NL2001553C2 - New Receptor for Advanced Glycated Endproducts (RAGE) fusion protein and nucleic acids, useful for treating a RAGE-mediated disorder, e.g. amyloidosis, Alzheimer's disease, cancer, kidney failure, or inflammation - Google Patents

Abstract

A RAGE fusion protein comprising fully defined 447 or 326 amino acids (SEQ ID NO. 56 or 57), or a sequence at least 90% identical to it, is new. Independent claims are: (1) an isolated DNA molecule that encodes the RAGE fusion protein; (2) an expression vector that encodes the RAGE fusion protein; (3) a cell transfected with the expression vector, so that the cell expresses a RAGE fusion protein comprising SEQ ID NO. 56 or 57, or a sequence at least 90% identical to it; (4) a formulation comprising a lyophilized mixture of a lyoprotectant, a RAGE fusion protein, and a buffer; (5) a reconstituted formulation comprising a lyophylized RAGE fusion protein reconstituted in a diluent, where the RAGE fusion protein concentration in the reconstituted formulation is 1-400 mg/ml; (6) an article of manufacture comprising a container, which holds a lyophylized RAGE fusion protein, and instructions for reconstituting the lyophilized formulation with a diluent; (7) a method for preparing a stable reconstituted formulation of a RAGE fusion protein; and (8) a method of treating a RAGE-mediated disorder in a subject. ACTIVITY : Antidiabetic; Cardiovascular-Gen; Neuroprotective; Nootropic; Cytostatic; Nephrotropic; Antiinflammatory; Gastrointestinal-Gen; Antiulcer; Antirheumatic; Antiarthritic; Antipsoriatic; Vasotropic; Cerebroprotective; Cardiant; Antibacterial; Immunosuppressive; Vulnerary; Osteopathic. The effects of administering TTP-4000 on rejection of syngeneic transplanted islets in diabetic NOD mice were studied. Results showed an increase in the time before detection of graft failure for animals treated with TTP-4000 and TTP-3000 as opposed to animals that are not treated at all. MECHANISM OF ACTION : RAGE-Antagonist.

Description

RAGE FUSION PROTEINS, PREPARATIONS AND METHODS FOR IT

USE IT

5 CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119 (e) of U.S. Provisional Patent Application Serial Number 60 / 798,455, filed May 5, 2006. The description of U.S. Provisional Patent Application 60 / 798,455 is hereby incorporated by reference in its entirety.

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FIELD OF THE INVENTION

The present invention relates to regulation of the Receptor for Advanced Glycated Endproducts (RAGE). More particularly, the present invention describes fusion proteins comprising a RAGE polypeptide, methods of making such fusion proteins and compositions of such RAGE fusion proteins, and the use of such RAGE fusion proteins for treatment of RAGE-based disorders.

BACKGROUND

Incubation of proteins or lipids with aldose sugars results in non-enzymatic glycation and oxidation of amino groups on proteins to form Amadori adducts. Over time, the adducts undergo additional rearrangements, dehydrations, and cross-linking with other proteins to form complexes known as Advanced Glycation End Products (AGEs).

Factors that promote the formation of AGEs include delayed protein conversion (for example as with amyloidosis), accumulation of macromolecules that have a high lysine content and high blood glucose levels (such as for example in diabetes) (Hori et al, J Biol Chem 270: 25752-761 (1995). AGEs are involved in a variety of disorders including complications associated with diabetes and normal aging.

AGEs exhibit specific and saturable binding to cell surface receptors on monocytes, macrophages, microvasculature endothelial cells, smooth muscle cells, mesengial cells and neurons. The Receptor for Advanced 2

Glycated Endproducts (RAGE) is a member of the immunoglobulin supergene family of molecules. The extracellular (N-terminal) domain of RAGE comprises three immunoglobulin-type regions: one V (variable) type domain followed by two C-type (constant) domains (Neeper et al., J. Biol. Chem., 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) composed of the V and C-10 domains.

RAGE is expressed on multiple cell types including leukocytes, neurons, microglial cells and vascular endothelium (e.g. Hori et al, J. Biol. Chem., 270: 25752-761 (1995)). Elevated levels of RAGE are also found in tissues undergoing an aging process (Schleicher et al., J. Clin. Invest. 99 (3): 457-468 (1997)) and the diabetic retina, vasculature and kidney (Schmidt et al. , Nature Med. 1: 1002-1004 (1995)).

In addition to AGEs, other compounds can bind to and modulate RAGE. RAGE binds to several functionally and structurally diverse ligands, including amyloid beta (Αβ), serum amyloid A (SAA), Advanced Glycation End 20 products (AGEs), SI00 (an anti-inflammatory member of the Calgranulin family), carboxymethyllysine (CML), amphoterin and CD11b / CD18 (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. Immunol., 61: 1-9 (2005); Schmidt et al, J. Clin. Invest., 108: 949-955 (2001); Rocken et al, Am. J. Pathol, 162: 1213-1220 (2003) ).

It has been shown that binding of ligands such as AGEs, S100 / calgranulin, β-amyloid, CML (N? -Carboxymethyl lysine) and amphoterin to RAGE modifies expression of a variety of genes. These interactions can then initiate signal transduction mechanisms including p38 activation, p21ras, MAP kinases, Erkl-2 phosphorylation and the activation of the transcriptional mediator of inflammation signaling, NF-κΒ (Yeh et al, Diabetes, 50: 1495-1504 (2001)). For example, in many cell types, interaction between RAGE and its ligands can generate oxidative stress resulting in activation of the free radical sensitive transcription factor NF-κΒ and the activation of NF-icB-regulated genes, such as the cytokines IL-Ιβ and TNF -α. In addition, expression of RAGE is up-regulated by NF-kB and shows increased expression at sites of inflammation or oxidative stress (Tanaka et al., J. Biol. Chem., 275: 25781-25790 (2000)). A downward and often harmful spiral can thus be fed by a positive feedback loop that is initiated by ligand binding.

Activation of RAGE in different tissues and organs can lead to a number of pathophysiological consequences. RAGE is involved in a variety of conditions including: acute and chronic inflammation (Hofmann et al, Cell 97: 889-901 (1999)), the development of late complications in diabetes such as increased vascular permeability (Wautier et al., J. Clin Invest. 97: 238-243 (1995), nephropathy (Teillet et al., J. Am. 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 is also involved 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 widespread expression of RAGE and its apparent pleiotropic role in multiple models of various diseases, RAGE does not appear to be essential for normal development. For example, knockout mice for RAGE do not have a clearly abnormal phenotype suggesting that while RAGE may play a role in pathology of disease when chronically stimulated, the inhibition of RAGE does not appear to contribute to any undesirable acute phenotype (Liliensiek et al. , J. Clin. Invest. 113: 1641-50 (2004).

Counteracting the binding of physiological ligands to RAGE can down-regulate the pathophysiological changes brought about by excessive concentrations of AGEs and other RAGE ligands. By lowering the binding of endogenous ligands to RAGE, symptoms associated with RAGE-mediated disorders can be reduced. Soluble RAGE (sRAGE) is capable of effectively countering the binding of RAGE ligands to RAGE. However, sRAGE, when administered in vivo, may have a half-life that may be too short to be therapeutically useful for one or more disorders. Thus, there is a need to develop compounds that oppose the binding of AGEs and other physiological ligands to the RAGE-4 receptor, the compound having a desired pharmacokinetic profile.

SUMMARY

Embodiments of the present invention include fusion proteins from RAGE and methods for using such proteins. The present invention can be carried out in a variety of ways. Embodiments of the present invention may include a RAGE fusion protein comprising a RAGE polypeptide linked to a second non-RAGE polypeptide. In one embodiment, the RAGE fusion protein comprises a binding site for a RAGE ligand. The RAGE fusion protein may further comprise a RAGE polypeptide that is directly linked to a polypeptide comprising the C 1 domain of an immunoglobulin or a portion of the C n 2 domain. In certain embodiments, the RAGE fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 56 or SEQ ID NO: 57 or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% , 96%, 97%, 98% or 99% identical to that. For example, in some embodiments, a sequence that is at least 90% identical to SEQ ID NO: 56 or SEQ ID NO: 57 includes the sequence of SEQ ID NO: 56 or SEQ ID NO: 57 without the C-terminal lysine.

The present invention also includes a method for making 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 binding site for a RAGE ligand. The method may include linking a RAGE polypeptide directly to a polypeptide comprising the CH2 domain of an immunoglobulin or a portion of the CH2 domain. In certain embodiments, the RAGE fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 56 or SEQ ID NO: 57 or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to that. In some embodiments, a sequence that is at least 90% identical to SEQ ID NO: 56 or SEQ ID NO: 57 includes, for example, the sequence of SEQ ID NO: 56 or SEQ ID NO: 57 without the C-terminal lysine.

In other embodiments, the present invention may include methods and compositions for treating a RAGE-mediated disorder in a patient. The method may include administering a RAGE fusion protein of the present invention to the patient. The composition may comprise a RAGE fusion protein of the present invention in a pharmaceutically acceptable carrier.

In other embodiments, the present invention also provides compositions comprising a freeze-dried mixture of a protective agent for freeze-drying, a RAGE fusion protein, and a buffer. In certain embodiments, the present invention may comprise, for example, a stable reconstituted preparation comprising a RAGE fusion protein in an amount of at least 50 mg / ml and a diluent, the reconstituted preparation being prepared from a freeze-dried mixture of the RAGE fusion protein and a protective agent for freeze-drying.

Embodiments of the present invention may also include industrially produced articles. In certain embodiments, the industrially produced articles may comprise a container that contains a composition comprising a freeze-dried RAGE fusion protein. The article of manufacture may also include instructions for reconstituting the freeze-dried preparation with a diluent.

In other embodiments, the present invention may also include methods for preparing a stably reconstituted preparation of a RAGE fusion protein. In certain embodiments, the method may comprise reconstituting a freeze-dried mixture of a RAGE fusion protein and a protective agent for freeze-drying in a diluent such that the concentration of the RAGE fusion protein in the reconstituted preparation is at least 50 mg / ml is. For example, in one embodiment, the method may include the steps of freeze-drying a mixture comprising a RAGE fusion protein and a freeze-drying amount of a freeze-drying protective agent and reconstituting the freeze-dried mixture into a diluent.

There are several advantages that may be associated with specific embodiments of the present invention. In one embodiment, the RAGE fusion proteins of the present invention can be metabolically stable when administered to a patient. The RAGE fusion proteins of the present invention can also exhibit binding to high affinity RAGE ligands.

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In certain embodiments, the RAGE fusion proteins of the present invention bind to RAGE ligands with affinities in the range of high nanomolar to low micromolar. By binding with high affinity to physiological ligands of RAGE, the RAGE fusion proteins of the present invention can be used to inhibit binding of endogenous ligands to RAGE, thereby providing means for attenuating RAGE-mediated diseases.

The RAGE fusion proteins of the present invention can also be provided in the form of protein or nucleic acid. In one example of an embodiment, the RAGE fusion protein can be systemically administered and remain in the vasculature to treat potentially vascular diseases partially mediated by RAGE. In another example of an embodiment, the RAGE fusion protein can be administered white locally to treat diseases in which RAGE ligands contribute to the pathology of the disease. Alternatively, a nucleic acid construct encoding the RAGE fusion protein can be delivered to a site using a suitable carrier such as a virus or as a bare DNA, with transient local expression locally interacting between RAGE ligands and receptors. Thus, administration can be transient (e.g., when the RAGE fusion protein is administered) or more permanent in nature (e.g., such as when the RAGE fusion protein is administered as a recombinant DNA).

There are additional features of the invention that will be described below. It should 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 can be used in other embodiments and can be used and practiced in various ways.

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BRIEF DESCRIPTION OF THE FIGURES

Various features, aspects and advantages of the present invention will become more apparent with reference to the following figures.

Figure 1 shows various sequences of RAGE and immunoglobulin sequences consistent with other 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 5 without the signal sequence of amino acids 1-23; Panel D, SEQ TD NO: 7, an amino acid sequence comprising the V domain of human RAGE; SEQ ID NO: 8, another amino acid sequence comprising 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, another N-terminal fragment of the V domain of human RAGE; SEQ ID NO: 10 11, the amino acid sequence for amino 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 amino acids 23-226 of human RAGE; SEQ ID NO: 18, the amino acid sequence for amino 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 20 human RAGE; SEQ ID NO: 21, an interdomain linker from RAGE; SEQ ID NO: 22, a second cross-domain linker from RAGE; SEQ ID NO: 23, a third cross-domain linker from RAGE; SEQ ID NO: 24, a fourth cross-domain linker from RAGE; Panel G, SEQ ID NO: 25, DNA encoding amino acids 1-118 of human RAGE; SEQ ID NO: 26, DNA encoding amino acids 1-123 of human RAGE; and SEQ ID NO: 27, DNA encoding amino acids 1-136 of human RAGE; Panel H, SEQ ID NO: 28, DNA encoding amino acids 1-230 of human RAGE; SEQ ID NO: 29, DNA encoding amino acids 1-251 of human RAGE; 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 human IgG; SEQ ID NO: 40, an amino acid sequence for the Ch 2 and C 4 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 8 amino acid sequence for the CH3 domain of human IgG; and SEQ ID NO: 44, a fifth cross-domain linker from RAGE; Panel K, SEQ ID NO: 45, the amino acid sequence of human sRAGE without the signal sequence of amino acids 1-23, wherein the glutamine residue at the N-terminus is cyclized to form pyroglutamic acid, SEQ TD NO: 46, another amino acid sequence comprising the V domain of human sRAGE where the glutamine residue is cyclized at the N-terminus to form pyroglutamic acid, SEQ ID NO: 47, another N-terminal fragment of the V domain of human RAGE where the glutamine residue at the N-terminus is cyclized to form pyroglutamic acid, SEQ ID NO: 48, the amino acid sequence for amino acids 24-123 of human RAGE, wherein the glutamine residue at the N-terminal is cyclized to form pyroglutamic acid; Panel L, SEQ ID NO: 49, the amino acid sequence of amino acids 24-136 of human RAGE, wherein the glutamine residue at the N-terminus is cyclized to form pyroglutamic acid, SEQ ID NO: 50, the amino acid sequence for amino acids 24 -226 of human RAGE, where the glutamine residue is cyclized at the N-terminus to form pyroglutamic acid, SEQ ID NO: 51, the amino acid sequence for amino acids 24-251 of human RAGE, where the glutamine residue is at the N- end is cyclized to form pyroglutamic acid; Panel M, SEQ ID NO: 52, another DNA sequence coding for a portion of the human Ch2 and CH3 domains of human IgG in SEQ ID NO: 38 and SEQ ID NO: 53, a different DNA sequence which encodes the human Ch2 and CH3 domains of human IgG in SEQ ID NO: 40.

Figure 2 shows other DNA sequences SEQ ID NO: 30 (Panel A) and SEQ ID NO: 54 (Panel B) that encodes a first coding region for a RAGE fusion protein (TTP-4000) that is consistent with an embodiment of the present invention. Coding sequence 1-753 that is highlighted in bold letters encodes the N-terminal protein sequence of RAGE while sequence 754-1386 encodes the protein sequence of human IgG (γΐ) without the hinge region.

Figure 3 shows other DNA sequences SEQ ID NO: 31 (Panel A) and SEQ ID

NO: 55 (Panel B) encoding the coding region of a second RAGE fusion protein (TTP-3000) in accordance with an embodiment of the present invention. Coding sequence 1-408 marked in bold letters 9 codes for N-terminal protein sequence of RAGE while sequence 409-1041 encodes protein sequence of human IgG (γΐ) without the hinge region.

Figure 4 shows the amino acid sequences, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 56 that each encode a RAGE fusion protein with four domains that is consistent with other embodiments of the present invention. RAGE sequence is highlighted in bold letters.

Figure 5 shows the amino acid sequences, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 57 each encoding a three-domain RAGE fusion protein that is consistent with other embodiments of the present invention. RAGE sequence is highlighted in bold letters.

Figure 6, Panel A, shows a comparison of the protein domains of human RAGE and human Ig gamma-1 Fc protein and splicing points used to make TTP-3000 (at position 136) and TTP-4000 (at position 251) in accordance with other embodiments of the present invention; and Panel B shows the domain structure for TTP-3000 and TTP-4000 that is in accordance with other embodiments of the present invention.

Figure 7 shows results of an in vitro binding test for sRAGE and a first RAGE fusion protein TTP-4000 (TT4) and a second RAGE fusion protein TTP-3000 (TT3), on the RAGE ligands amyloid beta (A beta), SI00b (SI00) and amphoterin (Ampho), in accordance with an embodiment of the present invention.

Figure 8 shows results of an in vitro binding test for a first RAGE fusion protein TTP-4000 (TT4) (“Protein”) to amyloid beta as compared to a negative control that only includes the immune detection reagent (“complex only”) ) and antagonism of such binding by an RAGE 25 antagonist ("RAGE ligand") in accordance with an embodiment of the present invention.

Figure 9 shows results of an in vitro binding test for a second RAGE fusion protein TTP-3000 (TT3) (“Protein”) to amyloid beta as compared to a negative control that only includes the immune detection reagent (“complex only” ") And antagonism of such binding by an antagonist of RAGE (" RAGE ligand ") in accordance with an embodiment of the present invention.

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Figure 10 shows the results of a cell-based assay that measures the inhibition of SlOOb-RAGE-induced production of TNF-α by RAGE fusion proteins TTP-3000 (TT3) and TTP-4000 (TT4) and sRAGE in accordance with an embodiment of the present invention.

Figure 11 shows the results of a cell-based assay that measures the inhibition of HMGB1-RAGE-induced production of TNF-α by RAGE fusion protein TTP-4000 and anti-RAGE antibody in accordance with an embodiment of the present invention .

Figure 12 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.

Figure 13 shows relative levels of TNF-α release from THP-1 cells by stimulation by RAGE fusion protein TTP-4000 and human IgG stimulation as a measure of an inflammatory response in accordance with an embodiment of the present invention.

Figure 14 shows the use of RAGE fusion protein TTP-4000 for reducing restenosis in diabetic animals in accordance with other embodiments of the present invention, wherein Panel A shows that RAGE fusion protein TTP-4000 4000 intima / media ratio decreased compared to a negative control (IgG) and Panel B shows that RAGE fusion protein reduced TTP-4000 proliferation of vascular smooth muscle cells in a dose-responsive manner.

Figure 15 shows the use of RAGE fusion protein TTP-4000 for reducing amyloid formation and cognitive dysfunction in animals with Alzheimer's disease (AD) in accordance with other embodiments of the present invention, where Panel A shows that RAGE TTP-4000 fusion protein reduced the amount of amyloid in the brain and Panel B shows that RAGE fusion protein TTP-4000 improved cognitive function.

Figure 16 shows the TTP-4000 binding saturation curves to various immobilized known RAGE ligands in accordance with an embodiment of the present invention.

Figure 17 shows the use of RAGE fusion protein TTP-4000 for reducing allogeneic transplant rejection of cells from the islets of the 11 pancreas in accordance with other embodiments of the present invention, where open (unfilled) ) circles of non-treated control animals; circles with diagonal hatching indicate animals treated with TTP-4000 at a first dose; circles with wavy shading indicate animals treated with TTP-4000 at a second dose; diamonds filled with diamonds indicate animals treated with control PBS; and solid circles indicate animals treated with control IgG.

Figure 18 shows the use of TTP-4000 RAGE fusion proteins to reduce the rejection of syngeneic grafts from pancreatic islet cells in accordance with other embodiments of the present invention, with open (unfilled) circles indicate untreated control animals; and solid circles indicate animals treated with TTP-4000.

DETAILED DESCRIPTION

Unless indicated to the contrary, the numerical parameters set forth in the following specification are approximations that may vary depending on the desired properties sought to be obtained by the present invention. At the very least and not as an attempt to limit the use of doctrine of equivalents of the scope of the claims, each numerical parameter should be considered at least with respect to the number of significant numbers reported and by applying the usual techniques of completion.

