyloid was determined over the ten sections.
A single operator blinded to treatment status performed all ineasurements.
Summing the amyloid volumes of the sections and dividing by the total number of sections was done to calculate the amyloid volume.
For quantitative analysis, an enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of liuman total A(3, A(3t tal and AP1_42 in the brains of APP transgenic mice (Biosource International, Camarillo, CA). A(3t tal and A(31_42 were extracted from mouse brains by guanidine hydrochloride and quantified as described by the manufacturer. This assay extracts the total A(3 peptide from the brain (both soluble and aggregated).
2. Cognitive Functioia The Morris water-maze testing was performed as follows:. All mice were tested once in the Morris water maze test at the end of the experiment. Mice were trained in a 1.2 m open field water maze. The pool was filled to a depth of 30 cm with water and maintained at 25 C.
The escape platform (10 cm square) was placed 1 cm below the surface of the water. During the trials, the platform was reinoved from the pool. The cued test was carried out in the pool surrounded with white curtains to hide any extra-maze cues. All animals underwent non-spatial pretraining (NSP) for three consecutive days. These trials are to prepare the animals for the final behavioral test to determine the retention of memory to find the platform. These trials were not recorded, but were for training purposes only. For the training and learning studies, the curtains were removed to extra maze cues (this allowed for identification of animals with swiinming impairments). On day 1, the mice were placed on the hidden platform for 20 seconds (trial 1), for trials 2-3 animals were released in the water at a distance of 10 cm from the cued-platform or hidden platfonn (trial 4) and allowed to swim to the platform. On the second day of trails, the hidden platform was moved randomly between the center of the pool or the center of each quadrant. The animals were released into the pool, randomly facing the wall and were allowed 60 seconds to reach the platform (3 trials). In the third trial, animals were given three trials, two with a hidden platform and one with a cued platform. Two days following the NSP, animals were subjected to final behavioral trials (Morris water maze test). For these trials (3 per animal), the platform was placed in the center of one quadrant of the pool and the animals released facing the wall in a random fashion. The animal was allowed to fmd the platform or swim for 60 seconds (latency period, the time it takes to find the platform). All aiiimals were tested within 4-6 hours of dosing and were randomly selected for testing by an operator blinded to the test group.
The results are expressed as the mean standard deviations (SD). The significance of differences in the amyloid and behavioral studies were analyzed using a t-test. Comparisons were made between the 6-month-old APP control group and the TTP-4000 treated animals, as well as, the 9-month-old APP vehicle treated group and the TTP-4000 treated animals.
Differences below 0.05 were considered significant. Percent changes in amyloid and behavior were determined by taking the summation of the data in each group and dividing by the coinparison (i.e., 1, i.p./6 month control= % change).
FIGS. 14A a.nd 14B show that mice treated for 3 months with either TTP-4000 or mouse sRAGE had fewer AD plaques and less cognitive dysfunction than vehicle and negative control human IgGI (IgGl) treated animals. This data indicates that TTP-4000 is effective in reducing AD pathology in a transgenic mouse model. It was also found that like sRAGE, TTP-4000 can reduce the inflammatory cytokines IL-1 and TNF-a (data not shown).
C. Efficacy of TTP-4000 in an animal model of stroke TTP-4000 was also compared to sRAGE in a disease relevant animal model of stroke.
In this model, the middle carotid artery of a mouse was ligated for 1 hour followed by 23 hours of reperfusion at which point the mice were sacrificed and the area of the infarct in the brain was assessed. Mice were treated with sRAGE or TTP-4000 or control immunoglobulin just prior to reperfusion.