Although the numerical ranges and parameters explaining the broad scope of the invention are approximations, the numerical values set forth in the specific examples are stated as precisely as possible. However, each numeric value inherently contains certain errors that necessarily result from the standard deviation found in the respective measurements of tests. Moreover, it is to be understood that all ranges described herein include any and all sub ranges included therein. For example, a set range of "1 to 10" should be considered to include any and all sub-ranges between (and including) the minimum value of 1 and the maximum value of 10; that is, all sub-ranges starting with a minimum value of 1 or more, for example 1 to 6.1 and ending with 12, a maximum value of 10 or lower, for example 5.5 to 10. In addition, any reference made herein referred to as "incorporated herein" is understood as included in its entirety.

It should further be noted that, as used in this specification, the singular forms "a," "an," and "it" include plural referents unless expressly and unambiguously are limited to one. The term "or" is used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

The terms "part" and "fragment" are also used interchangeably to refer to parts of a polypeptide, nucleic acid or other molecular construct.

"Polypeptide" and "protein" are used interchangeably herein to describe protein molecules that may include either partial proteins or full-length proteins.

As is known in the art, "proteins", "peptides", "polypeptides" and "oligopeptides" are chains of amino acids (typically L-amino acids) whose alpha carbons are linked by peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. The 20 amino acids that form a protein are typically numbered in sequence starting from the amino terminal residue and increasing toward the carboxy terminal residue of the protein.

As used herein, the term "upstream" refers to a residue that is N-terminal to a second residue when the molecule is a protein or 5 "to a second residue when the molecule is a nucleic acid. As used herein, the term "downstream" refers to a residue that is C-terminal to a second residue when the molecule is a protein, or 3 "to a second residue when the molecule is a nucleic acid.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is generally understood by those skilled in the art. Experts are particularly referred to Current Protocols in Molecular Biology (see, for example, Ausubel, F. M. et al., Short Protocols in Molecular Biology, 4th Edition, Chapter 2, John Wiley & Sons, 13 N.Y.) for definitions and terms of the art. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 general L-amino 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 analogs.

The term "vector" refers to a nucleic acid molecule that can be used to transport a second nucleic acid molecule into a cell. In one embodiment, the vector allows replication of DNA sequences that are inserted into the vector. The vector may include a promoter for increasing the expression of the nucleic acid molecule in at least some host cells. Vectors can replicate autonomously (extrachromosomally) or can be integrated into a chromosome of the host cell. In one embodiment, the vector may comprise an expression vector capable of producing a protein derived from at least a portion 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. High stringent hybridization conditions generally refer to washing of hybrids in low salt buffer at high temperatures. Hybridization can be filter-bound DNA using hybridization solutions that are standard in the art, such as 0.5 M NaHPO 4, 7% sodium dodecyl sulfate (SDS), at 65 ° C and washing in 0.25 M NaHPQ *, 3.5% SDS followed by washing in 0.1 x SSC / 0.1% SDS at a temperature ranging from room temperature to 68 ° C depending on the length of the probe. A high stringency wash includes, for example, 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 an oligonucleotide probe from 20 bases, or at 60 ° C for an oligonucleotide probe of 25 bases, or at 65 ° C for a nucleotide probe about 250 nucleotides in length. Nucleic acid probes can be labeled with radionucleotides by terminal labeling with, for example, [γ-32P] ATP or incorporation of radiolabeled nucleotides such as [α-32P] dCTP by 14 random labeling with primers. Alternatively, probes can be labeled by incorporation of biotinylated or fluorescein-labeled nucleotides and the probe is detected using Streptavidin or anti-fluorescein antibodies.

As used herein, "small organic molecules" are molecules with a molecular weight of less than 2,000 Daltons that contain at least one carbon atom.

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 10 may also include linking regions of amino acids between portions of amino acids that are derived from individual 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 immunoglobulin peptides, dimerizing polypeptides, stabilizing polypeptides, amphiphilic peptides or polypeptides that include amino acid sequences that provide "markers" for targeting or purifying the protein.

As used herein, "immunoglobulin peptides" may include an immunoglobulin heavy chain or a portion thereof. In one embodiment, the part of the heavy chain may be the Fc fragment or a part thereof. As used herein, the Fc fragment comprises the heavy chain hinge polypeptide and the Ch2 and Ch3 domains of the immunoglobulin heavy chain in either monomeric or dimeric form. The ChI and Fc fragment can also be used as the immunoglobulin polypeptide. The heavy chain (or part thereof) can originate from any of the known heavy chain isotypes: IgG (γ), IgM (μ), IgD (δ), IgE (ε) or IgA (a). In addition, the heavy chain (or part thereof) may come from any of the known heavy chain subtypes: IgG1 (γΐ), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgAl (a1), IgA2 ( a2), or mutations of these isotypes or subtypes that alter biological activity. An example of biological activity that can be changed includes reduction of the ability of an isotype to bind to some Fc receptors such as, for example, by modification of the hinge region.

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The terms "match" or "percentage match" refers to sequence similarity between two amino acid sequences or between two nucleic acid sequences. Percentage similarity can be determined by positioning two sequences and refers to the number of identical residues (i.e., amino acid or 5 nucleotides) at positions shared by the sequences being compared. Sequence positioning and comparison can be performed using standard algorithms 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) that are publicly available as BLAST and FASTA. Also, ENTREZ, available through the National Institutes of Health, Bethesda MD, can be used for the sequence comparison. In one embodiment, the percent similarity of two sequences can be determined by using GCG with a gap weight of 1, such that each gap of an amino acid is weighted as if it is a single mismatch of amino acids between the two sequences.

As used herein, the term "conserved residues" refers to amino acids that are the same among a variety of proteins that have the same structure and / or function. A region of conserved residues may be important for protein structure and function. Consecutive conserved residues as they are identified in a three-dimensional protein can thus be important for the structure and function of a protein. To find conserved residues or conserved regions of a 3-D structure, a comparison of sequences for the same or similar proteins of different species or of individuals of the same species can be performed.

As used herein, the term "homologue" means a polypeptide that has a degree of homology to the wild-type amino acid sequence. Comparisons on homology can be performed by eye or more commonly with the help of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of 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 considered to include 16 amino acid sequences which in other embodiments are at least 70% identical, 75% identical, 85% identical, 90% identical, 95% identical, 97% identical, or 98% identical, or 99% identical to each other to be.

As used herein, the term encompasses at least 90% identically thereto sequences ranging from 90 to 99.99% identity with the indicated sequences and includes all ranges between them. The term at least 90% identical therewith thus comprises sequences that include 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97 5, 98, 98.5, 99, 99.5 percent are identical to the indicated sequence. Similarly, the term "at least 70% identical" includes sequences ranging from 70 to 99.99% identical, with all ranges in between. The determination of percentage agreement is determined by the use of algorithms described here.

As used herein, a polypeptide or protein "domain" includes a region along a polypeptide or protein that comprises an independent entity. Domains can 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 of the rest of the protein. Domains can be identified through the use of domains databases such as but not limited to PFAM, PRODOM, PROSITE, BLOCKS, PRINTS, SBASE, ISREC PROFILES, SAMRT and PROCLASS.

As used herein, an "immunoglobulin domain" is a sequence of amino acids that is structurally homologous or identical to an immunoglobulin domain. The length of the amino acid sequence of an immunoglobulin domain can be of any length. In one embodiment, an immunoglobulin domain can be smaller than 250 amino acids. In an exemplary embodiment, an immunoglobulin domain can be about 80-150 amino acids in length. The variable region and the Ch1, Ch2 and Cn3 regions of an IgG are each, for example, immunoglobulin domains. In another example, the variable, the CH1, Ch2, Ch3, and Cn4 regions of an IgM are each immunoglobulin domains.

As used herein, a "RAGE immunoglobulin domain" is a sequence of amino acids of RAGE protein that is structurally homologous or identical to an immunoglobulin domain. For example, an RAGE immunoglobulin domain may be the RAGE V domain, the RAGE Ig-like C1 type 1 domain

17 ("C1 domain) or the Jg-like C2 type 2 domain (" C2 domain ") of RAGE.

As used herein, an "interdomain linker" includes a polypeptide that connects two domains together. An Fc hinge region is an example of an interdomain linker in a TgG.

As used herein, "directly linked" identifies a covalent link between two different groups (e.g., nucleic acid sequences, polypeptides, polypeptide domains) that has no intervening atoms between the two groups being linked.

As used herein, "ligand binding domain" refers to a domain of a protein that is responsible for binding a ligand. The term ligand binding domain includes homologues of a ligand binding domain or parts thereof. In this regard, intentional amino acid substitutions in the ligand binding site can be made based on 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" includes residues in a protein that interact directly with a ligand or residues involved in positioning the ligand in close proximity to those residues directly associated with the ligand interact. The interaction of residues in the ligand binding site can be determined by the spatial proximity of the residues with a ligand in the model or structure. The term ligand binding site includes homologues of a ligand binding site or parts thereof. In this regard, intentional amino acid substitutions in the ligand binding site can be made based on 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 can exist in one or more ligand binding domains of a protein or polypeptide.

As used herein, the term "interaction" refers to a condition of proximity between a ligand or compound or parts or fragments thereof and a part of a second molecule of interest. The interaction can be non-covalent, for example as a result of hydrogen bonding, van der Waals interactions or electrostatic or hydrophobic interactions or it can be covalent.

18

As used herein, "ligand" refers to a molecule or compound or moiety that interacts with a ligand binding site, including substrates or analogs or portions thereof. As described herein, the term "ligand" may refer to compounds that bind to the protein of interest. A ligand can be an agonist, an antagonist or a modulator. Or a ligand cannot have a biological effect. Or a ligand can block the binding of other ligands, thereby inhibiting a biological effect. Ligands can include, but are not limited to, small molecule inhibitors. These small molecules can include peptides, peptidomimetics, organic compounds and the like. Ligands may also include polypeptides and / or proteins.

As used herein, a "modulating compound" refers to a molecule that alters or exchanges the biological activity of a molecule of interest. A modulating compound can increase or decrease activity or change the physical or chemical characteristics or functional or immunological properties of the molecule of interest. For RAGE, a modulating compound may increase or decrease activity or change the characteristics or functional or immunological properties of the RAGE or part thereof. A modulating compound may comprise 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 modulating compound can be an endogenous physiological compound or it can be a natural or synthetic compound. Or the modulating compound can be a small organic molecule. The term "modulating compound" also includes a chemically modified ligand or compound and includes isomers and racemic forms.

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

An "antagonist" includes a compound that binds to an agonist or to a receptor to form a complex that does not elicit a substantial pharmacological response and can inhibit the biological response induced by an agonist.

RAGE agonists can therefore bind to RAGE and stimulate RAGE-mediated cellular processes, and RAGE antagonists can inhibit RAGE-mediated processes to be stimulated by a RAGE agonist. In one embodiment, the cellular process that is stimulated by RAGE agonists comprises, for example, activation of transcription of the TNF-α gene.

The term "peptidomimetics" refers to structures that serve as substituents for peptides in interactions between molecules (Morgan et al., 1989, Ann. Reports Med. Chem., 24: 243-252). Peptidomimetics may include synthetic structures that may or may not contain amino acids and / or peptide bonds but which retain the structural and functional characteristics of a peptide or agonist or antagonist. Peptidomimetics 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 "treatment" or "treats" refers to improving a symptom of a disease or disorder and may include curing the disorder, substantially preventing the onset of the disorder, or improving the condition of the patient. The term "treatment" as used herein refers to the full spectrum of treatments for a particular disorder that the patient is suffering from, including alleviating one symptom or most of the symptoms that result from that disorder, a cure for the specific disorder or the prevent 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 that has a measurable biological effect can include the value at which the agent exhibits 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 binding to RAGE may include the value at which the antagonist reduces binding of the ligand to the ligand-binding site of RAGE by 50%.

20

As used herein, an "effective amount" means the amount of an agent effective to produce a desired effect in a patient. The term "therapeutically effective amount" indicates that amount of a drug or pharmaceutical that will elicit the therapeutic response in an animal or human being that one desires. The actual dose comprising the effective amount may depend on the route of administration, the size and health of the patient, the disorder being treated and the like.

The term "pharmaceutically acceptable carrier" as used herein may refer to compounds and compositions suitable for use in human or animal patients, such as, for example, therapeutic compositions administered for the treatment of a RAGE-mediated disorder or disease.

The term "pharmaceutical composition" is used herein to indicate a composition that can be administered to a mammalian host, for example, orally, parenterally, topically, by inhalation spray, intranasally or rectally, in unit dosage formulations that are conventional contain non-toxic carriers, diluents, adjuvants, excipients and the like.

The term "parenteral" as used herein includes subcutaneous injections, intravenous, intramuscular, intracistemal injection, or infusion techniques.

As used herein, "rejection" refers to the immune or inflammatory response to tissue that leads to destruction of cells, tissues or organs or that leads to damage to cells, tissues or organs. The shed cells, tissue, or organ may be from the same patient who induces the immune response or may have been transplanted from another patient in the shedding patient.

As used herein, the term "cell" refers to the structural and functional units of a mammalian living system, each comprising an independent living system. As is known in the art, cells include a nucleus, cytoplasm, intracellular organelles, and a cell wall that surrounds the cell and allows the cell to be independent of other cells.

As used herein, the term "tissue" refers to an aggregate of cells that have a similar structure and function or that act together to perform a specific function. A tissue may comprise a collection of corresponding cells and the intercellular components that surround the cells. Tissues include, but are not limited to, muscle tissue, nerve tissue, and bone.

As used herein, an "organ" refers to a fully differentiated structural or functional unit in an animal that specializes for a particular specific function. An organ may comprise a group of tissue that performs a specific function or group of functions. Organs include, but are not limited to, the heart, lungs, brain, eye, stomach, spleen, pancreas, kidneys, liver, small intestines, skin, uterus, bladder, and bone.

A "stable" preparation is one in which the RAGE fusion protein therein retains essentially its physical and chemical stability and biological activity during storage. Various analytical techniques for measuring protein stability are available in the art and are summarized in Peptide and Protein Drug Delivery, 247-301, Vincent Lee editor, Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. For rapid screening, the preparation can be held at 40 ° C for 1 week to 1 month after which the stability is measured. For example, the degree of aggregation following lyophilization and storage can be used as an indicator of stability of the RAGE fusion protein (see the Examples herein). For example, a "stable" composition may be one in which less than about 10% and preferably less than about 5% of the RAGE fusion protein is present as an aggregate in the composition. In other embodiments, an increase in aggregate formation following lyophilization and storage of the lyophilized preparation can be determined. For example, a "stable" freeze-dried preparation may be one in which the increase in aggregates in the freeze-dried preparation is less than about 5% or less than about 3% when the freeze-dried preparation is incubated at 40 ° C for at least one week. In other embodiments, stability of the RAGE fusion protein preparation can be measured using a biological activity test such as a binding test as described herein.

A "reconstituted" preparation is one prepared by dissolving a preparation of a freeze-dried RAGE fusion protein in a diluent such that the RAGE fusion protein is dispersed and / or dissolved in the reconstituted preparation. The reconstituted preparation may be suitable for administration 22 (e.g., parenteral administration) to a patient to be treated with the fusion protein, and in certain embodiments of the invention may be one suitable for subcutaneous administration.

By "isotonic" it is meant that the preparation of interest has an osmotic pressure of about 240 to about 340 mOsm / kg. In an embodiment, an isotonic composition is one that has an osmotic pressure that is essentially the same as human blood (285-310 mOsm / kg). Isotonicity can be measured by using a vapor pressure or a freezing point type osmometer.

A "lyoprotectant protective" agent is a molecule that, when combined with a RAGE fusion protein, significantly prevents or reduces chemical and / or physical instability of the protein during lyophilization and subsequent storage. Illustrative freeze drying protective agents include sugars such as sucrose or trehalose; a polyol such as sugar alcohols, for example erythritol, arabitol, xylitol, sorbitol and mannitol; or combinations thereof. In one embodiment, the freeze drying protective agent may comprise a sugar. In another embodiment, the freeze drying protective agent may comprise a non-reducing sugar. In another embodiment, the freeze drying protective agent may comprise a non-reducing sugar such as sucrose. The freeze-drying protective agent can be added to the preparation before freeze-drying at a "freeze-drying amount" which means that following freeze-drying of the protein in the presence of a freeze-drying amount of the freeze-drying protective agent RAGE fusion protein essentially retains its physical and chemical stability and biological activity during lyophilization and storage.

The "diluent" for a freeze-dried preparation is one herein that is pharmaceutically acceptable (safe and non-toxic for administration to a human) and useful for the preparation of a reconstituted preparation. Illustrative diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In one embodiment, the diluent provides a reconstituted preparation suitable for injection. In another embodiment, where the diluent 23 provides a reconstituted preparation suitable for injection, the diluent may comprise water for injection (WFI).

A "preservative" for a reconstituted preparation is a compound that can be added to the diluent or reconstituted preparation to substantially reduce bacterial activity in the reconstituted preparation. In one embodiment, the amount of the preservative can be added in an amount useful for effecting the production of a reconstituted multi-use preparation. Examples of possible preservatives include octadecyldimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides where the alkyl groups are long chain compounds) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, allyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol.

A "bulking agent" for a freeze-dried preparation is a compound that adds mass to the freeze-dried mixture and contributes to the physical structure of the freeze-dried cake (for example, produces a substantially uniform freeze-dried cake that retains a structure of open pores). Illustrative volume-forming agents include, but are not limited to, mannitol, glycine, and xorbitol.

RAGE fusion proteins

Embodiments of the present invention include RAGE fusion proteins, methods of making such fusion proteins, and methods of using such fusion proteins. The present invention can be carried out in a variety of ways.

For example, embodiments of the present invention provide RAGE fusion proteins comprising a RAGE polypeptide linked to a second non-RAGE polypeptide. In one embodiment, the RAGE fusion protein may comprise a binding site for a RAGE ligand. In one embodiment, the ligand binding site comprises the most N-terminal domain of the RAGE fusion protein. The binding site for the RAGE ligand may comprise the V domain of RAGE or a portion 24 thereof. In one embodiment, the binding site for a ligand of RAGE includes SEQ ID NO: 9 or a sequence that is at least 90% identical to it, or SEQ ID NO: 10 or a sequence that is at least 90% identical to it, or SEQ ID NO: 9 : 47 or a sequence that is at least 90% identical to that (Figure 1).

In one 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 comprising an immunoglobulin domain comprises at least a portion of at least one of the Ch2 or Ch3 domains of a human IgG.

In certain embodiments, the RAGE fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 56 or SEQ ID NO: 57 or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that. In some embodiments, a sequence that is at least 90% identical to SEQ ID NO: 56 or SEQ ID NO: 57 includes, for example, the sequence of SEQ ID NO: 56 or SEQ ID NO: 57 without the C-terminal lysine.

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 has a length of at least 5 amino acids, it can be longer than a length of 30 amino acids, but is shorter than the complete amino acid sequence. In other embodiments of the phytosis proteins, compositions and methods of the present invention, the RAGE polypeptide may comprise a sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% is 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 instead of a Methionine (see, for example, Neeper et al., (1992)). Or the human RAGE can include full-length RAGE with the signal sequence removed (e.g., SEQ ID NO: 2 or SEQ ID NO: 3) (Figures 1A and 1B) or a portion of that amino acid sequence.

The RAGE fusion proteins of the present invention can also be sRAGE

(e.g. SEQ ID NO: 4), a polypeptide that is at least 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 can include human sRAGE or a fragment thereof, with Glycine as the first residue instead of a Methionine (see, for example, Neeper et al., (1992)). Or a RAGE polypeptide can include human sRAGE with the signal sequence deleted (see, for example, SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ TD NO: 45 in Figure 1) or a portion of that amino acid sequence.

In other embodiments, the RAGE protein may comprise a V domain of RAGE (see, for example, SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 46 in Figure 1) (Neeper et al, (1992); Schmidt et al. al. (1997)). Or a sequence that is at least 90% identical to the V domain of RAGE or a fragment thereof can be used.

Or the RAGE protein may comprise a fragment of the V-domain of RAGE (e.g., SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 47 in Figure 1). In one embodiment, the RAGE protein may comprise a ligand binding site. In an embodiment, the ligand binding site can be SEQ ID NO: 9, or a sequence that is at least 90% identical to it, or SEQ ID NO: 10, or a sequence that is at least 90% identical to it, or SEQ ID NO: 9 : 47, or a sequence that is at least 90% identical to that. In yet another embodiment, the RAGE fragment is a synthetic peptide.

In another embodiment, the ligand binding site may comprise amino acids 23-53 of SEQ ID NO: 1 (Figure 1). In another embodiment, the ligand binding site may comprise amino acids 24-52 of SEQ ID NO: 1. In another embodiment, the ligand binding site may comprise amino acids 31-52 of SEQ ID NO: 1. In another embodiment, the ligand binding site may comprise amino acids 31-25 116 of SEQ ID NO: 1. In another embodiment, the ligand binding site may comprise amino acids 19-52 of SEQ ID NO: 1. The ligand binding site may include, for example, a RAGE V domain or part thereof, such as the RAGE ligand binding domain (e.g., amino acids 1-118, 23-118, 24-118, 31-118, 1-116, 23- 116, 24-116, 31-116, 1-54, 23-54, 24-54, 31-54, 1-53, 23-53, 30-24-53 or 31-53 of SEQ ID NO: 1 or fragments thereof). Or fragments of the polypeptides that functionally bind a RAGE ligand can be used. Or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the V-domain of RAGE or a fragment thereof (e.g. as above 26 described) can be used. In embodiments where the N-terminus of the fusion protein is glutamine, such as, for example, removal of the signal sequence comprising residues 1-23 of SEQ ID NO: 1 (e.g., Q24 for a polypeptide comprising amino acids 24-118 or SEQ ID NO: 1) can further, as is known in the art, cyclize the glutamine to form pyroglutamic acid (pE).

The RAGE polypeptide used in the RAGE fusion proteins of the present invention can thus comprise a fragment of full-length RAGE. As is known in the art, RAGE comprises three immunoglobulin-like polypeptide domains, the V domain, and the C1 and C2 domains each linked by an interdomain linker. Full-length RAGE also includes a transmembrane polypeptide and a cytoplasmic tail downstream (C-terminal) of the C2 domain and linked to the C2 domain.

In one embodiment, the RAGE polypeptide does not include residues of a signal sequence. The signal sequence of RAGE can include either full-length residues 1-22 or residues 1-23. As is known in the art, in embodiments where the N-terminus of the fusion protein is glutamine (e.g., the signal sequence comprises residues 1-23), the N-terminal glutamine (Q24) can cyclize to form pyroglutamic acid (pE). Constructs as an example of such molecules are polypeptides that have the amino acid sequences as set forth in SEQ ID NOs: 45, 46, 47, 48, 49, 50 and 51 (Figure 1), as well as RAGE fiction proteins that have the amino acid sequences as set forth in SEQ ID NOs: 56 and 57 (Figure 4).

As is known in the art, the Cn3 region of the RAGE fixation protein of the present invention may be cleaved off the C-terminal amino acid by post-translational modification when expressed in certain recombinant systems (see, for example, Li, et al., BioProcessing J2005; 4, 23-30). In one embodiment, the C-terminal amino acid being cleaved is lysine (K). Thus, in other embodiments, the RAGE fusion protein of the present invention may comprise a polypeptide having the amino acid sequence as set forth in SEQ ID NOs: 32-37, 56 and 57 without the C-terminal lysine (K).

Thus, in various embodiments, the RAGE polypeptide can be amino acids 23-116 of human RAGE (SEQ ID NO: 7) or a sequence that is at least 90% identical to it, or amino acids 24-116 of human RAGE (SEQ ID NO: 8) ) or a sequence that is at least 90% identical to it, or amino acids 24-116 of human RAGE where Q24 cyclizes to form pE (SEQ ID NO: 46) or a sequence that is at least 90% identical to it, corresponding to the V domain 5 of RAGE. Or the RAGE polypeptide can include human RAGE amino acids 124-221 (SEQ ID NO: 11) or a sequence that is at least 90% identical to that corresponding to the RAGE C1 domain. In another embodiment, the RAGE polypeptide may comprise amino acids 227-317 of human RAGE (SEQ ID NO: 12) or a sequence that is at least 90% identical to that corresponding to the C2-10 domain of RAGE. Or the RAGE polypeptide can be amino acids 23-123 of human RAGE (SEQ ID NO: 13) or a sequence that is at least 90% identical to it, or amino acids 24-123 of human RAGE (SEQ ID NO: 14) or a sequence which is at least 90% identical to that corresponding to the V-domain of RAGE and includes a downstream inter-domain linker. Or the RAGE polypeptide can comprise 15 amino acids 24-123 of human RAGE where Q24 cyclizes to form pE (SEQ ID NO: 48) or a sequence that is at least 90% identical to it. Or the RAGE polypeptide can be amino acids 23-226 of human RAGE (SEQ ID NO: 17) or a sequence that is at least 90% identical to it, or amino acids 24-226 of human RAGE (SEQ ID NO: 18) or a sequence which is at least 90% identical to 20, corresponding to the V domain, the Cl domain, and the interdomain linker that links these two domains. Or the RAGE polypeptide can include human RAGE amino acids 24-226 where Q24 cyclizes to form pE (SEQ ID NO: 50), or a sequence that is 90% identical to it. Or the RAGE polypeptide can be amino acids 23-339 of human RAGE (SEQ ID NO: 5) or a sequence that is at least 90% identical to it, or amino acids 24-339 of human RAGE (SEQ ID NO: 6) or a sequence that is at least 90% identical to that corresponding to sRAGE (ie, encodes the V, C1, and C2 domains and include interdomain linkers). Or the RAGE polypeptide can include human RAGE amino acids 24-339 where Q24 cyclizes to form pE (SEQ ID NO: 45) or a sequence that is at least 90% identical to it. Or fragments of any of these sequences can be used. See Figure 1 for the amino acid sequences of these polypeptides.

28

The RAGE fusion protein can include various types of peptides that are not derived from RAGE or a fragment thereof. The second polypeptide of the RAGE fusion protein may comprise a polypeptide that is derived from an immunoglobulin. In one embodiment, the immunoglobulin polypeptide may comprise an immunoglobulin heavy chain or a portion (i.e., a fragment) thereof. For example, the heavy chain fragment may include a polypeptide derived from the Fc fragment of an immunoglobulin, the Fc fragment comprising the heavy chain hinge polypeptide and the Ch2 and Cu3 domains of the immunoglobulin heavy chain as a monomer. The heavy chain (or part thereof) can originate from one of the known isotopes of the heavy chain: IgG (γ), IgM (μ), IgD (δ), IgE (ε), or IgA (α). In addition, the heavy chain (or part thereof) can be obtained from one of the known subtypes of the heavy chain: IgG1 (γΐ), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgAl (a1), IgA2 (a2), or mutations of these isotypes or subtypes that alter biological activity. The second polypeptide can comprise the Ch2 and CH3 domains of a human IgG1 or parts of each or both of these domains. As an example of an embodiment, the polypeptide comprising the CH2 and CH3 domains of a human IgG1 or a portion thereof may comprise SEQ ID NO: 40 (Figure 1) or a portion thereof. In one embodiment, the polypeptide comprising the Ch2 and CH3 domains of a human IgG1 or a portion thereof may comprise SEQ ID NO: 38 or a fragment thereof. The immunoglobulin peptide can be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 41 (Figure 1). The immunoglobulin sequence in SEQ ID NO: 38 or SEQ ID NO: 40 can also be encoded by SEQ ID NO: 52 or SEQ ID NO: 53 (Figure 1), wherein silent base changes for the codons encoding proline (CCG to CCC) ) and glycine (GGT to GGG) remove a cryptic RNA cleavage site near the terminal codon at the C-terminus of the sequence (ie nucleotides 622-627 of SEQ ID NO: 39 are modified to generate SEQ ID NO: 52 or nucleotides 652-657 of SEQ ID NO: 41 are modified to generate SEQ ID NO: 53).

The hinge region of the Fc portion of the immunoglobulin chain can be anti-inflammatory 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 certain embodiments, the RAGE fusion protein may comprise a RAGE polypeptide that is directly linked to a polypeptide comprising a Cn2 domain of an immunoglobulin or a fragment or a portion of the Ch2 domain of an immunoglobulin. In one embodiment, the Ch2 domain or a fragment thereof comprises SEQ ID NO: 42 (Figure 1). In one embodiment, the fragment of SEQ ID NO: 42 comprises SEQ ID NO: 42 with the first ten amino acids comprising at least a portion of the Fc hinge region removed. In one embodiment, the RAGE polypeptide may comprise a ligand binding site. The binding site for the RAGE ligand may include the RAGE V domain or part thereof. In one embodiment, the binding site for a ligand of RAGE SEQ ID NO: 9 or a sequence that is at least 90% identical to it, or SEQ ID NO: 10 or a sequence that is at least 90% identical to it, or SEQ ID NO: 47, or a sequence that is at least 90% identical to that.

The RAGE polypeptide used in the RAGE fusion proteins of the present invention may comprise an RAGE immunoglobulin domain. In addition or alternatively, the RAGE fragment may comprise a linker domain. Or, the RAGE polypeptide may include an RAGE immunoglobulin domain linked to an upstream (i.e., closer to 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) immunoglobulin domains of RAGE that are each linked to each other by an interdomain linker. The RAGE polypeptide may further comprise multiple RAGE immunoglobulin domains linked to one another by one or more inter-domain linkers and having a terminal inter-domain linker linked to the RAGE N-terminal immunoglobulin domain and / or the C-terminal immunoglobulin domain domain of RAGE. Additional combinations of immunoglobulin domains and inter-domain linkers of RAGE are within the scope of the present invention.

In one embodiment, the RAGE polypeptide comprises an RAGE inter-domain linker that is linked to the 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 linker inter-domain and the C-terminal amino acid of the inter-domain linker of RAGE is directly linked to the N-terminal amino acid of a polypeptide comprising a C 1 domain of an immunoglobulin or a fragment thereof. The polypeptide comprising an immunoglobulin Cn2 domain can include the Ch2 and Cn3 domains of a human IgG1 or a portion of each, or both, of these domains. As an example of embodiments, the polypeptide comprising the Ch2 and Cn3 domains or a portion thereof of a human IgG1 may comprise SEQ ID NO: 40 or a portion thereof. In one embodiment, the polypeptide comprising the Ch2 and CH3 domains of a human IgG1 or a portion thereof may comprise SEQ ID NO: 38 or a fragment thereof. Or the human IgG1 may comprise SEQ ID NO: 38 or SEQ ID NO: 40 with the terminal lysine (K) removed.

As described above, the RAGE fusion protein of the present invention can comprise a single or multiple domains of RAGE. The RAGE polypeptide comprising an inter-domain linker that is linked to a domain of a RAGE polypeptide may also include a fragment of the full-length RAGE protein. For example, the RAGE polypeptide can be amino acids 23-136 of human RAGE (SEQ ID NO: 15) or a sequence that is at least 90% identical to it or 20 amino acids 24-136 of human RAGE (SEQ ID NO: 16) or a sequence that is at least 90% identical to it, or amino acids 24-136 of human RAGE where Q24 cyclizes to form pE (SEQ ID NO: 49), or a sequence that is at least 90% identical to it, corresponding to the V domain of RAGE and a downstream interdomain linker. Or the RAGE polypeptide can be 25 amino acids 23-251 of human RAGE (SEQ ID NO: 19) or a sequence that is at least 90% identical to it, or amino acids 24-251 of human RAGE (SEQ ID NO: 20) or a sequence that is at least 90% identical to it, or amino acids 24-251 of human RAGE where Q24 cyclizes to form pE (SEQ ID NO: 51), or a sequence that is at least 90% identical to it, corresponding to the V domain, the C1 domain, the interdomain linker that links these two domains and a second interdomain linker downstream of C1.

For example, in one embodiment, the RAGE fusion protein may comprise two immunoglobulin domains derived from RAGE protein and two immunoglobulin domains derived from a human Fc polypeptide. The RAGE fusion protein may comprise a first immunoglobulin domain from RAGE and a first inter-domain linker from RAGE linked to a second immunoglobulin domain from RAGE and a second inter-domain linker from RAGE 5 such that the N-terminal amino acid of the first linker interdomain 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 the C-terminal amino acid of the first linker left, the N-terminal amino acid of the second linker domain is linked to the C-terminal amino acid of the second immunoglobulin domain of RAGE and the C-terminal amino acid of the second linker domain of RAGE is directly linked to the N-terminal amino acid of the Ch2- immunoglobulin domain. In other embodiments, the four domain RAGE fusion protein is encoded by SEQ ID NO: 30 or SEQ ID NO: 54 (Figure 2). In one embodiment, a four domain RAGE fusion protein can comprise SEQ ID NO: 32 (Figure 4). In other embodiments, a four-domain RAGE fusion protein comprises SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 56 (Figure 4).

Alternatively, a three-domain RAGE fusion protein may comprise one immunoglobulin domain from RAGE and two immunoglobulin domains from a human Fc polypeptide. For example, the RAGE fusion protein may comprise a single immunoglobulin domain from RAGE linked through an inter-domain linker from RAGE to the N-terminal amino acid of a CH2 immunoglobulin domain or a portion of a CH2 immunoglobulin domain. In other embodiments, the three-domain RAGE fusion protein is encoded by SEQ ID NO: 31 or SEQ ID NO: 55 (Figure 3). In one embodiment, the three domain RAGE fusion protein may comprise SEQ ID NO: 35 (Figure 5). In other embodiments, a three-domain RAGE fusion protein may comprise SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 57 (Figure 5).

A fragment of an RAGE interdomain linker may include a peptide sequence 30 that is naturally downstream from, and thus linked to, an RAGE immunoglobulin domain. For example, for the V domain of RAGE, the interdomain linker may include amino acid sequences that are naturally downstream of the V domain. In one embodiment, the left SEQ ID

32 include NO: 21 corresponding to amino acids 117-123 of full-length RAGE. Or the linker may include a peptide that has additional portions of the natural sequence of RAGE. For example, an interdomain linker comprising several amino acids (e.g., 1-3, 1-5, or 1-10, or 1-15 amino acids) upstream or downstream of SEQ 1D NO: 21 can be used. In one embodiment, the interdomain linker thus comprises SEQ ID NO: 23 which comprises amino acids 117-136 of full-length RAGE. Or fragments of SEQ ID NO: 21 wherein, for example, 1, 2 or 3 amino acids are removed from each end of the linker can be used. In other embodiments, the linker may comprise a peptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21 or SEQ ID NO: 23.

For the RAGE C1 domain, the linker may include a peptide sequence that is naturally downstream of the C1 domain. In one embodiment, the left-hand SEQ ID may include NO: 22 corresponding to amino acids 222-251 of RAGE with the full length. Or the linker may include a peptide that has additional portions of the natural sequence of RAGE. For example, a linker comprising several amino acids (1-3, 1-5, or 1-10, or 1-15 amino acids) upstream and downstream of SEQ ID NO: 22 can be used. Or fragments of SEQ ID NO: 22 can be used, wherein for example 1-3, 1-5, or 1-10, or 1-15 amino acids have been removed from each end of the linker. In one embodiment, an inter-domain linker from RAGE may include, for example, SEQ ID NO: 24 corresponding to amino acids 222-226. In other embodiments, the linker may comprise a peptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 22 or SEQ ID NO: 24.

Or an interdomain linker may include SEQ ID NO: 44 corresponding to amino acids 318-342 of RAGE. In other embodiments, the linker may comprise a peptide that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 44.

Moreover, it will be apparent to one skilled in the art that individual substitutions, deletions, or additions that change a single amino acid or a small percentage of amino acids (typically less than about 5%, more typically less than about 1%) into a coded sequence , adding or removing are conservatively modified variations where the 33 changes result in the substitution of an amino acid with a chemically similar amino acid. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. The following examples of groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

A conservative substitution is a substitution in which the substituting amino acid (naturally occurring or modified) is structurally related to the amino acid being substituted, i.e. has about the same size and electronic properties as the amino acid being substituted. The substituting amino acid would thus have the same or a similar functional group in the side chain as the original amino acid. A "conservative substitution" also refers to the use of a substituting amino acid that is identical to the amino acid being replaced except that a functional group in the side chain is protected with a suitable protecting group.

As is known in the art, amino acids can be chemically modified from their natural structure by either enzymatic or non-enzymatic reaction mechanisms. For example, in one embodiment, an N-terminal glutamic acid or glutamine can cyclize with loss of water to form pyroglutamic acid (pyroE or pE) (Chelius et al, Anal.Chem, 78: 2370-2376 (2006) and Burstein et al. ., Proc. National Acad. Sci., 73: 2604-2608 (1976)). Alternatively, a fusion protein that has an N-terminal pyroglutamic acid could possibly be obtained by a nucleic acid sequence encoding glutamic acid at the position in the protein that becomes the N-terminus by post-translational processing (e.g. when residue 24 of SEQ ID NO: 1 is glutamate instead of a glutamine).

Methods for producing RAG £ fusion proteins 34

The present invention also includes a method for making a RAGE fusion protein. Thus, in one embodiment, the present invention comprises a method for making a RAGE fusion protein comprising the step of covalently linking a RAGE polypeptide that is linked to a second non-RAGE polypeptide, wherein the RAGE polypeptide comprises a binding site for a RAGE ligand. For example, the linked RAGE polypeptide and the second non-RAGE polypeptide can be encoded by a recombinant DNA construct. The method may further include the step of incorporating the DNA construct into an expression vector. The method may also include the step of inserting the expression vector into a host cell.

For example, embodiments of the present invention provide RAGE fusion proteins that include a RAGE polypeptide linked to a second non-RAGE polypeptide. In one embodiment, the RAGE fusion protein may comprise a binding site for a RAGE ligand. In one embodiment, the ligand binding site comprises the most N-terminal domain of the RAGE fusion protein. The binding site for the RAGE ligand may include the RAGE V domain or part thereof. In one embodiment, the binding site for the ligand of RAGE includes SEQ ID NO: 9 or a sequence that is at least 90% identical to it, or SEQ ID NO: 10 or a sequence that is at least 90% identical to it, or SEQ ID NO: 9 NO: 47, or a sequence that is at least 90% identical to that.

In one 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 comprising an immunoglobulin domain comprises at least a portion of at least one of the Ch2 or CH3 domains of a human IgG.

Thus, embodiments of the present invention can include isolated DNA molecules that encode the RAGE fusion proteins of the present invention. In certain embodiments, the DNA molecules encode a RAGE fusion protein comprising an amino acid sequence as set forth in SEQ ID NO: 56 or SEQ ID NO: 57 or a sequence that is at least 70%, 75%, 80%, 85 %, 90%, 95%, 96%, 97%, 98% or 99% identical to this. For example, in some embodiments, a sequence that is at least 90% identical to SEQ ID NO: 56 or SEQ ID NO: 57 includes the sequence of SEQ ID NO: 56 or SEQ ID NO: 57 without the C-terminal lysine. Thus, in certain embodiments, the present invention may comprise a DNA molecule that has the sequence set forth in SEQ ID NO: 54 or SEQ ID NO: 55 or a sequence that is at least 90% identical thereto.

The RAGE fusion protein can be manipulated by recombinant DNA techniques. For example, in one embodiment, the present invention may include an isolated nucleic acid sequence comprising, complementarity to, or substantial similarity to, a polynucleotide sequence encoding a RAGE polypeptide coupled to a second non-RAGE polypeptide. In one embodiment, the RAGE polypeptide may comprise a binding site for a RAGE ligand.

The RAGE protein or polypeptide may comprise full-length human RAGE (e.g., SEQ ID NO: 1) or a fragment of human RAGE. In one embodiment, the RAGE polypeptide does not include residues of a signal sequence. The signal sequence of RAGE can include either residues 1-22 or residues 1-23 of full-length RAGE (SEQ ID NO: 1). In other embodiments, the RAGE polypeptide may comprise a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% 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 instead of a Methionine (see, for example, Neeper et al., (1992)). Or the human RAGE can include full-length RAGE with the signal sequence removed (e.g., SEQ ID NO: 2 or SEQ ID NO: 3) (Figures 1A and 1B) or include a portion of that amino acid sequence. The RAGE fusion proteins of the present invention may also include sRAGE (e.g., SEQ ID NO: 4), a polypeptide that is at least 90% identical to sRAGE, or a fragment of sRAGE. For example, the RAGE polypeptide can include human sRAGE or a fragment thereof, with Glycine as the first residue instead of a Methionine (see, for example, Neeper et al., (1992)). Or the human RAGE may include sRAGE with the signal sequence removed (See, for example, SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 45 in Figure 1) or a portion of that amino acid sequence. In other embodiments, the RAGE protein may comprise a V domain (see, for example, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 46 in Figure 1). Or a sequence that is at least 90% identical to the 36 V domain or a fragment thereof can be used. Or the RAGE protein may comprise a fragment of RAGE that includes a portion of the V domain (see, for example, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 47 in Figure 1). In one embodiment, the binding site for the ligand can be SEQ ID NO: 9 or a sequence 5 that is at least 90% identical to it, or SEQ TD NO: 10, or a sequence that is at least 90% identical to it, or SEQ ID NO: 9 : 47, or a sequence that is at least 90% identical to that. In yet another embodiment, the RAGE fragment is a synthetic peptide.