In these experiments, male C57BL/6 were injected with vehicle at 250 l/mouse or TTP test articles (TTP-3000, TTP-4000 at 250 l/mouse). Mice were injected intraperitoneally, 1 hour after the initiation of ischemia. Mice were subjected to one hour of cerebral ischemia followed by 24 hours of reperfusion. To induce ischemia, each mouse was anesthetized and body temperature was maintained at 36-37 C by external warming. The left common carotid artery (CCA) was exposed through a midline incision in the neclc. A microsurgical clip was placed around the origin of the internal carotid artery (ICA). The distal end of the ECA was ligated with sillc and transected. A 6-0 silk was tied loosely around the ECA stump. The fire-polished tip of a nylon suture was gently inserted into the ECA stump. The loop of the 6-0 sillc was tightened around the stump and the nylon suture was advanced into and tlirough the internal carotid artery (ICA), until it rested in the anterior cerebral artery, thereby occluding the anterior communicating and middle cerebral arteries. After the nylon suture had been in place for 1 hour, the animal was re-anesthetized, rectal temperature was recorded and the suture was removed and the incision closed.
Infarct volume was determined by anesthetizing the animals with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and then removing the brains. The brains were then sectioned into four 2-mm sections through the infracted region and placed in 2%
triphenyltetrazolium chloride (TTC) for 30 minutes. After, the sections were placed in 4%
paraformaldehyde over night. The infaret area in each section was detennined with a computer-assisted image analysis systein, consisting of a Power Macintosh computer equipped with a Quick Capture frame grabber card, Hitachi CCD cainera mounted on a camera stand. NIH Image Analysis Software, v. 1.55 was used. The images were captured and the total area of infarct was determined over the sections. A single operator blinded to treatment status performed all measurements. Summing the infarct volumes of the sections calculated the total infarct volume. The results are expressed as the mean =L
standard deviation (SD). The significance of difference in the infaret volume data was asialyzed using a t-test.
As illustrated by the data in Table 2, TTP-4000 was more efficacious than sRAGE in limiting the area of infarct in these animals suggesting that TTP-4000, because of its better half-life in plasma, was able to maintain greater protection in these mice.
Example 6: Detection of RAGE Fusion Protein by ELISA .
Initially, 50 uL of the RAGE specific monoclonal antibody 1HB1011at a concentration of 10 ug/mL in 1X PBS pH 7.3 is coated on plates via overnight incubation.
When ready for use, plates are washed three times with 300 uL of 1X Imidazole-Tween wash buffer and blocked with 1% BSA. The samples (diluted) and standard dilutions of known TTP-4000 dilutions are added at 100 uL final vohxme. The samples are allowed to incubate at room temperature for one hour. After incubation, the plates are plates are washed three times. A Goat Anti-human IgGl 1(Sigma A3312) AP conjugate in 1XPBS with 1% BSA
is added and allowed to incubate at room temperature for 1 hour. The plates are washed three times. Color was elucidated with paranitrophenylphosphate.
Example 7: Quantification of RAGE Ligand Binding to RAGE Fusion Protein Figure 15 shows saturation-binding curves with TTP-4000 to various immobilized known RAGE ligands. The ligands are immobilized on a microtiter plate and incubated in the presence of increasing concentrations of fusion protein from 0 to 360 nM.
The fusion protein-ligand interaction is detected using a polyclonal antibody conjugated with alkaline phosphatase that is specific for the IgG portion of the fusion chimera.
Relative Kds were calculated using Graphpad Prizm software and match with established literature values of RAGE-RAGE ligand values. HMG1B = Ampoterin, CML= Carboxymethyl Lysine, A beta =
Amyloid beta 1-40.
The foregoing is considered as illustrative only of the principal of the invention.
Since numerous modifications and changes will readily occur to those slcilled in the art, it is not intended to limit the invention to the exact embodiments shown and described, and all suitable modifications and equivalents falling within the scope of the appended claims are deemed within the present inventive concept.

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JUMBO APPLICATIONS / PATENTS

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THAN ONE VOLUME.

NOTE: For additional volumes please contact the Canadian Patent Office.

Claims

1. A fusion protein comprising a RAGE polypeptide directly linked to a polypeptide comprising a C H2 domain of an immunoglobulin or a portion of a C H2 domain of an immunoglobulin.

2. The fusion protein of claim 1, wherein the RAGE polypeptide comprises a RAGE
interdomain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-terminal amino acid of the interdomain linker, and the C-terminal amino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising a C
H2 domain of an immunoglobulin, or a portion thereof.

3. The fusion protein of claim 1, wherein the RAGE polypeptide comprises a ligand binding site.

4. The fusion protein of claim 3, wherein the RAGE ligand binding site comprises SEQ
ID NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%
identical thereto.

5. The fusion protein of claim 2, wherein the RAGE polypeptide comprising an interdomain linker linked to a RAGE immunoglobulin domain comprises a fragment of a full-length RAGE protein.