In one 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 can be used to encode amino acids 1-116 of human RAGE. Or the nucleic acid may comprise SEQ ID NO: 26 to encode amino acids 1-123 of human RAGE. Or the nucleic acid can comprise SEQ ID NO: 27 to encode 15 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 can be used to encode fragments of the RAGE polypeptide.

The RAGE fusion protein can include various types of peptides that are not derived from RAGE or a fragment thereof. The second polypeptide of the RAGE fusion protein may comprise a polypeptide that is derived from an immunoglobulin. The heavy chain (or part thereof) can come from one of the known heavy chain isotypes: IgG (γ), IgM (μ), IgD (5), IgE (ε) or IgA (a). In addition, the heavy chain (or part thereof) may be from one of the known subtypes of the heavy chain: IgG1 (γΐ), IgG2 (y2), IgG3 (γ3), IgG4 (γ4), IgAl (a1), IgA2 (a2), or mutations of these isotypes or subtypes that alter biological activity. The second polypeptide may comprise the Ch2 and CH3 domains of a human IgG1 or a portion of each or both of these domains. As an example of embodiments, the polypeptide comprising the Ch2 and CH3 domains of a human IgG1 or a part thereof may comprise SEQ ID NO: 38 or SEQ ID NO: 40. In one embodiment, the polypeptide comprising the Ch2 and Cn3 domains of a human IgG1 or a portion thereof may comprise SEQ ID NO: 38 or a fragment thereof.

37

The immunoglobulin peptide can be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 41. In other embodiments, the immunoglobulin sequence in SEQ ID NO: 38 or SEQ ID NO: 40 can also be encoded by SEQ ID NO: 52 or SEQ ID NO: 53.

The hinge region of the Fc portion of the immunoglobulin chain can be anti-inflammatory in vivo. The RAGE fusion protein of the present invention can thus include an interdomain linker that is derived from RAGE instead of an interdomain hinge polypeptide that is from an immunoglobulin.

Thus, in one embodiment, the present invention comprises a method of making a RAGE fusion protein which comprises the step of covalently linking a RAGE polypeptide to a polypeptide comprising an immunoglobulin C ^ domain or a part of an CH2 domain of an immunoglobulin. In one embodiment, the RAGE fusion protein may comprise a binding site for a RAGE ligand. The binding site for the RAGE ligand may comprise the V-15 domain of RAGE or a portion thereof. In one embodiment, the binding site for the ligand of RAGE includes SEQ ID NO: 9 or a sequence that is at least 90% identical to it, or SEQ ID NO: 10 or a sequence that is at least 90% identical to it, or SEQ ID NO: 9 : 47, or a sequence that is at least 90% identical to that.

For example, in one embodiment, the RAGE fusion protein can be encoded by a recombinant DNA construct. The method may also include the step of incorporating the DNA construct into an expression vector. The method may also include transfecting the expression vector into a host cell. Thus, embodiments of the present invention also include expression vectors encoding DNA molecules that encode the RAGE fusion proteins of the present invention. In certain embodiments, the DNA molecules encode a RAGE fusion protein comprising an amino acid sequence as set forth in SEQ ID NO: 56 or SEQ ID NO: 57 or a sequence that is at least 70%, 75%, 80%, 85% , 90%, 95%, 96%, 97%, 98% or 99% are identical to this. In some embodiments, a sequence that is at least 90% identical to SEQ ID NO: 56 or SEQ ID NO: 57 includes, for example, the sequence of SEQ ID NO: 56 or SEQ ID NO: 57 without the C-terminal lysine.

38

Still other embodiments of the present invention also include cells transfected with an expression vector encoding DNA molecules encoding the RAGE fusion proteins of the present invention. In certain embodiments, the DNA molecules encode a RAGE fusion protein comprising an amino acid sequence as set forth in SEQ TD NO: 56 or SEQ ID NO: 57 or a sequence that is at least 70%, 75%, 80%, 85 %, 90%, 95%, 96%, 97%, 98% or 99% identical to this. For example, in some embodiments, a sequence that is at least 90% identical to SEQ ID NO: 56 or SEQ ID NO: 57 includes the sequence of SEQ ID NO: 56 or SEQ ID NO: 57 without the C-10 terminal lysine.

For example, in one embodiment, the present invention comprises a nucleic acid that encodes a RAGE polypeptide that is directly linked to a polypeptide that comprises a C ^ domain of an immunoglobulin or a fragment thereof. In one embodiment, the Ch2 domain or a fragment thereof comprises SEQ ID NO: 42. In one embodiment, the fragment of SEQ ID NO: 42 comprises SEQ ID NO: 42 from which the first 10 amino acids have been removed. The second polypeptide can include the Ch2 and CH3 domains of a human IgG1. As an exemplary embodiment, the polypeptide comprising the Ch2 and CH3 domains of a human IgG1 may include SEQ ID NO: 38 or SEQ ID NO: 40. In one embodiment, the polypeptide comprising the Ch2 and CH3 domains of a human IgG1 or a portion thereof may comprise SEQ ID NO: 38 or a fragment thereof. The immunoglobulin peptide can be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 41. The immunoglobulin sequence in SEQ ID NO: 38 or SEQ ID NO: 40 can also be encoded by SEQ ID NO: 52 or SEQ ID NO: 53, wherein silent base changes for the codons encoding proline (CCG

to CCC) and glycine (GGT to GGG) remove a cryptic RNA cleavage site near the terminal codon at the C-terminus of the sequence (ie nucleotides 622-627 of SEQ ID NO: 39 are modified to generate SEQ ID NO: 52 or nucleotides 652-657 of SEQ ID NO: 41 are modified to generate SEQ ID NO: 53).

In one embodiment, the RAGE polypeptide may include an RAGE interdomain linker linked to the RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-39 terminal amino acid of the linker interdomain 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 Cn2 domain of an immunoglobulin domain, or a fragment thereof. The polypeptide comprising an CH 2 domain of an immunoglobulin or a part thereof may comprise a polypeptide comprising the CH 2 and C 1 domains of a human IgG 1 or a part of both or of each of these domains. As an exemplary embodiment, the polypeptide comprising the Ch2 and CH3 domains of a human IgG1 or a part thereof may comprise SEQ ID NO: 38 or SEQ ID NO: 40. In one embodiment, the polypeptide comprising the Ch2 and CH3 domains of a human IgG1 or a portion thereof may comprise SEQ ID NO: 38 or a fragment thereof. In certain embodiments, 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 with the C-terminal lysine (K) removed.

The RAGE fusion protein of the present invention can be a single or multiple

RAGE domains. The RAGE polypeptide comprising an inter-domain linker linked to an RAGE immunoglobulin domain may also comprise a fragment of a full-length RAGE protein. For example, in one embodiment, the RAGE fusion protein may comprise two immunoglobulin domains derived from a RAGE protein and two immunoglobulin domains derived from a human Fc polypeptide. The RAGE fusion protein may comprise a first immunoglobulin domain from RAGE and a first interdomain linker linked to a second immunoglobulin domain from RAGE and a second interdomain linker from RAGE such that the N-terminal amino acid of the first interdomain 25 is linked to the C-terminal amino acid of the first immunoglobulin domain of RAGE, the N-terminal amino acid of the second immunoglobulin domain of RAGE is linked to the C-terminal amino acid of the first interdomain linker, the N-terminal amino acid of the second interdomain linker is linked to the C-terminal amino acid of the second immunoglobulin domain 30 of RAGE and the C-terminal amino acid of the second interdomain linker of RAGE is directly linked to the N-terminal amino acid of the polypeptide which is a CH2 immunoglobulin domain or fragment thereof. The RAGE polypeptide can, for example, amino acids 23-251 of human RAGE (SEQ ID

40 NO: 19) or a sequence that is at least 90% identical to it, or amino acids 24-251 of human RAGE (SEQ ID NO: 20) or a sequence that is at least 90% identical to it, or amino acids 24-251 of human RAGE where Q24 cyclizes to form pE (SEQ ID NO: 51) or a sequence that is at least 90% identical to 5, corresponding to the V domain, the Cl domain, the interdomain linker that links these two domains and a second cross-domain left downstream of C1. In one embodiment, a nucleic acid construct comprising SEQ ID NO: 30 or a fragment thereof may encode a four-domain RAGE fusion protein (Figure 2A). In another embodiment, a nucleic acid construct comprising SEQ ID 10 NO: 54 (Figure 2B) can encode a four domain RAGE fusion protein, with silent base changes for the codons encoding a pro line (CCG to CCC) and glycine (GGT to GGG) at the C-terminus of the sequence are made to remove a cryptic RNA cleavage site near the terminal codon (ie nucleotides 1375-1380 of SEQ ID NO: 30 are modified to generate SEQ ID NO : 54).

Alternatively, a three-domain RAGE fusion protein may comprise one immunoglobulin domain derived from RAGE and two immunoglobulin domains derived from a human Fc polypeptide. For example, the RAGE fusion protein may comprise a single immunoglobulin domain from RAGE that is linked through an inter-domain linker of RAGE to the N-terminal amino acid of the polypeptide comprising a CH2 immunoglobulin domain or a fragment thereof. The RAGE polypeptide can, for example, amino acids 23-136 of human RAGE (SEQ ID NO: 15) or a sequence that is at least 90% identical to it or amino acids 24-136 of human RAGE (SEQ ID NO: 16) or a sequence that is at least 90% identical to that, or amino acids 24-136 of human RAGE where Q24 cyclizes to form pE (SEQ ID NO: 49) or a sequence that is at least 90% identical to that, corresponding to the V- RAGE domain and a downstream interdomain linker (Figure 1). In one embodiment, a nucleic acid construct comprising SEQ ID NO: 31 or a fragment 30 thereof may encode a three-domain RAGE fusion protein (Figure 3A). In another embodiment, the nucleic acid construct comprising SEQ ID NO: 55 may encode a three domain RAGE fusion protein, silent base changes for the codons encoding proline (CCG to CCC) and glycine 41 (GGT to GGG) the C-terminus of the sequence removing a cryptic RNA cleavage site near the terminal codon (ie nucleotides 1030-1035 of SEQ ID NO: 31 are modified to generate SEQ ID NO: 55) (Figure 3B).

A fragment of the RAGE interdomain linker may include a peptide sequence that is naturally downstream from and thus linked to an RAGE immunoglobulin domain. For the V domain of RAGE, the interdomain linker may include, for example, amino acid sequences that are naturally downstream of the V domain. In one embodiment, the left SEQ ID 10 may include NO: 21 corresponding to amino acids 117-123 of full-length RAGE. Or the linker may include a peptide that has additional portions of the natural sequence of RAGE. For example, an 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 can be used. Thus, in one embodiment, the interdomain linker comprises SEQ ID NO: 23 which comprises amino acids 117-136 of full-length RAGE. Or fragments of SEQ ID NO: 21 wherein, for example, 1, 2, or 3 amino acids have been removed from each end of the linker, can be used. In other embodiments, the linker may comprise a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21 or SEQ ID NO: 23.

For the RAGE C1 domain, the linker may include a peptide sequence that is naturally downstream of the C1 domain. In one embodiment, the linker can include SEQ ID NO: 22 corresponding to amino acids 222-251 of full-length RAGE. Or the linker may include a peptide that has additional portions of the natural sequence of RAGE. For example, a linker that has several (1-3, 1-5, or Ι-ΙΟ, or 1-15 amino acids) amino acids upstream and downstream of SEQ ID NO: 22 can be used. Or fragments of SEQ ID NO: 22 can be used with, for example, 1-3, 1-5, or 1-10, or 1-15 amino acids removed from one of the ends of the linker. In one embodiment, an inter-domain linker from RAGE may include, for example, SEQ ID NO: 24 corresponding to amino acids 222-226. In other embodiments, the linker may comprise a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 22 or SEQ ID NO: 24.

42

Or an interdomain linker may include SEQ ID NO: 44 corresponding to amino acids 318-342 of RAGE. In other embodiments, the linker may comprise a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 44.

The method may further comprise the step of incorporating the DNA construct into an expression vector. Thus, in one embodiment, the present invention comprises an expression vector encoding a RAGE fusion protein comprising a RAGE polypeptide that is directly linked to a polypeptide comprising an CH2 domain of an immunoglobulin or a portion of a Cn2 domain of a immunoglobulin. In one embodiment, the RAGE polypeptide comprises constructs such as those described herein that have an RAGE inter-domain linker linked to an RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is N-terminal amino acid of the linker cross-domain and the C-terminal amino acid of the linker cross-domain of RAGE is directly linked to the N-terminal amino acid of a polypeptide comprising a C ^ domain of an immunoglobulin or a portion thereof. For example, the expression vector used to transfect the cells may include the nucleic acid sequence SEQ ID NO: 30 or a fragment thereof, SEQ ID NO: 54, or a fragment thereof, SEQ ID NO: 31, or a fragment thereof, or SEQ ID NO: 55, 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 one embodiment, the present invention comprises a cell transfected with the expression vector expressing the RAGE fusion protein of the invention, such that the cell expresses a RAGE fusion protein comprising a RAGE polypeptide which is directly linked to a polypeptide comprising an Ch2 domain of an immunoglobulin or a portion of a CH2 domain of an immunoglobulin. In one embodiment, the RAGE polypeptide comprises constructs, such as those described herein, that have an inter-domain linker of RAGE linked to an immunoglobulin domain of RAGE, such that the C-terminal amino acid of the immunoglobulin domain of RAGE is linked the N-terminal amino acid of the linker interdomain and the C-terminal amino acid of the 43 interdomain linker of RAGE is directly linked to the N-terminal amino acid of a polypeptide which is a C ^ domain of an immunoglobulin or a part thereof includes. For example, the expression vector may be the nucleic acid sequence SEQ ID NO: 30 or a fragment thereof, SEQ ID NO: 54, or a fragment thereof, SEQ ID NO: 31, or a fragment thereof, or SEQ ID NO: 55, or a fragment thereof include.

For example, plasmids can be constructed to express RAGE-IgG fusion proteins by fusing different lengths of a human RAGE 5 'cDNA sequence with a human IgG1 (γΐ) 3' cDNA sequence. The sequences of the expression cassette can be inserted into an expression vector such as expression vector pcDNA3.1 (Invitrogen, CA) using standard recombinant techniques.

The method may also include transfecting the expression vector into a host cell. RAGE fusion proteins can be expressed in mammalian expression systems including systems in which the expression constructs are introduced into the mammalian cells by the use of a virus such as retrovirus or adenovirus. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include Chinese hamster ovary (CHO), NSO, SP2 cells, Hela cells, baby hamster kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines can be selected by determining which cell lines have high expression levels of a RAGE fusion protein. Other cell lines that can be used are insect cell lines such as Sf9 cells. For example, plant host cells include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, and so on. Bacterial host cells include species of E. coli and Streptomyces. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris. When recombinant expression vectors encoding RAGE fusion protein genes 30 are introduced into mammalian host cells, the RAGE fusion proteins are produced by culturing the host cells for a period of time sufficient to express RAGE fusion protein in to allow the host cells or secretion of the RAGE fusion protein in the 44 culture medium in which the host cells are grown. RAGE fusion proteins can be recovered from the culture medium using standard protein purification methods.

Nucleic acid molecules encoding RAGE fusion proteins and expression vectors comprising these nucleic acid molecules can be used for transfection of a suitable host cell derived from mammals, plants, bacteria or yeasts. Transformation can be by any known method of introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes and direct micro injection of the DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods for transforming plant cells are well known in the art including, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial cells and yeast cells are also well known in the art.

An expression vector can also be delivered to an expression system using biolostic techniques with DNA, wherein the plasmid is precipitated on microscopic particles, preferably of gold, and the particles are shot into a target cell or expression system. Biolistic techniques with DNA are well known in the art and devices, for example a "gene gun" are commercially available for delivering the microparticles into a cell (e.g. Helios Gene Gun, Bio-Rad Labs., Hercules, CA) and in the skin (PMED Device, PowderMed Ltd., Oxford, UK).

Expression of RAGE fusion proteins from production cell lines can be increased by the use of a number of known techniques. For example, the expression system with the glutamine synthetase gene (the GS system) and the plasma-encoded neomycin resistance system are general approaches for increasing expression under certain conditions.

RAGE fusion proteins expressed by different cell lines may have glycosylation patterns that differ from each other. All RAGE fusion proteins encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the RAGE fusion protein.

In one embodiment, a recombinant expression vector can be transfected into Chinese hamster ovary cells (CHO) and expression optimized. In other 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 known in the art, such nucleic acid constructs can be modified by mutation, such as, for example, by PCR amplification of a nucleic acid template with primers comprising the mutation of interest. In this way, polypeptides that include varying affinity for ligands of RAGE can be designed. In one embodiment, the mutated sequences may be 90% or more identical to the starting DNA. As such, 15 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 can be expressed by transfecting the expression vector into a suitable host. For example, the recombinant vectors can be stably transfected into Chinese hamster ovary (CHO) cells and cells expressing the RAGE fusion protein are selected and cloned. In one embodiment, the cells expressing the recombinant construct are selected for plasmid-encoded neomycin resistance by adding the antibiotic G418. Individual clones can be selected and clones expressing high levels of recombinant protein, as detected by Westem blot analysis of the supernatant of the cells, can be expanded and the gene product can be purified by affinity chromatography using Protein A columns.

Examples of embodiments of recombinant nucleic acids encoding the RAGE fusion proteins of the present invention are shown in Figures 2 and 3. As described, for example, above, the RAGE fusion protein produced by the recombinant DNA construct can be a RAGE polypeptide linked to a second non-RAGE polypeptide. The RAGE fusion protein can comprise two 46 domains derived from RAGE protein and two domains derived from an immunoglobulin. As an example of nucleic acid constructs encoding a RAGE fusion protein, TTP-4000 (TT4) having this type of structure is shown in Figure 2 (SEQ ID NO: 30 and SEQ ID NO: 54). As shown for SEQ ID 5 NO: 30 and SEQ TD NO: 54, the coding sequence 1-753 (marked in bold) encodes the N-terminal protein sequence of RAGE while the sequence of 754-1386 codes for the Fc protein sequence of Encodes IgG without the hinge.

When it is obtained from SEQ ID NO: 30 or SEQ ID NO: 54 or a sequence that is at least 90% identical thereto, the RAGE fusion protein may be the amino acid sequence of SEQ ID NO: 32 with four domains or the polypeptide wherein the signal sequence is deleted include (see, for example, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 56 in Figure 4). In SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 56, the amino acid sequence for RAGE is marked with 15 characters in bold. The immunoglobulin sequence are the Ch2 and Ch3 immunoglobulin domains of IgG without the hinge region.

Figure 6 shows a comparison of the polypeptide domains found in RAGE and IgG (Figure 6A) and the domain structure of the RAGE fusion proteins TTP-3000 and TTP-4000. As shown in Figure 6B, the first 251 amino acids of the full-length RAGE fusion protein TTP-4000 contain 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 inter-domain linker comprising amino acids 117 to 123, a second immunoglobulin domain (C1), comprising amino acids 124-221 and a downstream inter-domain linker comprising amino acids 222-251 includes.

In one embodiment, the RAGE fusion protein need not necessarily include the second RAGE immunoglobulin domain. For example, the RAGE fusion protein may comprise one immunoglobulin domain that is derived from RAGE and two immunoglobulin domains that are derived from a human Fc polypeptide. An example of nucleic acid constructs encoding this type of RAGE fusion protein is shown in Figure 3 (SEQ ID NO: 31 and SEQ ID NO: 55). As shown in SEQ ID NO: 31 and SEQ ID NO: 55, the coding sequence of nucleotides 1 to 408 (marked in bold) encodes the N-terminal 47 protein sequence of RAGE, while the sequence of 409-1041 is for the protein sequence of IgG1 (γΐ).

When it is obtained from SEQ ID NO: 31 or SEQ ID NO: 55, or a sequence that is at least 90% identical thereto, the RAGE fusion protein may be the amino acid sequence of three domain SEQ TD NO: 35 or the polypeptide from which the signal sequence has been deleted include (see, for example, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 57 in Figure 5). In SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 57 in Figure 5, the amino acid sequence of RAGE is marked with a bold font. As shown in Figure 6B, the first 136 amino acids of the full-length RAGE fusion protein TTP-3000 contain as the RAGE polypeptide a signal sequence comprising amino acids 1-22 / 23, the V immunoglobulin domain (including the ligand-binding site) which comprises amino acids 23 / 24-116 and an inter-domain linker that comprises amino acids 117 to 136. The sequence of 137-346 includes the Ch2 and CH3 immunoglobulin domains of IgG without the hinge region.

The RAGE fusion proteins of the present invention may include improved in vivo stability over RAGE polypeptides that do not include a second polypeptide. The RAGE fusion protein can be further modified to increase stability, efficacy, potency and bioavailability. The RAGE fusion proteins of the present invention can thus be modified by post-translational processing or by chemical modification. For example, the RAGE fusion protein can be prepared synthetically to include L, D or non-natural amino acids, alpha-disubstituted amino acids or N-alkyl amino acids. In addition, proteins can be modified by acetylation, acylation, ADP ribosylation, amidation, connection of lipids such as phosphatidyl inositol, formation of disulfide bonds and the like. Polyethylene glycol can additionally be added to enhance the biological stability of the RAGE fusion protein.

Binding of RAGE antagonists to RAGE fusion proteins

The RAGE fusion proteins of the present invention can include a number of applications. For example, the RAGE fusion protein of the present invention can be used in a binding test to identify ligands for RAGE, such as agonists, antagonists or modulators of RAGE.

For example, in one embodiment, the present invention provides a method for detecting modulators of RAGE, which comprises (a) providing a RAGE fusion protein comprising a RAGE polypeptide linked to a second non-RAGE polypeptide, wherein the RAGE polypeptide comprises a ligand binding site; (b) mixing a compound of interest and a ligand that has a known binding affinity for RAGE with the RAGE 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 one embodiment, the ligand binding site comprises the most N-terminal domain of the RAGE iusion protein.

The RAGE fusion proteins can 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 that has a known binding affinity for 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 binding site for a RAGE ligand; and (c) instructions for use. In one embodiment, the ligand binding site comprises the most N-terminal domain of the RAGE fusion protein.

For example, the RAGE fusion protein can be used in a binding test to identify potential ligands of RAGE. In one example of an embodiment of such a binding assay, a known RAGE ligand can be coated on a solid substrate (e.g., Maxisorb plates) at a concentration of about 5 micrograms per well, each well having a total volume of about 25 100 microliters (μΐ ). The plates can be incubated overnight at 4 ° C to allow the ligand to absorb or bind to the substrate. Alternatively, shorter incubation periods at higher temperatures (e.g., room temperature) can be used. After a period of time to allow the ligand to bind to the substrate, the test wells can be aspirated and a blocking buffer (e.g., 1% BSA in 50 mM imidizole buffer, pH 7.2) can be added to allow non-specific block binding. For example, the blocking buffer can be added to the plates for 1 hour at room temperature. The plates can then be suctioned off 49 and / or washed with a wash buffer. In one embodiment, a buffer containing 20 mM imidazole, 150 mM NaCl, 0.05% Tween-20, 5 mM CaCE and 5 mM MgCb, pH 7.2 can be used as a wash buffer. The RAGE fusion protein can then be added to the test wells with increasing dilutions. The RAGE fusion protein can then be allowed to incubate with the immobilized ligand in the test well, such that binding can reach equilibrium. In one embodiment, the RAGE fusion protein is allowed to incubate with the immobilized ligand for about one hour at 37 ° C. In other embodiments, longer incubation periods at lower temperatures can be used. After the RAGE fusion protein and the immobilized ligand have been incubated, the plate can be washed to remove any unbound RAGE fusion protein. The RAGE fusion protein bound to the immobilized ligand can be detected in a variety of ways. In one embodiment, the detection uses ELISA. In one embodiment, an immune detection complex containing a mouse monoclonal anti-human IgG1, biotinylated goat anti-mouse IgG and an avidin-linked alkaline phosphatase can be added to the RAGE fusion protein immobilized in the test well. The immune detection complex may be allowed to bind to the immobilized RAGE fusion protein such that the binding between the RAGE fusion protein and the immune detection complex 20 reaches equilibrium. For example, the complex may be allowed to bind to the RAGE fusion protein for one hour at room temperature. At that point, any unbound complex can be removed by washing the test well with wash buffer. The bound complex can be detected by adding the substrate for alkaline phosphatase, α-ara-nitrophenyl phosphate (PNPP) and measuring the conversion of PNPP to para-nitrophenol (PNP) as an increase in absorbance at 405 nm.

In one embodiment, the RAGE ligand binds to the RAGE fusion protein with an affinity in the nanomolar (nM) or micromolar (μΜ) range. An experiment illustrating binding of RAGE ligands to RAGE fusion proteins of the present invention is shown in Figure 7. Solutions of TTP-3000 (TT3) and TTP-4000 (TT4) having initial concentrations of 1.082 mg / ml and 370 pg / ml, respectively, were prepared. As shown in Figure 7, the RAGE fusion proteins TTP-3000 and TTP-4000 are capable, at various dilutions, of immobilized 50 RAGE ligands Amyloid Beta (Abeta) (Amyloid Beta (1-40) from Biosource), To bind SlOOb (SI00) and amphoterine (Ampho), which results in an increase in absorption. In the absence of ligand (i.e. coating with BSA only) there was no increase in absorption.

The binding test of the present invention can be used to quantify ligand binding to RAGE. In other embodiments, RAGE ligands can bind to the RAGE 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 RAGE fusion protein of the present invention can also be used to identify compounds that have the ability to bind to RAGE. As shown in Figures 8 and 9, respectively, a RAGE ligand can be tested for its ability to compete with immobilized amyloid beta to bind to RAGE fusion proteins TTP-4000 (TT4) or TTP-3000 (TT3) . It can thus be seen that a RAGE ligand at a final test concentration (FAC) of 10 μΜ binding of RAGE fusion protein to amyloid beta at concentrations of 1: 3, 1:10, 1:30 and 1: 100 of can displace the initial TTP-4000 solution (Figure 8) or TTP-3000 (Figure 9).

Modulation of cellular effectors

Embodiments of the RAGE fusion proteins of the present invention can be used to modulate a biological response mediated by RAGE. For example, the RAGE fusion proteins can be designed to modulate RAGE-induced increase in gene expression. Thus, in one embodiment, RAGE fusion proteins of the present invention can be used to modulate the function of biological enzymes. For example, the interaction between RAGE and its ligands can generate oxidative stress and activation of NF-κΒ and NF-icB-regulated genes, such as the cytokines IL-Ιβ, TNF-α and the like. In addition, it has been shown that several other regulatory routes, such as those involving p21ras, MAP kinases, ERK1 and ERK2, are activated by binding of AGEs and other ligands to RAGE.

The use of the RAGE fusion proteins of the present invention to modulate the expression of the TNF-α cellular effector is shown in Figure 10.

51 THP-1 myeloid cells can be grown in RPMM640 medium supplemented with 10% FBS and induced to secrete TNF-α by stimulation of RAGE with SI00b. When such stimulation takes place in the presence of a RAGE fusion protein, induction of TNF-α 5 by binding of SI00b to RAGE can be inhibited. Thus, as shown in Figure 10, the addition of 10 µg of RAGE fusion protein TTP-3000 (TT3) or TTP-4000 (TT4) reduces induction of TNF-α by SlOOb by about 50% to 75%. RAGE fusion protein TTP-4000 can be at least as effective in blocking induction of TNF-α by SlOOb as sRAGE (Figure 10). Specificity of the inhibition for the RAGE sequences of TTP-4000 and TTP-3000 has been shown by the experiment in which only IgG was added to SlOOb-stimulated cells. Addition of IgG and SlOOb to the test shows the same levels of TNF-α as SlOOb alone.

In another cell-based assay, the ability of TTP-4000 to prevent the RAGE ligand HMGB1 from interacting with RAGE and other HMGB1 receptors was assessed. Unlike anti-RAGE antibodies that bind to RAGE and prevent the interaction of a RAGE ligand with RAGE, TTP-4000 can block the interaction of a RAGE ligand with RAGE by binding to the RAGE ligand. HMGB1 has been reported to be a ligand for RAGE and Toll-like receptors 2 and 4 (Park et al., J. Biol. Chem., 2004; 279 (9): 7370-7). These three receptors (RAGE, Toll-like receptor 2 and Toll-like receptor 4) are all expressed on THP-1 cells (Parker et al., J. Immunol., 2004, 172 (8): 4977- 86).

In this experiment, THP-1 cells were stimulated to produce TNF-α by HMGB1 (50 mg / ml) in the presence or absence of either TTP4000 or anti-RAGE antibodies. Under the conditions used in the test, HMGB1 should be the only inducer of TNF-α. The results in Figure 11 show that the anti-RAGE antibody and RAGE fusion protein blocks TTP-4000 HMGB1 from interacting with RAGE expressed on the THP-1 cells and that TTP-4000 HMGB 1-induced production of TNF -α inhibits to a greater extent than the anti-RAGE antibody. The data thus indicates that TTP-4000 can inhibit the activity of HMGB1 to a greater extent than the anti-RAGE antibody by inhibiting HMGB1 from interacting with Toll-like receptors 2 and 4 as well as RAGE present on THP-1 cells .

52

Physiological characteristics of RAGE fusion proteins

While sRAGE may have a therapeutic benefit in the modulation of RAGE-mediated diseases, human sRAGE may have limitations as an independent therapeutic agent based on the relatively short half-life of sRAGE in plasma. For example, while rodent sRAGE has a half-life of approximately 20 hours in normal and diabetic rats, human sRAGE has a half-life of less than 2 hours when determined by retention of sRAGE immunoreactivity (Renard et al., J. Pharmacol. Exp. 10 Ther., 290: 1458-1466 (1999)).

To generate a therapeutic agent from RAGE that has similar binding characteristics to sRAGE but has a more stable pharmacokinetic profile, a RAGE fusion protein that has a binding site for a RAGE ligand linked to one or more human immunoglobulin domains be used. As is known in the art, the immunoglobulin domains may comprise the Fc portion of the immunoglobulin heavy chain.

The Fc portion of the immunoglobulin can confer various properties to a RAGE fusion protein. For example, the Fc fusion protein can extend 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 the Ch2 and CH3 regions of the Fc fragment with the FcRn receptor (Wines et al., J. Immunol., 164: 5313- 5318 (2000)).

Although fusion proteins comprising an immunoglobulin Fc polypeptide may provide the benefit of increased stability, immunoglobulin fusion proteins may elicit an inflammatory response when introduced into a host. The inflammatory response can to a large extent be due to the Fc portion of the immunoglobulin of the fusion protein. The inflammatory response may be a desirable feature if the target is expressed on a cell type that is sick to be destroyed (e.g., a cancer cell or a population of lymphocytes causing an autoimmune disease). The inflammatory response can be a neutral trait if the target is a soluble protein, since most soluble proteins do not activate 53 immunoglobulins. However, the inflammatory response may be a negative feature if the target is expressed on cell types whose destruction would lead to undesirable side effects. The inflammatory response can also be a negative feature if an inflammatory cascade arises at the site of a fusion protein binding to a target tissue because many inflammation mediators may be harmful to the surrounding tissue and / or cause systemic effects.

The primary anti-inflammatory site on immunoglobulin Fc fragments is present in the hinge region between Ch1 and Ch2. This hinge region 10 interacts with the FcR1-3 on various leukocytes and causes these cells to attack the target. (Wines et al., J. Immunol, 164: 5313-5318 (2000)).

As therapeutic agents for RAGE-mediated diseases, RAGE fusion proteins may not require the generation of an inflammatory response. Thus, embodiments of RAGE fusion proteins of the present invention may include a RAGE fusion protein comprising a RAGE polypeptide linked to an immunoglobulin domain (s), wherein the Fc hinge region of the immunoglobulin has been removed and is replaced with a RAGE polypeptide. In this way, the interaction between the RAGE iusion protein and Fc receptors on inflammatory cells can be minimized. However, it may be important to maintain proper stacking and other three-dimensional structural interactions between the various immunoglobulin domains of the RAGE fusion protein. Thus, embodiments of the RAGE fusion proteins of the present invention may be the biologically inert, but structurally similar, inter-domain linker of RAGE that separates the V and C1 domains from RAGE or the linker that cleaves the C1 and C2 domains of RAGE. separates substitute the normal hinge region from the heavy chain of the immunoglobulin. The RAGE polypeptide of the RAGE iusion protein can thus include a sequence of an inter-domain linker that is naturally found downstream of an RAGE immunoglobulin domain to form an RAGE immunoglobulin domain / linker fragment. In this way, the three-dimensional interactions between the immunoglobulin domains contributed by either RAGE or the immunoglobulin can be maintained.

In one embodiment, a RAGE fusion protein of the present invention can have a significant increase in pharmacokinetic stability compared to sRAGE

54. For example, Figure 12 shows that if the RAGE fusion protein TTP-4000 is saturated with its ligands, it can reach a half-life of more than 300 hours. This can be compared to the half-life of sRAGE of just a few hours in human plasma.

Thus, in one embodiment, the RAGE fusion proteins of the present invention can be used to counteract the binding of physiological ligands to RAGE as a means of treating RAGE-mediated diseases without generating an unacceptable extent of inflammation. The RAGE fusion proteins of the present invention can exhibit a substantial decrease in the generation of an inflammatory response compared to IgG. For example, as shown in Figure 13, the RAGE fusion protein TTP-4000 does not stimulate release of TNF-α from cells under conditions where stimulation of release of TNF-α by human IgG is detected.

15

Treatment of disease with RAGE fusion proteins

The present invention may also include methods for the treatment of RAGE-mediated disorder in a human patient. In one embodiment, the method may comprise administering to a patient a RAGE fusion protein comprising a RAGE polypeptide comprising a binding site for a ligand of RAGE linked to a second non-RAGE polypeptide.

In certain embodiments, the RAGE preparation comprises a freeze-dried RAGE fusion protein. In certain embodiments, the present invention may include methods for treating a RAGE-mediated disorder in a patient, which comprises administering to a person a therapeutically effective amount of a reconstituted composition comprising a RAGE fusion protein, a protective agent for freeze-drying and a buffer.

Any of the embodiments of the RAGE fusion proteins described herein can be used to treat disease in the therapeutic compositions and compositions of the present invention. The RAGE fusion protein can thus comprise a sequence derived from a binding site for a ligand of RAGE that is linked to an immunoglobulin polypeptide. Embodiments of the RAGE fusion protein may include a RAGE polypeptide 55 that is directly linked to a polypeptide comprising an immunoglobulin Cn2 domain or a portion of an immunoglobulin Cn2 domain. In certain embodiments, the RAGE polypeptide may include an RAGE interdomain linker linked to an 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 linker interdomain and the C- RAGE terminal amino acid is directly linked to the N-terminal amino acid of a polypeptide comprising an immunoglobulin C1 domain, or part thereof. For example, certain embodiments of the fusion protein may comprise a first immunoglobulin domain from RAGE and a first interdomain linker from RAGE linked to a second immunoglobulin domain from RAGE and a second interdomain linker from RAGE, such that the N-terminal amino acid of the first interdomain linker is linked to the C-terminal amino acid of the first immunoglobulin domain of RAGE, the N-terminal amino acid of the second immunoglobulin domain of RAGE is linked to the C-terminal amino acid of the first interdomain linker, the N-terminal amino acid of the second interdomain linker is linked to the C-terminal amino acid of the second immunoglobulin domain of RAGE and the C-terminal amino acid of the second interdomain linker of RAGE is directly linked to the N-terminal amino acid of the Ch2 immunoglobulin domain or part of a CH2 domain of an immunoglobulin.

In other embodiments of this multi-domain fusion protein, the RAGE polypeptide may comprise the amino acid sequence as set forth in SEQ ID NO: 10 or a sequence that is at least 90% identical thereto or the amino acid sequence as set forth in SEQ ID NO. : 47 or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to it. In other alternative embodiments, the RAGE fusion protein may comprise the amino acid sequence as set forth in at least one of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 56 or 57 or a sequence that is at least 70%, 75% 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% is identical to this. In certain embodiments, a sequence that is at least 90% identical to SEQ ID NOs: 32, 33, 34, 56, 35, 36, 37 or 57 includes, for example, the polypeptide of SEQ ID NOs: 32, 33, 34, 56, 35, 36, 37 or 57 without the C-terminal lysine. Or other embodiments as described herein 56 may include the RAGE fusion protein used in the compositions of the present invention.

A variety of freeze-drying protective agents can be used in the freeze-dried RAGE fusion protein preparation. In some embodiments, the freeze drying protective agent may comprise a non-reducing sugar. The non-reducing sugar can include, for example, sucrose, mannitol or trehalose. A variety of buffers can also be used in the freeze-dried RAGE fusion protein preparation. In certain embodiments, the buffer may include histidine.

The freeze-dried RAGE fusion protein may comprise additional ingredients.

In certain embodiments, the RAGE fusion protein composition may further comprise at least one of a surfactant, a chelating agent, or a volume-forming agent. In one embodiment, the preparation of the reconstructed RAGE fusion protein comprises about 40-100 mg / ml RAGE fusion protein comprising the sequence set forth in SEQ ID NOs: 32, 33, 34, 56, 35, 36 , 37, or 57; about 2 mM to about 50 mM histidine; about 60 mM to about 65 mM sucrose; about 0.001% to about 0.05% Tween 80; and a pH of about 6.0 to 6.5. The composition of the reconstituted RAGE fusion protein may, for example, in some embodiments be about 40-50 mg / ml RAGE fusion protein comprising the sequence set forth in SEQ ID NOs: 32, 33, 34, 56, 35, 36, 37 or 57; about 10 mM histidine, about 65 mM sucrose, about 0.01% Tween 80 and at a pH of about 6.0. Or other concentrations of the RAGE fusion protein can be used in the compositions for treatment of RAGE-mediated disorders as needed.

The RAGE fusion protein preparation may comprise a stable therapeutic agent prepared for use in a hospital or as a prescribed drug. For example, in certain embodiments, the RAGE fusion protein preparation may exhibit less than 10% or less than 5% or less than 3% degradation after one week at 40 degrees Celsius.

The preparation of the RAGE fusion protein can also be stable upon reconstitution into a diluent. In certain embodiments, less than about 10%, 5%, 4%, 3%, 2%, or 1% of the RAGE fusion protein is present as an aggregate in the preparation of the RAGE fusion protein.

57

The preparation of the reconstituted RAGE fusion protein may be suitable for administration by various routes and is necessary for the treatment of the RAGE-mediated disorder of interest. Administration of the RAGE fusion protein of the present invention can use intraperitoneal (IP) injection. Alternatively, the RAGE fusion protein can be administered orally, intranasally or as an aerosol. In another embodiment, administration is intravenous (IV). The RAGE fusion protein can also be injected subcutaneously. In another embodiment, administration of the RAGE fusion protein is intra-arterial. In another embodiment, administration is sublingual. Administration can also use a capsule with release in time. In yet another embodiment, administration may be transrectal, such as by a suppository or the like. Subcutaneous administration may be useful, for example, for treating chronic disorders when self-administration is desired.

As described in more detail herein, RAGE is involved in the pathogenesis of a variety of disease states and it has been found that the RAGE fusion proteins of the present invention are effective in improving such disease states. Thus, compositions of the RAGE fusion protein of the present invention can be used to treat a variety of RAGE-mediated disorders.