6. The fusion protein of claim 5, further comprising a first RAGE
immunoglobulin domain and a first RAGE interdomain linker linked to a second RAGE
immunoglobulin domain and a second RAGE interdomain linker, such that the N-terminal amino acid of the first interdomain linker is linked to the C-terminal amino acid of the first RAGE
immunoglobulin domain, the N-terminal amino acid of the second RAGE
immunoglobulin domain is linked to C-terminal amino acid of the first interdomain linker, the N-terminal amino acid of the second interdomain linker is linked to C-terminal amino acid of the second RAGE immunoglobulin domain, and the C-terminal amino acid of the RAGE second interdomain linker is directly linked to the N-terminal amino acid of the C H2 immunoglobulin domain or a portion of a C H2 domain of an immunoglobulin.

7. The fusion protein of claim 6, comprising the amino acid sequence SEQ ID
NO. 33 or SEQ ID NO: 34.

8. The fusion protein of claim 6, comprising the amino acid sequence SEQ ID
NO: 32.

9. The fusion protein of claim 6, wherein the RAGE interdomain linker directly linked to the immunoglobulin C H2 domain or a portion thereof comprises SEQ ID NO: 22 or a sequence 90% identical thereto, or SEQ ID NO: 24, or a sequence 90% identical thereto.

10. The fusion protein of claim 5, comprising a single RAGE immuglobulin domain linked via a RAGE interdomain linker to the N-terminal amino acid of a polypeptide comprising a C H2 immunoglobulin domain or a portion of a C H2 domain of an immunoglobulin.

11. The fusion protein of claim 10, comprising the amino acid sequence SEQ ID
NO: 36 or SEQ ID NO: 37.

12. The fusion protein of claim 10, comprising the amino acid sequence SEQ ID
NO: 35.

13. The fusion protein of claim 10, wherein the RAGE linker directly linked to the immunoglobulin C H2 comprises SEQ ID NO: 21 or a sequence 90% identical thereto or SEQ
ID NO: 23 or a sequence 90% identical thereto.

14. An isolated nucleic acid sequence encoding a fusion protein comprising a RAGE
polypeptide directly linked a polypeptide comprising a C H2 domain of an immunoglobulin or a portion of a C H2 domain of an immunoglobulin.

15. The isolated nucleic acid sequence of claim 14, wherein the RAGE
polypeptide comprises a RAGE interdomain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-terminal amino acid of the interdomain linker, and the C-terminal amino acid of the RAGE
interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising a C H2 domain of an immunoglobulin, or a portion thereof.

16. The nucleic acid of claim 15, comprising SEQ ID NO:30, or a fragment thereof, or SEQ ID NO: 31, or a fragment thereof.

17. An expression vector that encodes for a fusion protein comprising a RAGE
polypeptide directly linked to a polypeptide comprising a C H2 domain of an immunoglobulin or a portion of a C H2 domain of an immunoglobulin.

18. The expression vector of claim 17, wherein the RAGE polypeptide comprises a RAGE interdomain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-terminal amino acid of the interdomain linker, and the C-terminal amino acid of the RAGE
interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising a C H2 domain of an immunoglobulin, or a portion thereof.

19. The expression vector of claim 17, comprising the nucleic acid sequence SEQ ID
NO:30, or a fragment thereof, or SEQ ID NO: 31, or a fragment thereof.

20. A cell transfected with the expression vector of claim 17, such that the cell expresses a fusion protein comprising a RAGE polypeptide directly linked to a polypeptide comprising a C H2 domain of an immunoglobulin or a portion of a C H2 domain of a.n immunoglobulin.

21. A composition comprising a therapeutically effective amount of a RAGE
fusion protein in a pharmaceutical carrier, wherein the RAGE fusion protein comprises a RAGE
polypeptide directly linked to a polypeptide comprising a C H2 domain of an immunoglobulin or a portion of a C H2 domain of an immunoglobulin.

22. The composition of claim 21, wherein the RAGE polypeptide comprises a RAGE

interdomain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-terminal amino acid of the interdomain linker, and the C-terminal amino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising a C
H2 domain of an immunoglobulin, or a portion thereof.

23. The composition of claim 21, wherein the RAGE polypeptide comprises a ligand binding site.

24. The composition of claim 23, wherein the RAGE ligand binding site comprises SEQ
ID NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%
identical thereto.

25. The composition of claim 22, wherein the RAGE polypeptide comprising an interdomain linker linked to a RAGE immunoglobulin domain comprises a fragment of a full-length RAGE protein.

26. The composition of claim 25, wherein the RAGE polypeptide comprises a first RAGE
immunoglobulin domain and a first RAGE interdomain linker linked to a second RAGE
immunoglobulin domain and a second RAGE interdomain linker, such that the N-terminal amino acid of the first interdomain linker is linked to the C-terminal amino acid of the first RAGE immunoglobulin domain, the N-terminal amino acid of the second RAGE
immunoglobulin domain is linked to C-terminal amino acid of the first interdomain linker, the N-terminal amino acid of the second interdomain linker is linked to C-terminal amino acid of the second RAGE immunoglobulin domain, and the C-terminal amino acid of the RAGE second interdomain linker is directly linked to the N-terminal amino acid of the C H2 immunoglobulin domain or a portion of a C H2 domain of an immunoglobulin.

27. The composition of claim 26, wherein the RAGE fusion protein comprises the amino acid sequence SEQ ID NO: 33 or SEQ ID NO: 34.

28. The composition of claim 25, wherein the fusion protein comprises a single RAGE
immunoglobulin domain linked via a RAGE interdomain linker to the N-terminal amino acid of a polypeptide comprising a C H2 immunoglobulin domain or a portion of a C
H2 domain of an immunoglobulin.

29. The composition of claim 28, wherein the RAGE fusion protein comprises the amino acid sequence SEQ ID NO: 36 or SEQ ID NO: 37.

30. The composition of claim 21, wherein the RAGE fusion protein is formulated as an injectable solution.

31. The composition of claim 21, wherein the RAGE fusion protein is formulated as a sterile lyophilized powder.

32. A method of making a RAGE fusion protein comprising the step of covalently linking a RAGE polypeptide to a polypeptide comprising a C H2 domain of an immunoglobulin or a portion of a C H2 domain of an immunoglobulin.

33. The method of claim 32, wherein the RAGE polypeptide comprises a RAGE
interdomain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-terminal amino acid of the interdomain linker, and the C-terminal amino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising a C
H2 domain of an immunoglobulin, or a portion thereof.

34. The method of claim 32, wherein the RAGE polypeptide comprises a ligand binding site.

35. The method of claim 34, wherein the RAGE ligand binding site comprises SEQ
ID
NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%
identical thereto.

36. The method of claim 33, wherein the RAGE polypeptide comprising an interdomain linker linked to a RAGE immunoglobulin domain comprises a fragment of a full-length RAGE protein.

37. The method of claim 36, wherein RAGE polypeptide comprises a first RAGE
immunoglobulin domain and a first RAGE interdomain linker linked to a second RAGE
immunoglobulin domain and a second RAGE interdomain linker, such that the N-terminal amino acid of the first interdomain linker is linked to the C-terminal amino acid of the first RAGE immunoglobulin domain, the N-terminal amino acid of the second RAGE
immunoglobulin domain is linked to C-terminal amino acid of the first interdomain linker, the N-terminal amino acid of the second interdomain linker is linked to C-terminal amino acid of the second RAGE immunoglobulin domain, and the C-terminal amino acid of the RAGE second interdomain linker is directly linked to the N-terminal amino acid of the C H2 immunoglobulin domain or a portion of a C H2 domain of an immunoglobulin.

38. The method of claim 36, wherein the RAGE polypeptide comprises a single RAGE
immuglobulin domain linked via a RAGE interdomain linker to the N-terminal amino acid of a polypeptide comprising a C H2 immunoglobulin domain or a portion of a C H2 domain of an immunoglobulin.

39. The method of claim 32, wherein the fusion protein is encoded by a recombinant DNA construct.

40. The method of claim 32, further comprising the step of incorporating the DNA
construct into an expression vector.

41. The method of claim 40, further comprising transfecting the expression vector into a host cell.

42. A method of treating a RAGE-mediated disorder in a subject comprising administering to a subject a RAGE fusion protein comprising a RAGE polypeptide directly linked a polypeptide comprising a C H2 domain of an immunoglobulin or a portion of a C H2 domain of an immunoglobulin.