In certain embodiments, a composition of a reconstituted RAGE fusion protein of the present invention can be used to treat a symptom of diabetes or a symptom of late complications of diabetes. The symptom of diabetes or late complications of diabetes include, for example, at least one of diabetic nephropathy, diabetic retinopathy, a diabetic foot ulcer, a cardiovascular complication, or diabetic neuropathy.

In other embodiments, a composition of a reconstituted RAGE fusion protein of the present invention can be used to treat at least one of amyloidosis, Alzheimer's disease, cancer, renal failure or inflammation associated with autoimmunity, inflammatory bowel disease, rheumatoid arthritis, psoriasis, multiple sclerosis, hypoxia, stroke, heart attack, hemorrhagic shock, sepsis, organ transplantation or weakened wound healing.

Or, the preparation of the reconstituted RAGE fusion protein can be used to treat osteoporosis. For example, in certain embodiments 58, administration of a preparation of the RAGE fusion protein of the present invention increases the bone density of the subject or lowers the rate of a decrease in the bone density of a subject.

In some embodiments, the autoimmunity treated by the use of the RAGE fusion protein compositions of the present invention may rejection of at least one of skin cells, pancreatic cells, nerve cells, muscle cells, endothelial cells, heart cells, liver cells, kidney cells, a heart, bone marrow cells, bone, blood cells, arterial cells, venous cells, cartilage cells, thyroid cells or stem cells. Or the reconstituted RAGE fusion protein preparation can be used to treat renal failure.

In certain embodiments, the reconstituted RAGE fusion protein preparation can be used to treat inflammation and / or rejection associated with transplantation of at least one of an organ, a tissue or a variety of cells from a first site to a second place. The first 15 and second places can either be with different people or with the same person. Transplantation of a variety of different cell types can be improved by the use of preparations of the RAGE fusion protein of the present invention. The transplanted cells, tissue, or organ may include, for example, a pancreatic cell, tissue or organ, skin, liver, kidney, heart, bone marrow, blood, bone, muscle, artery, vein, cartilage, thyroid, nervous system, or stem cells.

Examples of the use of RAGE fusion proteins of the present invention in the treatment of such diseases and disorders are described herein.

A variety of animal models have been used, for example, to validate the use of the compounds that modulate RAGE as therapeutic agents. Examples of these models are as follows: a) sRAGE inhibited neointima formation in a restenosis rat model following arterial damage in both diabetic and normal rats by inhibiting endothelial, smooth muscle and macrophage activation by RAGE ( Zhou et al, Circulation 107: 2238-2243 (2003)); b) Inhibition of RAGE / ligand interactions by using either sRAGE or an anti-RAGE antibody, reduced the formation of amyloid plaques in a mouse systemic amyloidosis model 59 (Yan et al, Nat. Med., 6: 643-651 (2000)). Along with the reduction of amyloid plaques, there was a reduction in inflammatory cytokines, interleukin-6 (1L-6) and macrophage-colony stimulating factor (M-CSF) as well as reduced activation of NF-κΒ in the treated animals; C) RAGE transgenic mice (mice overexpressing RAGE and mice expressing RAGE dominantly negative) exhibit plaque formation and cognitive deficits in a mouse model of AD (Arancio et al, EMBOJ., 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 nephropathy in db / db mice (Hudson et al., Arch. Biochem. Biophys., 419: 80-88 (2003) )); and f) sRAGE attenuated the severity of inflammation in a mouse model of collagen-induced arthritis (Hofmann 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 (Hofmann et al., Cell, 97: 889-901 (1999)).

Thus, in one embodiment, the RAGE fusion proteins of the present invention can be used to treat a symptom of diabetes and / or complications resulting from diabetes mediated by RAGE. In other embodiments, the symptom of diabetes or late complications of diabetes may include 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 for the pathophysiology of diabetic complications. In vivo, it has been shown that inhibition of interaction of RAGE with its ligand (s) is 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 renal function and reduced abnormal kidney histopathology in diabetic mice (Flyvbjerg et al, Diabetes 53: 166-60 172 (2004)). Moreover, treatment with a soluble form of RAGE (sRAGE) that binds to RAGE ligands and inhibits RAGE / ligand interactions, reduced atherosclerotic lesions in diabetic apolipoprotein E-null mice and weakened the functional and morphological pathology of diabetic nephropathy at 5 db / db mice (Bucciarelli et al, Circulation 106: 2827-2835 (2002)).

It has also been shown that non-enzymatic glycoxidation of macromolecules, which ultimately results in the formation of advanced glycation endproducts (AGEs), is increased at sites of inflammation, in renal failure, in the presence of hyperglycemia and other conditions associated with systemic or local oxidant stress (Dyer et al, J. 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, such as in the amyloid of joints composed of AGE-p2 microglobulin found in patients with dialysis-related amyloidosis (Miyata et al, J. Clin. Invest., 92: 1243-1252 (1993); Miyata et al., J. Clin. Invest, 98: 1088-1094 (1996)), or generally as illustrated by the vasculature and tissues of patients with diabetes (Schmidt et al, Nature Med. , 1: 1002-1004 (1995)). The progressive accumulation of AGEs over time in patients with diabetes suggests that endogenous clearance mechanisms are unable to function effectively at sites of AGE deposition. Such accumulated AGEs have the ability to change cellular properties through a number of mechanisms. Although RAGE is expressed at low levels in normal tissues and vasculature, it is shown that in an environment where the ligands of the receptor accumulate, RAGE is up-regulated (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)). Expression of RAGE is increased in endothelium, smooth muscle cells and infiltrating mononuclear phagocytes in diabetic vasculature. Studies in cell culture have also shown that interaction of AGE-RAGE causes changes in cellular properties that are important in vascular homeostasis.

The use of the RAGE fusion proteins in the treatment of diabetes related pathology is illustrated in Figure 14. The RAGE-fiction protein TTP-4000 was evaluated in a diabetic model of rat from restenosis that involved measuring 61 proliferation of smooth muscle cells and intima extension following vascular damage. As illustrated in Figure 14, treatment with TTP-4000 can significantly reduce the intima / media (I / M) ratio (Figure 14A; Table 1) in diabetes-associated restenosis in a dose-responsive manner. Treatment with TTP-5 4000 can also significantly reduce restenosis-associated proliferation of vascular smooth muscle cells in a dose-dependent manner (Figure 14B).

Table 1

Effect of TTP-4000 in a model of rat from 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)

Intima area (mm 2) 0.2 ± 0.03 0.18 ± 0.04 0.16 ± 0.02

Media area (mm 2) 0.12 ± 0.01 0.11 ± 0.02 0.11 ± 0.01 1 / M ratio 1.71 ± 0.27 1.61 ± 0.26 1.44 * ± 0.15 * P <0.05; ** A loading dose of 3 mg / animal was used for both high and low doses.

In other embodiments, the RAGE fusion proteins of the present invention can also be used to treat or reverse amyloidosis and Alzheimer's disease. RAGE is a receptor for amyloid beta (Αβ) as well as other amyloidogenic proteins including SAA and amyline (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)). The RAGE ligands, including AGEs, SlOOb and Αβ proteins, are also found in tissue surrounding the senile plaque in humans (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 Neurosci., 12: 167-173 (1998)). RAGE β-sheet fibrillary material has been shown to bind regardless of the composition of the subunits (amyloid β peptide, amyline, 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, it has been shown that amyloid deposition results in increased expression of RAGE. For example, in the brains of patients with Alzheimer's disease (AD), expression of RAGE increases in neurons and glia (Yan, et al., Nature 382: 685-691 (1996)).

62

Simultaneously with expression of RAGE ligands, RAGE is regulated up in astrocytes and microglial cells in the hippocampus of individuals with AD but is not regulated up in individuals who do not have AD (Lue et al, Exp. Neurol, 171: 29-45 (2001 )). These findings suggest that cells expressing RAGE are activated through interactions of RAGE / RAGE ligand in the vicinity of the senile plaques. Aβ-mediated microglial cell activation can also be blocked in vitro with antibodies directed against the RAGE ligand binding domain (Yan et al., Proc. Natl. Acad. Sci., USA, 94: 5296-5301 (1997 )). It has also been shown that RAGE can serve as a focal point for collection of fibrils (Deane et al., Nat. Med. 9: 907-913 (2003)).

Inhibition of RAGE / ligand interactions by using either sRAGE or an anti-RAGE antibody can also reduce formation of amyloid plaques in vivo in a mouse model of systemic amyloidosis (Yan et al, Nat. Med., 6: 643 -651 (2000)). Double transgenic mice overexpressing human RAGE and human amyloid precursor protein (APP) with the Swedish and London mutations (mutant hAPP) in neurons develop learning disorders and neuropathological abnormalities rather than their transgenic counterparts with a single mutant hAPP. In contrast, double transgenic mice with decreased Αβ signaling capacity show neurons expressing a dominant negative form of RAGE in the same mutant hAPP background, delayed onset of neuropathological abnormalities and learning abnormality compared to its transgenic counterparts from a single APP (Arancio et al., EMBOJ., 23: 4096-4105 (2004)).

In addition, it has been shown that inhibition of interaction of RAGE amyloid reduces cellular RAGE expression and cell stress markers (as well as NF-κ activation) and nullifies amyloid deposition (Yan et al., Nat. Med., 6: 643-651 (2000)) that suggests a role for RAGE amyloid interaction in both perturbation of cellular properties in an amyloid-enriched environment (even in early phases) as well as in amyloid accumulation.

The RAGE fusion proteins of the present invention can thus also be used in treatment for reducing amyloidosis and for reducing amyloid plaques and cognitive dysfunction associated with Alzheimer's disease (AD). As described above, it has been shown that 63 sRAGE both lowers the formation of amyloid plaques in the brain and subsequently increases the markers for inflammation in an animal model of AD. Figures 15A and 15B show that mice that have AD and are treated with either TTP-4000 or mouse sRAGE for 3 months had less amyloid beta 5 (Αβ) plaques and less cognitive dysfimction than animals that had an excipient or human IgG as a negative control (IgG1). Like sRAGE, TTP-4000 can also lower the inflammatory cytokines IL-1 and TNF-α associated with AD (data not shown).

RAGE fusion proteins of the present invention can also be used to treat atherosclerosis and other cardiovascular disorders. Thus, it is shown that ischemic heart disease is particularly prevalent 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 accelerated and expanded more than in patients who do not suffer from diabetes (see, for example, Waller et al., Am. J. Med., 69: 498-506 (1980); Crall et al, Am. 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 there are many reasons for the accelerated atherosclerosis related to diabetes, it has been shown that lowering AGEs can reduce plaque formation.

For example, the RAGE fusion proteins of the present invention can also be used to treat stroke. When TTP-4000 was compared with sRAGE in an animal model of stroke relevant to the disease, it was found that TTP-4000 provides a considerably stronger reduction in the volume of the infarction. In this model, the middle carotid artery of a mouse is ligated and then treated by reperfusion to form an infarction. To determine the efficacy of RAGE fusion proteins for treating or preventing 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 effective than sRAGE in limiting the area of an infarction in these animals suggesting that, due to the better half-life in plasma, TTP-4000 was able to maintain better protection then sRAGE.

64

Table 2

Reduction of infarction in stroke% reduction in infarction ** sRAGE 15% * 1ΤΡ ^ 4000 (300 | μ ^ 38% * TTP-4000 (300 pg) 21% * TTP-4000 (300 Hg) 10% *

IgG Isotype control (300 pg) 4% * Significant to p <0.001; ** In comparison with saline. In another embodiment, the RAGE fusion proteins of the present invention can be used to treat cancer. In one embodiment, the cancer treated by the use of the RAGE fusion proteins of the present invention comprises cancer cells that express RAGE. Types of cancer that can be treated, for example, with the RAGE fusion protein of the present invention include some types of lung cancer, some gliomas, some papillomas, and the like. Amphoterin is a high mobility I group non-histone chromosomal DNA binding protein (Rauvala et al., J. Biol. Chem., 262: 16625-16635 (1987); Parkikinen et al., J. Biol. Chem. 268: 19726 -19738 (1993)) that has been shown to interact with RAGE. Amphoterin has been shown to promote neurite outgrowth as well as serve as a surface for collecting protease complexes in the fibrinolytic system (which is also known to contribute to cell mobility). In addition, a local inhibitory effect of the growth of RAGE blocking tumors has been observed in a model of a primary tumor (C6 glioma) the model of Lewis lung metastasis (Taguchi et al, Nature 405: 354-360 (2000) ) and spontaneously emerging papillomas in mice expressing the v-Ha ras transgene (Leder et al, Proc. Natl. Acad. Sci, 87: 9178-9182 (1990)).

In yet another embodiment, the RAGE fusion proteins of the present invention can be used to treat inflammation. In other embodiments, the RAGE fusion proteins of the present invention can be used to treat inflammation associated with inflammatory bowel disease, inflammation associated with rheumatoid 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 hemorrhagic shock, inflammation associated with sepsis, inflammation associated with organ transplantation, inflammation associated with associated with weakened wound healing or inflammation associated with rejection of self (e.g., autoimmune) or non-self (e.g., transplanted) cells, tissue, or organs.

For example, following thrombolytic treatment, inflammatory cells such as granulocytes infiltrate the ischemic tissue and produce oxygen radicals that can destroy more cells than were killed by hypoxia. Inhibition of the receptor on the neutrophil, which is responsible for neutrophils being able to invade the tissues, with antibodies or other protein antagonists, has been shown to weaken this response. Because RAGE is a ligand for this neutrophil receptor, a RAGE fusion protein containing a fragment of RAGE can act as a bait and prevent the neutrophil from moving to the site of reperfusion and thus prevent further tissue destruction. The role of RAGE in preventing inflammation can be indicated by studies showing that sRAGE inhibits neointimal enlargement in a model of rat restenosis following arterial damage in both diabetic and normal rats, presumably by inhibiting proliferation of endothelial smooth muscle cells and activation of macrophages by RAGE (Zhou et al., Circulation, 107: 2238-2243 (2003)). In addition, sRAGE inhibited models of inflammation including delayed type of hypersensitivity, experimental autoimmune encephalitis, and inflammatory bowel disease (Hofman et al, Cell, 97: 889-901 (1999)).

In one embodiment, the RAGE fusion proteins of the present invention can be used to treat autoimmune-based disorders. For example, in one embodiment, the RAGE fusion proteins of the present invention can be used to treat kidney failure. The RAGE fusion proteins of the present invention can thus be used to treat systemic lupus nephritis or inflammatory lupus nephritis. For example, it has been shown that the S100 / calgranulins comprise a family of 66 closely related calcium-binding polypeptides characterized by two EF-hand regions joined by a linking peptide (Schafer et al, TIBS, 21: 134-140 (1996) Zimmer et al, Brain Res Bull, 37: 417-429 (1995); Rammes et al, J. Biol. Chern., 272: 9496-9502 (1997); Lugering et al, Eur. J. Clin.

5 Invest, 25: 659-664 (1995)). Although they lack signal peptides, it has long been known that S100 / calgranulins gain access to the extracellular space, particularly at sites of chronic immune / inflammatory responses, such as cystic fibrosis and rheumatoid arthritis. RAGE is a receptor for many members of the S100 / calgranulin family, which mediates its anti-inflammatory effects on cells such as lymphocytes and mononuclear phagocytes. Studies on models of delayed type of hypersensitivity reaction, colitis in IL-10 null mice, collagen-induced arthritis and experimental autoimmune encephalitis also suggest that RAGE-Igigand interaction (presumably with S100 / calgranulins) has a major role in the inflammatory cascade.

Type I diabetes is an autoimmune disorder that can be prevented or weakened by treatment with the RAGE fusion proteins of the present invention. For example, sRAGE has been shown to allow the transfer of splenocytes from non-obese diabetic (NOD) mice to NOD mice with severe combined immunodeficiency (NOD scid mice). NOD-scid mice do not display diabetes spontaneously but require the presence of immunocytes capable of destroying the islet cells, such that diabetes is subsequently induced. It was found that recipient NOD scid mice treated with sRAGE exhibited a delayed onset of diabetes induced by splenocytes transferred from a diabetic (NOD) mouse compared to recipient NOD scid mice not treated with sRAGE have been treated (U.S. Patent Publication No. 2002/0122799). As stated in US 2002/0122799, the experimental results with the use of sRAGE in this model are relevant to human disease such as clinical backgrounds in which future immunotherapy and islet transplantation can occur.

Thus, in one embodiment, a RAGE fission protein of the present invention can be used to treat inflammation associated with transplantation of at least one of an organ, a tissue, or a variety of cells from a first site to a second site . The first and second places 67 can be in different people or in the same person. In other embodiments, the transplanted cells, tissue or organ cells include pancreatic, skin, liver, kidney, heart, lung, bone marrow, blood, bone, muscle, endothelial cells, artery, vein, cartilage, thyroid, nervous system, or stem cells. For example, administration of the RAGE fusion proteins of the present invention can be used to effect cell transplantation from the islets of a first non-diabetic person to a second diabetic person.

In another embodiment, the present invention can provide a method for treating osteoporosis by administering a therapeutically effective amount of a RAGE fusion protein of the present invention to a subject (Zhou et al, J. Exp. Med. ., 203: 1067-1080 (2006)). In one embodiment, the method of treating osteoporosis may further comprise the step of increasing the bone density of the person or decreasing the rate of decrease of bone density of a person.

Thus, in various selected embodiments, the present invention can provide a method for inhibiting the interaction of an AGE with RAGE in a subject by administering to the subject a therapeutically effective amount of a RAGE fusion protein of the present invention. The person treated using the RAGE fusion proteins of the present invention can be an animal. In one embodiment, the person is a human. The person may suffer from an AGE-related disease such as diabetes, diabetic complications such as nephropathy, neuropathy, retinopathy, foot ulcers, amyloidoses, or renal failure and inflammation. Or the person can be an individual with Alzheimer's disease. In an alternative embodiment, the person may be an individual with cancer. In still other embodiments, the person may suffer from systemic lupus erythematosis or inflammatory lupus neffitis. Other diseases can be mediated by RAGE and thus treated using the RAGE fusion proteins of the present invention. In additional alternative embodiments of the present invention, the RAGE fusion proteins can be used to treat Crohn's disease, arthritis, vasculitis, nephropathies, retinopathies and neuropathies in human or animal patients. In other embodiments, inflammation involving both autoimmune responses (e.g., rejection of self) and non-autoimmune responses (e.g., 68 rejection of self) may be mediated by RAGE and thus treated by the use of RAGE fusion proteins of the present invention.

A therapeutically effective amount may include an amount capable of preventing the interaction of RAGE with an AGE or other types of RAGE endogenous ligands in a subject. Accordingly, the amount will vary with the person being treated. Administration of the connection can be every hour, daily, weekly, monthly, annually or as a single event. In various alternative embodiments, the effective amount of the RAGE fusion protein can range from about 1 ng / kg of body weight to about 100 mg / kg of body weight or from about 10 µg / kg of body weight to about 50 mg / kg of body weight or from about 100 pg / kg body weight to about 20 mg / kg body weight. The actual effective amount can be determined by dose / response testing using methods that are standard in the art (Johnson et al., Diabetes. 42: 1179, (1993)). As is known to those skilled in the art, the effective amount may thus depend on the bioavailability, bioactivity and biodegradability of the compound.

Compositions The present invention may comprise a composition comprising a RAGE fusion protein of the present invention in admixture with a pharmaceutically acceptable carrier. The RAGE fusion protein may comprise a RAGE polypeptide linked to a second non-RAGE polypeptide. In one embodiment, the RAGE fusion protein may comprise a binding site for a RAGE ligand. In one embodiment, the ligand binding site comprises the most N-terminal domain of the RAGE fusion protein. The binding site for a RAGE ligand may include the RAGE V domain or part thereof. In one embodiment, the binding site for a ligand of RAGE SEQ includes 1D NO: 9 or a sequence that is at least 90% identical to it, or SEQ ID NO: 10 or a sequence that is at least 90% identical to it, or SEQ ID NO. : 47 or a sequence that is at least 90% identical to that.

In one 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 69 polypeptide comprising an immunoglobulin domain comprises at least a portion of at least one of the Ch2 or CH3 domains of a human IgG.

In certain embodiments, the RAGE fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 56 or SEQ ID NO: 57 or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% 96% is 97%, 98% or 99% identical to that. For example, in some embodiments, a sequence that is at least 90% identical to SEQ ID NO: 56 or SEQ ID NO: 57 includes the sequence of SEQ ID NO: 56 or SEQ ID NO: 57 without the C-terminal lysine.