43. The method of claim 42, wherein the RAGE polypeptide comprises a RAGE
interdomain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-terminal amino acid of the interdomain linker, and the C-terminal amino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising a C
H2 domain of an immunoglobulin, or a portion thereof.

44. The method of claim 42, wherein the RAGE ligand binding site comprises SEQ
ID
NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%
identical thereto.

45. The method of claim 43, wherein the RAGE polypeptide comprising an interdomain linker linked to a RAGE immunoglobulin domain comprises a fragment of a full-length RAGE protein.

46. The method of claim 45, further comprising a first RAGE immunoglobulin domain and a first RAGE interdomain linker linked to a second RAGE immunoglobulin domain and a second RAGE interdomain linker, such that the N-terminal amino acid of the first interdomain linker is linked to the C-terminal amino acid of the first RAGE
immunoglobulin domain, the N-terminal amino acid of the second RAGE immunoglobulin domain is linked to C-terminal amino acid of the first interdomain linker, the N-terminal amino acid of the second interdomain linker is linked to C-terminal amino acid of the second RAGE
immunoglobulin domain, and the C-terminal amino acid of the RAGE second interdomain linker is directly linked to the N-terminal amino acid of the C H2 immunoglobulin domain or a portion of a C H2 domain of an immunoglobulin.

47. The method of claim 46, wherein the RAGE fusion protein comprises the amino acid sequence SEQ ID NO: 33 or SEQ ID NO: 34.

48. The method of claim 46, wherein the RAGE fusion protein comprises the amino acid sequence SEQ ID NO: 35.

49. The method of claim 46, wherein the RAGE interdomain linker directly linked to the immunoglobulin C H2 domain or a portion thereof, comprises SEQ ID NO: 22 or a sequence 90% identical thereto, or SEQ ID NO: 24 or a sequence 90% identical thereto.

50. The method of claim 45, comprising a single RAGE immuglobulin domain linked via a RAGE interdomain linker to the N-terminal amino acid of a polypeptide comprising a C H2 immunoglobulin domain or a portion of a C H2 domain of an immunoglobulin.

51. The method of claim 50, wherein the RAGE fusion protein comprises the amino acid sequence SEQ ID NO: 36 or SEQ ID NO. 37.

52. The method of claim 50, wherein the RAGE fusion protein comprises the amino acid sequence SEQ ID NO: 35.

53. The method of claim 50, wherein the RAGE interdomain linker directly linked to the immunoglobulin C H2 or a portion thereof, comprises SEQ ID NO: 21 or a sequence 90%
identical thereto, or SEQ ID NO: 23 or a sequence 90% identical thereto.

54. The method of claim 42, comprising intravenous administration of the RAGE
fusion protein to the subject.

55. The method of claim 42, comprising intraperitoneal administration of the RAGE
fusion protein to the subject.

56. The method of claim 42, comprising subcutaneous administration of the RAGE
fusion protein to the subject.

57. The method of claim 42, wherein the fusion protein is used to treat a symptom of diabetes or a symptom of diabetic late complications.

58. The method of claim 57, wherein the symptom of diabetes or diabetic late complications comprises diabetic nephropathy.

59. The method of claim 57, wherein the symptom of diabetes or diabetic late complications comprises diabetic retinopathy.

60. The method of claim 57, wherein the symptom of diabetes or diabetic late complications comprises a diabetic foot ulcer.

61. The method of claim 57, wherein the symptom of diabetes or diabetic late complications comprises a cardiovascular complication.

62 The method of claim 57, wherein the symptom of diabetes or diabetic late complications comprises diabetic neuropathy.

63. The method of claim 42, wherein the fusion protein is used to treat amyloidosis.

64. The method of claim 42, wherein the fusion protein is used to treat Alzheimer's disease.

65. The method of claim 42, wherein the fusion protein is used to treat cancer.

66 The method of claim 42, wherein the fusion protein is used to treat inflammation associated with autoimmunity.

67. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with inflammatory bowel disease.

68. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with rheumatoid arthritis.

69. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with psoriasis.

70. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with multiple sclerosis.

71. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with hypoxia.

72. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with stroke.

73. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with heart attack..

74. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with hemorraghic shock.

75. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with sepsis.

76. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with organ transplantation.

77. The method of claim 42, wherein the fusion protein is used to treat inflammation associated with impaired wound healing.

78. The method of claim 42, wherein the fusion protein is used to treat kidney failure.