The RAGE protein or polypeptide may comprise full-length human RAGE (e.g., SEQ ID NO: 1) or a fragment of human RAGE. In one embodiment, the RAGE polypeptide does not include residues of the signal sequence. The signal sequence of RAGE can include either full-length residues 1-22 or residues 1-23 (SEQ ID NO: 1). In other embodiments, the RAGE polypeptide may comprise a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% 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 being the first residue instead of a Methionine (see, for example, Neeper et al., (1992)). Or the human RAGE can include full-length RAGE with the signal sequence removed (e.g., SEQ ID NO: 2 or SEQ ID NO: 3) (Figures 1A and 1B) or a portion of that amino acid sequence.

The RAGE fusion proteins of the present invention may also include sRAGE (e.g., SEQ ID NO: 4), a polypeptide that is at least 90% identical to sRAGE, or a fragment of sRAGE. The RAGE polypeptide can include, for example, human sRAGE, or a fragment thereof, wherein Glycine is the first residue instead of a Methionine (see, for example, Neeper et al., (1992)). Or the human RAGE may include sRAGE with the signal sequence removed (see, for example, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 45 in Figure 1) or a portion of that amino acid sequence. In other embodiments, the RAGE protein may comprise a V domain (see, for example, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 46 in Figure 1). Or, a sequence that is at least 90% identical to the V domain or a fragment thereof can be used. Or the RAGE protein may comprise a fragment of RAGE that includes a portion of the V domain (see, for example, 70 SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 47 in Figure 1). In one embodiment, the ligand binding site can be SEQ ID NO: 9, or a sequence that is at least 90% identical to it, or SEQ ID NO: 10, or a sequence that is at least 90% identical to it, or SEQ ID NO: 9 47, or a sequence that is at least 90% identical to that. In yet another embodiment, the RAGE fagment is a synthetic peptide.

For example, the RAGE polypeptide can be amino acids 23-116 of human RAGE (SEQ ID NO: 7) or a sequence that is at least 90% identical to it, or amino acids 24-116 of human RAGE (SEQ ID NO: 8) or a sequence which is at least 10 to 90% identical thereto, or amino acids 24-116 of human RAGE where Q24

cyclizes to form pE (SEQ ID NO: 46), or include a sequence that is at least 90% identical to that corresponding to the V-domain of RAGE. Or the RAGE polypeptide can include human RAGE amino acids 124-221 (SEQ ID NO: 11) or a sequence that is at least 90% identical to that corresponding to the C1-15 domain of RAGE. In another embodiment, the RAGE polypeptide can include amino acids 227-317 of human RAGE (SEQ ID NO: 12) or a sequence that is at least 90% identical to that corresponding to the C2 domain of RAGE. Or the RAGE polypeptide can be amino acids 23-123 of human RAGE (SEQ ID NO: 13) or a sequence that is at least 90% identical to it, or 20 amino acids 24-123 of human RAGE (SEQ ID NO: 14) or a sequence that is at least 90% identical to that corresponding to the V-domain of RAGE and includes a downstream interdomain linker. Or the RAGE polypeptide can include amino acids 24-123 of human RAGE, wherein Q24 cyclizes to form pE (SEQ ID NO: 48), or a sequence that is at least 90% identical to that. Or the RAGE polypeptide can be amino acids 23-226 of human RAGE (SEQ ID

NO: 17) or a sequence that is at least 90% identical to it, or amino acids 24-226 of human RAGE (SEQ ID NO: 18) or a sequence that is at least 90% identical to it, corresponding to the V domain , the C1 domain and the interdomain linker that links these two domains. Or the RAGE polypeptide can be amino acids 24-30 226 of human RAGE where Q24 cycles to form pE (SEQ ID NO:

50), or a sequence that is 90% identical to that. Or the RAGE polypeptide can be amino acids 23-339 of human RAGE (SEQ ID NO: 5) or a sequence that is at least 90% identical to it, or amino acids 24-339 of human RAGE (SEQ

71 ID NO: 6) or a sequence that is at least 90% identical to that corresponding to sRAGE (i.e., encoding the V, C1, and C2 domains and include interdomain linkers). Or the RAGE polypeptide can include human RAGE amino acids 24-339 where Q24 cyclizes to form pE (SEQ ID NO: 45), or a sequence that is at least 90% identical to it. Or fragments of any of these sequences can be used.

In another embodiment, the ligand binding site may comprise amino acids 22-51 of SEQ ID NO: 1. In another embodiment, the ligand binding site may comprise amino acids 23-51 of SEQ ID NO: 1. In another embodiment, the ligand binding site may comprise amino acids 31-51 of SEQ ID NO: 1. In another embodiment, the ligand binding site may comprise amino acids 31-116 of SEQ ID NO: 1. The ligand binding site may include, for example, a RAGE V domain or a portion thereof such as the RAGE ligand binding domain (e.g., amino acids 1-118, 23-118, 24-118, 31-118, 1-116, 23- 116, 24-15 116.31-116.1-54.23-54.24-54.31-54, 1-53, 23-53, 24-53 or 31-53 of SEQ ID NO: 1 or fragments thereof) (Figure 1). Or fragments of the polypeptides that functionally bind a RAGE ligand can be used. Or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the V-domain of RAGE or a fragment thereof (e.g. as above) 20) can be used. As is known in the art, in embodiments where the N-terminus of the fusion protein is glutamine, such as, for example, removing the signal sequence comprising residues 1-23 of SEQ ID NO: 1 (e.g., Q24 for a polypeptide comprising amino acids 24-118 or SEQ ID NO: 1), further cyclize the glutamine to form pyroglutamic acid (pE).

The RAGE fusion protein can include various types of peptides that are not derived from RAGE or a fragment thereof. The second polypeptide of the RAGE fusion protein may comprise a polypeptide that is derived from an immunoglobulin. The heavy chain (or part thereof) can come from one of the known isotypes of the heavy chain: IgG (γ), IgM (μ), IgD (δ), IgE (ε), or IgA (a). In addition, the heavy chain (or part thereof) may come from one of the known subtypes of the heavy chain: IgG1 (γΐ), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgAl (a1), IgA2 (a2), or mutations of these isotypes or subtypes that alter biological activity 72. The second polypeptide may comprise the Ch2 and CH3 domains of a human IgG1 or a portion of one or both of these domains. As an exemplary embodiment, the polypeptide comprising the Ch2 and CH3 domains of a human IgG1 or a portion thereof may comprise SEQ ID NO: 40 or a portion thereof. In one embodiment, the polypeptide comprising the Ch2 and Cn3 domains of a human IgG1 or a portion thereof may comprise SEQ ID NO: 38 or a portion thereof. For example, 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, with the terminal lysine (K) removed. The immunoglobulin peptide can be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 41. The immunoglobulin sequence in SEQ ID NO: 38 or SEQ ID NO: 40 can also be encoded by SEQ ID NO: 52 or SEQ ID, respectively. NO: 53.

The Fc portion of the immunoglobulin chain can be anti-inflammatory 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 RAGE fusion protein may further comprise a RAGE polypeptide that is directly linked to a polypeptide comprising an CH2 domain of an immunogobulin or a fragment thereof. In one embodiment, the CH2 domain or a fragment thereof comprises SEQ ID NO: 42. In one embodiment, the fragment of SEQ ID NO: 42 comprises SEQ ID NO: 42 from which the first ten amino acids have been removed.

In one embodiment, the RAGE polypeptide comprises an RAGE interdomain linker that is linked to an 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 linker interdomain and the C-terminal amino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of a polypeptide comprising an CH2 domain of an immunoglobulin or a fragment thereof. The polypeptide comprising a CH2 domain of an immunoglobulin or a part thereof can comprise the Ch2 and Cn3 domains of a human IgG1 or a part of both or one of these domains. As an example of an embodiment, the polypeptide comprising the Ch2 and Ch3 domains of a human IgG1 or a portion thereof may comprise SEQ ID NO: 40 or a 73 portion thereof. In one embodiment, the polypeptide comprising the Ch2 and CH3 domains of a human IgG1 or a portion thereof may comprise SEQ ID NO: 38 or a portion thereof. For example, 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 with the terminal lysine (K) removed.

The RAGE fusion protein of the present invention can comprise a single or multiple domains of RAGE. The RAGE polypeptide comprising an inter-domain linker linked to an RAGE immunoglobulin domain may comprise a fragment of a full-length RAGE protein. For example, in one embodiment, the RAGE fusion protein may comprise two immunoglobulin domains derived from RAGE protein and two immunoglobulin domains derived from a human Fc polypeptide. The RAGE fusion protein can comprise a first immunoglobulin domain from RAGE and a first interdomain linker linked to a second immunoglobulin domain from RAGE and a second interdomain linker from RAGE, such that the N-terminal amino acid of the first interdomain linker is linked to the C-terminal amino acid of the first immunoglobulin domain of RAGE, the N-terminal amino acid of the second immunoglobulin domain of RAGE is linked to the C-terminal amino acid of the first interdomain linker, the N-terminal amino acid of the second interdomain linker is linked to the C-terminal amino acid of the second immunoglobulin domain of RAGE and the C-terminal amino acid of the second interdomain linker of RAGE is directly linked to the N-terminal amino acid of the polypeptide which is a CH2 immunoglobulin domain or a fragment thereof. For example, the RAGE polypeptide can be amino acids 23-251 of human RAGE (SEQ ID NO: 19) or a sequence that is at least 90% identical to it, or amino acids 24-251 of human RAGE (SEQ ID NO: 20) or a sequence that is at least 90% identical to it, or amino acids 24-251 of human RAGE where Q24 cycles to form pE, or a sequence that is at least 90% identical to it (SEQ ID NO: 51), corresponding to the V domain, the Cl domain, the interdomain linker that links these two domains and a second interdomain linker downstream of C1. In one embodiment, a nucleic acid construct comprising SEQ ID NO: 30 or a fragment thereof may encode a RAGE fusion protein with four domains. In another embodiment, a nucleic acid construct comprising SEQ ID NO: 54 74 can encode a four domain RAGE fusion protein, silent base changes for the codons encoding proline (CCG to CCC) and glycine (GGT to GGG) the C-terminus of the sequence are made to remove a cryptic RNA cleavage site near the terminal codon (ie nucleotides 1375-1380 of SEQ TD NO: 30 are modified to generate SEQ ID NO: 54).

Alternatively, a three-domain RAGE fusion protein may comprise one immunoglobulin domain derived from RAGE and two immunoglobulin domains derived from a human Fc polypeptide. For example, the RAGE-10 fusion protein may comprise a single immunoglobulin domain from RAGE linked through an inter-domain linker from RAGE to the N-terminal amino acid of the polypeptide comprising the C 1 immunoglobulin domain or a fragment thereof. For example, the RAGE polypeptide can be amino acids 23-136 of human RAGE (SEQ ID NO: 15) or a sequence that is at least 90% identical there to 15 or amino acids 24-136 of human RAGE (SEQ ID NO: 16) or a sequence that is at least 90% identical to it, or amino acids 24-136 of human RAGE where Q24 cycles to form pE, or a sequence that is at least 90% identical to it (SEQ ID NO: 49) corresponding to the Y domain of RAGE and a downstream interdomain linker. In one embodiment, a nucleic acid construct comprising SEQ ID NO: 31 or a fragment thereof may encode a three-domain RAGE fusion protein. In another embodiment, a nucleic acid construct comprising SEQ ID NO: 55 can encode a three domain RAGE fusion protein, silent base changes for the codons encoding praline (CCG to CCC) and glycine (GGT to GGG) on the C-terminus of the sequence are made to remove a cryptic RNA cleavage site near the terminal codon (ie nucleotides 1030-1035 of SEQ ID NO: 31 are modified to generate SEQ ID NO: 55).

A fragment of the RAGE interdomain linker may include a peptide sequence that is naturally downstream of, and thus linked to, an immunoglobulin domain of RAGE. For example, for the V domain of RAGE, the interdomain linker may include amino acid sequences that are naturally downstream of the V domain. In one embodiment, the linker can include SEQ ID NO: 21, which corresponds to amino acids 117-123 of RAGE with the full length 75. Or the linker may include a peptide that has additional portions of the natural sequence of RAGE. For example, an 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 can be used. Thus, in one embodiment, the interdomain linker comprises SEQ 1D NO: 23 which comprises amino acids 117-136 of full-length RAGE. Or fragments of SEQ ID NO: 21 wherein, for example, 1, 2, or 3 amino acids have been removed from one of the ends of the linker can be used. In other embodiments, the linker may comprise a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21 or SEQ ID NO: 23.

For the RAGE C1 domain, the linker may include a peptide sequence that is naturally downstream of the C1 domain. In one embodiment, the left-hand SEQ ID can include NO: 22 corresponding to amino acids 222-251 of full-length RAGE. Or the linker may include a peptide that has additional portions of the natural sequence of RAGE. For example, a linker comprising several (1-3, 1-5, or Ι-ΙΟ, or 1-15 amino acids) amino acids upstream and downstream of SEQ ID NO: 22 can be used. Or fragments of SEQ ID NO: 22 can be used, wherein for example 1-3, 1-5, or 1-10, or 1-15 amino acids have been removed from one of the ends of the linker. In one embodiment, an inter-domain linker from RAGE may include, for example, SEQ ID NO: 24 corresponding to amino acids 222-226. In other embodiments, the linker may comprise a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 22 or SEQ ID NO: 24.

Or an interdomain linker may include SEQ ID NO: 44 corresponding to 25 amino acids 318-342 of RAGE. In other embodiments, the linker may comprise a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 44.

Pharmaceutically acceptable carriers can include any of the standard pharmaceutically acceptable carriers known in the art. In one embodiment, the pharmaceutical carrier can be a liquid and the RAGE fusion protein or nucleic acid construct can be in the form of a solution. In another embodiment, the pharmaceutically acceptable carrier may be a solid in the form of a powder, a freeze-dried powder or a tablet. Or the pharmaceutical carrier can be a gel, suppository or cream. In other embodiments, the carrier may be a liposome, a microcapsule, a polymer-encapsulated cell, or a virus. The term pharmaceutically acceptable carrier thus includes, but is not limited to, any of the standard pharmaceutically acceptable carriers such as water, alcohols, phosphate buffered saline solutions, sugars (e.g., sucrose or mannitol), oils or emulsions such as oil / water emulsions or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules.

In certain embodiments, the RAGE fusion proteins may be present in a neutral form (including zwitterion forms) or as positively or negatively charged species. In some embodiments, the RAGE fusion proteins can be complexed with a counterion to form a pharmaceutically acceptable salt.

The terms "pharmaceutically acceptable salt" refers to a complex comprising one or more RAGE fusion proteins and one or more counterions, the counterions coming from pharmaceutically acceptable inorganic and organic acids and bases.

Pharmaceutically acceptable inorganic bases include metal ions. More preferred metal ions include, but are not limited to, suitable alkali metal salts, alkaline earth metal salts, and other physiologically acceptable metal ions. Salts derived from inorganic bases include aluminum, ammonium, calcium, cobalt, nickel, molybdenum, vanadium, manganese, chromium, selenium, tin, copper, iron, ferrous, lithium, magnesium, manganese salts, manogen, potassium, rubidium, sodium and zinc and at its usual valencies.

Pharmaceutically acceptable acid addition salts of the RAGE fusion proteins of the present invention can be prepared from the following acids, including, without limitation, formic acid, acetic acid, acetamidobenzoic acid, adipic acid, ascorbic acid, boric acid, propionic acid, benzoic acid, camphoric acid, carboxylic acid, cyclamic acid, dehydrocholic acid, malonic acid, edetic acid, ethyl sulfuric acid, phendizoic acid, metaphosphoric acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, tannic acid, citric acid, nitric acid, ascorbic acid, glucuronic acid, maleic acid, folic acid, propionic acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, pumoric acid , hydrochloric acid, hydrobromic acid, iodine hydrochloric acid, lysine, isocitric acid, trifluoroacetic acid, pamoic acid, propionic acid, 77 anthranilic acid, mesylic acid, orotinic acid, oxalic acid, oxalacetic acid, oleic acid, stearic acid, salicylic acid, aminosalicylic acid, silicic acid, p-benzoic acid, phenylacetic acid, p-benzoic acid, p-phenyl acid oic acid, sulfonic acid, methanesulfonic acid, phosphoric acid, phosphonic acid, ethanesulfonic acid, ethanedisulfonic acid, ammonium, benzenesulfonic acid, pan to an acid, naphthalenesulfonic acid, toluene sulfonic acid, 2-hydroxy-ethanesulfonic acid, sulfanilic acid, sulfuric acid, nitric acid, nitric sulfonic acid, sulfuric acid, sulphonic acid, sulfuric acid β-hydroxybutyric acid, glycine, glycylglycine, glutamic acid, cacodylate, diaminohexanoic acid, camphor sulphonic acid, gluconic acid, thiocyanic acid, oxoglutaric acid, pyridoxal-5-phosphate, chlorophenoxy-acetic acid, undecanoic acid, N-acetyl-l-lactaric acid, galactic acid

Pharmaceutically acceptable organic bases include trimethylamine, diethylamine, Ν, Ν'-dibenzylethylenediamine, chloroprocaine, choline, dibenzylamine, diethanolamine, ethylenediamine, meglumin (N-methylglucamine), procaine, cyclic amines, quaternary ammonium cationaine, arginine, caffeine , clemizole, 2-ethylamino-ethanol, 2-diethylamino-ethanol, 2-dimethylamino-ethanol, ethanediamine, butylamine, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, ethylglucamine, glucamine, glucosamine, histidine, hydrabamine, imidazlamine, isopropyl amine , methylglucamine, morpholine, piperazine, pyridine, pyridoxine, neodymium, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, tripropylamine, triethanolamine, tromethamine, methylamine, taurine, cholate, 6-amino-2-methyl-2- heptanol, 2-amino-2-methyl-1, 3-propanediol, 2-amino-2-methyl-1-propanol, aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy-alkanoic acids, flavoring atic acids, aliphatic and aromatic sulfonic acids, strontium, tricine, hydrazine, phenylcyclohexylamine, 2- (N-morphino) ethanesulfonic acid, bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methane, N- (2- acetamido) -2-aminoethanesulfonic acid, 1,4-piperazine diethanesulfonic acid, 3-morpholino-2-hydroxypropanesulfonic acid, 1,3-bis [tris (hydroxymethyl) methylamino] propane, 4-morpholinopropanesulfonic acid, 4- (2-hydroxyethyl ) piperazine 1-ethanesulfonic acid, 2-30 [(2-hydroxy-1,1-bis (hydroxymethyl) ethyl) amino] ethanesulfonic acid, N, N-bis (2-hydroxyethyl) -2-amino -ethanesulfonic acid, 4- (N-morpholino) butanesulfonic acid, 3- (N, N-bis [2-hydroxyethyl] amino) -2-hydroxypropanesulfonic acid, 2-hydroxy-3- [tris (hydroxy-methyl) methylamino] -1 - propanesulfonic acid, 4- (2-hydroxyethyl) piperazine-1- (2-hydroxypropanesulfonic acid), piperazine-1,4-bis (2-hydroxypropanesulfonic acid) dihydrate, 4- (2-hydroxyethyl) -1-piperazine propanesulfonic acid, N, V-bis (2-hydroxyethyl) glycine, N- (2-hydroxyethyl) piperazine-N '- (4-butanesulfonic acid), N- (t ris (hydroxymethyl) methyl] -3-aminopropanesulfonic acid, N-tris (hydroxymethyl) methyl-4-aminobutane-5-sulfonic acid, N- (1,1-dimethyl-2-hydroxyethyl) -3-amino-2-hydroxy-propanesulfonic acid, 2 - (cyclohexylamino) ethanesulfonic acid, 3- (cyclohexylamino) -2-hydroxy-1-propane sulfonic acid, 3- (cyclohexylamino) -1-propanesulfonic acid, A, - (2-acetamido) iminodiacetic acid, 4- (cyclohexylamino) - 1-butanesulfonic acid, A- [tris (hydroxymethyl) methyl] glycine, 2-amino-2- (hydroxymethyl) -1,3-propanediol and trometamol.

Administration of the RAGE fusion proteins of the present invention can use various routes. Thus, administration of the RAGE fusion protein of the present invention can use intraperitoneal (IP) injection. Alternatively, the RAGE fusion protein can be administered orally, intranasally or as an aerosol. In another embodiment, administration is intravenous (IV). The RAGE fusion protein can also be administered subcutaneously. In another embodiment, administration of RAGE fusion protein is intra-arterial. In another embodiment, administration is sublingual. Administration may also use a capsule with release over time. Subcutaneous administration may be useful, for example, for treating chronic disorders when it is desired that someone perform the administration themselves.

In another aspect of the present invention, the RAGE fusion proteins of the invention can be used in adjuvants in therapeutic or therapeutic combination treatments with other known therapeutic agents. The following is a non-exhaustive list of adjuvants and additional therapeutic agents that can be used in combination with the modulators of the RAGE fusion protein of the present invention:

Pharmacological classification of anti-cancer agents: 30 1. Alkylating agents: Cyclophosphamide, nitrosourea, carboplatin, cisplatin, procarbazine 2. Antibiotics: Bleomycin, Daunorubicin, Doxorubicin 79 3. Antimetabolites: Methotrexate, Cytarabine, Fluoruracil, Azathin, Azath

Mercaptopurine and cytotoxic chemotherapeutic agents against cancer 4. Plant alkaloids: Vinblastine, Vincristine, Etoposide, Paclitaxel, 5. Hormones: Tamoxifen, Octreotide acetate, Finasteride, Flutamide 5 6. Modifiers of the biological response: Interferons, Tnterleukins

Pharmacological classifications of rheumatoid arthritis treatment 1. Analgesics: Aspirin 2. NSAIDs (non-steroidal anti-inflammatory drugs): Ibuprofen, Naproxen 10, Diclofenac 3. DMARDs (disease-modifying antirheumatic drugs): Methotrexate, gold preparations, hydroxychloroquine Modified sulfersalazine of the biological response, DMARDs: Etanercept, Infliximab Glucocorticoids, such as beclomethasone, methyl prednisolone, beta-methasone, prednisone, dexamethasone and hydrocortisone

Pharmacological classifications of treatment of diabetes mellitus 1. Sulfonylureas: Tolbutamide, Tolazamide, Glyburide, Glipizide 2. Biguanides: Metformin 20 3. Versatile oral agents: Acarbose, Troglitazone 4. Insulin

Pharmacological classifications of Alzheimer's disease treatment 1. Cholinesterase inhibitor: Tacrine, Donepezil 2. Antipsychotics: Haloperidol, Thioridazine 3. Antidepressants: Desipramine, Fluoxetine, Trazodone, Paroxetine 4. Anticonvulsants: Carbamazepine, Valproic acid

In one embodiment, the compositions of the present invention may comprise a therapeutically effective amount of a RAGE fusion protein in combination with a single or more additional therapeutic agents. In addition to the agents described above, the following therapeutic agents may be used in combination with the RAGE fusion proteins of the present invention: immunosuppressants such as cyclosporin, tacrolimus, rapamycin and other FK-506 type immunosuppressants.

Therefore, in one embodiment, the present invention can provide a method of treating RAGE-mediated diseases, the method comprising administering to a patient in need thereof a therapeutically effective amount of a RAGE fusion protein in combination with therapeutic agents selected from the group consisting of alkylating agents, antimetabolites, plant alkaloids, antibiotics, hormones, biological response modifiers, analgesics, NSAIDs, DMARDs, biological reaction modifiers (eg glucocorticoids), sulfonylureas , biguanides, insulin, cholinesterase inhibitors, antipsychotics, antidepressants, anticonvulsants and immunosuppressants such as cyclosporin, tacrolimus, rapamycin and other FK-506 type immunosuppressants. In another 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, biological response modifiers, analgesics, NSAIDs, DMARDs, biological response modifiers (eg glucocorticoids), sulfonylureas, biguanides, insulin, cholinesterase inhibitors, antipsychotics, antidepressants, anticonvulsants and immunosuppressants such as cyclosporin, tacrolimus, rapamycin, rapamycin -506 type of immunosuppressive agents.

25

Freeze-dried preparations

In other embodiments, the present invention also provides compositions comprising a RAGE fusion protein. Embodiments of the compositions may comprise a freeze-dried mixture of a protective agent for freeze-drying, a RAGE-30 fusion protein, and buffer.

A variety of freeze-drying protective agents can be used in compositions of the freeze-dried RAGE fusion protein of the present invention. In some embodiments, the freeze-drying protective agent may comprise a non-reducing sugar. The non-reducing sugar can include, for example, sucrose, mannitol or trehalose. A variety of buffers can also be used in the freeze-dried RAGE fusion protein preparation. In certain embodiments, the buffer may include histidine.

The freeze-dried RAGE fusion protein may comprise additional ingredients.

In certain embodiments, the RAGE fusion protein composition may further comprise at least one of a surfactant, a chelating agent, or a volume-forming agent. In one embodiment, the reconstituted RAGE fusion protein composition comprises about 40-100 mg / ml RAGE fusion protein comprising the sequence set forth in SEQ ID NOs: 32, 33, 34, 56, 35, 36, 37 or 57; about 2 mM to about 50 mM histidine; about 60 mM to about 65 mM sucrose; about 0.001% to about 0.05% Tween 80; and a pH of about 6.0 to 6.5. The preparation of the reconstituted RAGE fusion protein may in certain embodiments comprise, for example, about 40-50 mg / ml RAGE fusion protein comprising the sequence set forth in SEQ ID NOs: 32, 33, 34, 56, 35, 36, 37 or 57; about 10 mM histidine; about 65 mM sucrose; about 0.01% Tween 80; and a pH of about 6.0. Whether other concentrations of the RAGE fusion protein can be used, as further described herein.

In one embodiment, the present invention comprises a reconstituted preparation comprising a freeze-dried RAGE fusion protein reconstituted in a diluent, wherein the concentration of the RAGE fusion protein in the reconstituted preparation is within the range of about 1 mg / ml to about 400 mg / ml. Or other concentrations of the RAGE fusion protein can be used, as described herein.

In other embodiments, the present invention may also include methods for preparing a stably reconstituted preparation of a RAGE fusion protein. The reconstituted preparation may comprise a concentration suitable for immediate use (e.g., direct administration to a subject) or which may be further diluted and / or mixed with a delivery agent.

In certain embodiments, the method may comprise reconstituting a freeze-dried mixture of the RAGE fusion protein and a protective agent for freeze-drying in a diluent such that the concentration of the RAGE fusion protein in the reconstituted preparation is in a range from about 1 82 mg / ml to about 400 mg / ml. Or other concentrations as described herein can be used as described herein.

A variety of freeze drying protective agents can be used in the compositions of the reconstituted RAGE fusion protein of the present invention. In some embodiments, the freeze drying protective agent may comprise a non-reducing sugar. The non-reducing sugar can include, for example, sucrose, mannitol or trehalose. A variety of buffers can also be used in the freeze-dried RAGE fusion protein preparation. In certain embodiments, the buffer may include histidine.

The preparation of the reconstituted RAGE fusion protein may comprise additional ingredients. In certain embodiments, the RAGE fusion protein preparation may further comprise at least one of a surfactant, a chelating agent, or a volume-forming agent. In one embodiment, the composition of the reconstituted RAGE fusion protein comprises about 40-100 mg / ml of RAGE-15 fusion protein comprising the sequence set forth in SEQ ID NOs: 32, 33, 34, 56, 35, 36, 37 or 57; about 2 mM to about 50 mM histidine; about 60 mM to about 65 mM sucrose; about 0.001% to about 0.05% Tween 80; and a pH of about 6.0 to 6.5. The preparation of the reconstituted RAGE fusion protein may, for example, comprise in some embodiments about 40-50 mg / ml RAGE fusion protein comprising the sequence set forth in SEQ ID NOs: 32, 33, 34, 56, 35, 36, 37 or 57; about 10 mM histidine, about 65 mM sucrose, about 0.01% Tween 80 and at a pH of about 6.0.

A variety of diluents suitable for pharmaceutical agents can be used to reconstitute the freeze-dried RAGE fusion protein. In one embodiment, the freeze-dried RAGE fusion protein is sterile. The diluent is also sterile in one embodiment. In one embodiment, the diluent may comprise water for injection (WFI). In certain embodiments, the amount of the diluent added is based on the therapeutic dosage and the pharmacokinetic profile of the RAGE fusion protein, as well as the biological compatibility of the composition and carrier being administered. In one embodiment, the reconstituted preparation is isotonic.

83

The preparation of the RAGE fusion protein may comprise a stable therapeutic agent prepared for use in a hospital or as a prescribed drug. In certain embodiments, the reconstituted RAGE fusion protein preparation may exhibit less than 10%, or less than 5%, or less than 3% degradation within one week at 40 degrees Celsius.

The preparation of the RAGE fusion protein can also be stable upon reconstitution into a diluent. In certain embodiments, less than about 10% or about 5% or about 4% or about 3% or about 2% or about 1% of the RAGE fusion protein is present as an aggregate in the preparation of the RAGE fusion 10 protein.

The preparation of the reconstituted RAGE fusion protein may be suitable for administration by various routes and as needed for treatment of the RAGE-mediated disorder of interest. In certain embodiments, the reconstituted RAGE fusion protein preparation is suitable for at least one of intravenous, intraperitoneal or subcutaneous administration of the preparation to a subject.

For example, in certain embodiments, the present invention may include a stably reconstituted composition comprising a RAGE fusion protein at a concentration of at least 10 mg / ml or at least 20 mg / ml or at least 50 mg / ml and a diluent, the reconstituted composition of a freeze-dried mixture of the RAGE fusion protein and a protective agent for freeze-drying has been prepared. In other embodiments, the concentration of the RAGE fusion protein in the reconstituted preparation may be at least 100 mg / ml, or at least 200 mg / ml or at least 400 mg / ml. In still other embodiments, the concentration of the RAGE fusion protein in the reconstituted preparation is in an amount within the range of about 0.5 mg / ml to about 400 mg / ml, or about 1 mg / ml to about 200 mg / ml, or about 40 mg / ml to about 400 mg / ml, about 40 to 100 mg / ml or about 40-50 mg / ml. The composition may also comprise a buffer.

In still other embodiments, the present invention can include industrially produced articles that include RAGE fusion proteins. In certain embodiments, the industrially produced article may comprise a container comprising a freeze-dried RAGE fusion protein and instructions for reconstituting the freeze-dried preparation with a diluent. In certain embodiments, the industrially produced articles may include a container 84 containing a composition comprising a freeze-dried mixture of a protective agent for freeze-drying, a RAGE fusion protein, and a buffer. The industrially produced article may also include instructions for reconstituting the freeze-dried preparation with a diluent.

A variety of freeze drying protective agents can be used in the industrially produced articles of the present invention. In some embodiments, the freeze drying protective agent may comprise a non-reducing sugar. The non-reducing sugar can include, for example, sucrose, mannitol or trehalose. A variety of buffers can also be used in the freeze-dried RAGE fusion protein preparation. In certain embodiments, the buffer may include histidine.

The RAGE fusion protein preparation of the industrially produced articles of the present invention may include additional ingredients. In certain embodiments, the freeze-dried RAGE fusion protein preparation may further comprise at least one of a surfactant, a chelating agent or a volume-forming agent. In one embodiment of the articles of manufacture of the present invention, the preparation of the RAGE fusion protein comprises about 40-100 mg / ml of RAGE fusion protein comprising the sequence as set forth by reconstitution according to the instructions provided. in SEQ ID 20 NOs: 32, 33, 34, 56, 35, 36, 37 or 57; about 2 mM to about 50 mM histidine; about 60 mM to about 65 mM sucrose; about 0.001% to about 0.05% Tween 80; and a pH of about 6.0 to 6.5. The preparation of the reconstituted RAGE fusion protein may, for example, comprise in certain embodiments about 40-50 mg / ml RAGE fusion protein comprising the sequence set forth in SEQ ID NOs: 32, 33, 34, 56, 35, 36, 37 or 57; about 10 mM histidine; about 65 mM sucrose; about 0.01% Tween 80; and a pH of about 6.0. Or other concentrations of the RAGE fusion protein can be used, as described herein.

A variety of diluents suitable for pharmaceutical agents can be provided for reconstituting the freeze-dried RAGE fusion protein. In one embodiment, the freeze-dried preparation may be sterile. Alternatively or additionally, the diluent may be sterile. In one embodiment, the diluent may comprise water for injection (WFI) 85. The industrially produced article can thus further comprise a second container containing a diluent for reconstituting the freeze-dried preparation, the diluent being water for injection (WF1). In one embodiment, the reconstituted preparation is isotonic.

In certain embodiments, the amount of the diluent added is also based on the therapeutic dosage and the pharmacokinetic profile of the RAGE fusion protein as well as the biological compatibility of the composition and carrier being administered. In an alternative embodiment, the instructions are for reconstituting the freeze-dried preparation so that the concentrations as described herein are obtained. For example, in certain embodiments, the instructions for reconstituting the freeze-dried formulation are such that the concentration of the RAGE fusion protein in the reconstituted formulation is in the range of about 40 mg / ml to about 100 mg / ml.

The RAGE fusion protein preparation provided as the industrially produced article may comprise a stable therapeutic agent prepared for use in a hospital or as a prescribed drug. In certain embodiments, when reconstituted according to the instructions provided, the RAGE fusion protein may exhibit less than 10%, or less than 5%, or less than 3% degradation after one week at 40 degrees Celsius. The preparation of the RAGE fusion protein can also be stable upon reconstitution into a diluent. In certain embodiments, less than about 10%, or about 5%, or about 4%, or about 3%, or about 2%, or about 1% of the RAGE fusion protein is present as an aggregate in the RAGE preparation. fusion protein.

In certain embodiments, the preparation of the reconstituted RAGE fusion protein, when reconstituted according to the instructions provided, may also be suitable for administration by various routes and as needed for treatment of the RAGE-mediated disorder of interest. In certain embodiments, the reconstituted RAGE fusion protein composition is suitable for at least one of intravenous, intraperitoneal, or subcutaneous administration of the composition to the subject.

In certain embodiments of the compositions, industrially produced articles and methods of making the compositions comprising a RAGE fusion protein, the concentration of the RAGE fusion protein in the reconstituted composition may be at least 10 mg / ml, or at least 20 mg / ml, or at least 50 mg / ml. In other embodiments, the concentration of the RAGE fusion protein in the reconstituted preparation may be at least 100 mg / ml, or 200 mg / ml or 400 mg / ml. For example, in other embodiments, the concentration of the RAGE fusion protein in the reconstituted preparation is at least about 0.5 to 400 mg / ml or about 1 to 200 mg / ml, 40 to 400 mg / ml, 50 to 400 mg / ml ml, 40 to 100 mg / ml, 50 to 100 mg / ml or about 40-50 mg / ml. For example, in one embodiment, the RAGE fusion protein is administered in a preparation as a sterile aqueous solution that has a pH ranging from about 5.0 to about 6.5 and from about 1 mg / ml to about 200 mg / ml RAGE fusion protein, from about 1 millimolar to about 100 millimolar histidine buffer, from about 0.01 mg / ml to about 10 mg / ml polysorbate 80, from about 100 millimolar to about 400 millimolar trehalose and from about 0.01 millimolar to about 1.0 millimolar disodium EDTA dihydrate.

Any of the embodiments described herein can be used as the RAGE fusion protein in the compositions of the present invention. For any of the freeze-dried compositions, the reconstituted freeze-dried compositions or the methods of making the freeze-dried compositions or reconstituted freeze-dried compositions or the industrially produced articles comprising either the freeze-dried compositions or the reconstituted freeze-dried compositions of the present invention, the RAGE may be fusion protein thus comprising a sequence derived from a binding site for a ligand of RAGE linked to an immunoglobulin polypeptide.

Thus, embodiments of the RAGE fusion protein may comprise a RAGE polypeptide that is directly linked to a polypeptide comprising an CH2 domain of an immunoglobulin or a portion of a Cn2 domain of an immunoglobulin as described herein. In certain embodiments, the RAGE polypeptide may comprise an RAGE immunodlobulin domain linked to an 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 linker inter-domain and the C- RAGE terminal amino acid is directly linked to the N-87 terminal amino acid of a polypeptide comprising an CH2 domain of an immunoglobulin or a portion thereof. For example, certain embodiments of the fusion protein may include a first immunoglobulin domain from RAGE and a first interdomain linker from RAGE linked to a second immunoglobulin domain from RAGE and a second interdomain linker from RAGE such that the N-terminal amino acid of the first interdomain linker is linked to the C-terminal amino acid of the first immunoglobulin domain of RAGE, the N-terminal amino acid of the second immunoglobulin domain of RAGE is linked to the C-terminal amino acid of the first linker domain, the N-10 terminal amino acid of the second interdomain linker is linked to the C-terminal amino acid of the second immunoglobulin domain of RAGE and the C-terminal amino acid of the second interdomain linker of RAGE is directly linked to the N-terminal amino acid of the CH2 immunoglobulin domain or part of an Ch2 domain of an immunoglobulin.

In other embodiments, the RAGE polypeptide of the RAGE fusion protein may comprise, for example, the amino acid sequence as set forth in SEQ ID NO: 10 or a sequence that is at least 90% identical thereto or the amino acid sequence as set forth in SEQ ID NO. : 47 or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to it. In other alternative embodiments, the RAGE fusion protein may comprise the amino acid sequence as set forth in at least one of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 56 or 57, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% are identical to this. In certain embodiments, a sequence that is at least 90% identical to SEQ ID NOs: 32, 33, 34, 56, 35, 36, 37 or 57 comprises the polypeptide of SEQ ID NOs: 32, 33, 34, 56, 35, 36, 37 or 57 without the C-terminal lysine.

Preparation of freeze-dried preparations

In another embodiment, the present invention provides a composition prior to freeze-drying, a freeze-dried preparation, a reconstituted preparation, and methods of preparation thereof.

After preparation of a RAGE fusion protein of interest as described above, a "preparation before freeze-drying" can be produced. The 88 amount of the RAGE fusion protein present in the preparation prior to lyophilization can be determined by taking into account the desired volumes of dosage, method (s) of administration, and so on. In one embodiment, the amount of the fusion protein in the composition prior to lyophilization may be higher than 1 mg / ml. In certain embodiments, the amount of fusion protein in the composition prior to lyophilization may also be lower than about 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml or 200 mg / ml.

In another embodiment, the composition may be a pH buffered solution at a pH of about 4-8 prior to freeze drying. In another embodiment, the composition may be a pH buffered solution at a pH of about 5-7 prior to freeze drying. In another embodiment, the composition may be a pH buffered solution at pH below 6.7 prior to freeze drying. In another embodiment, the composition may be a pH buffered solution at a pH of about 6.0 prior to freeze drying. Illustrative buffers include histidine, phosphate, Tris, citrate, succinate, and other organic acids as described herein. The concentration of the buffer may be from about 1 mM to about 100 mM or less than about 50 mM or from about 2 mM to about 50 mM or less than about 15 mM or from about 3 mM to about 15 mM, depending on, for example, the buffer and the desired isotonicity of the preparation (e.g. the reconstituted preparation). In one embodiment, the buffer is histidine.

The freeze drying protective agent can be added to the composition prior to freeze drying. In one embodiment, the freeze drying protective agent comprises a sugar. In another embodiment, the freeze-drying protective agent comprises a non-reducing sugar. In another embodiment, the freeze drying protective agent comprises the non-reducing sugar sucrose. Or the non-reducing sugar may comprise mannitol. Or the non-reducing sugar can include trehalose. The amount of the freeze drying protective agent in the composition prior to freeze drying is generally such that upon reconstitution, the resulting composition will be isotonic. However, a hypertonically reconstituted preparation may also be useful, for example, in preparations for peripheral parenteral administration. In addition, the amount of the freeze drying protective agent should not be so low that 89 an unacceptable amount of protein degradation and / or aggregation occurs due to freeze drying. In other embodiments, an unacceptable amount of aggregation can be when 20%, or 10%, or 5% or more of the RAGE fusion protein is present as an aggregate in a composition. An illustrative range of freeze-drying protective agent concentrations in the composition prior to freeze-drying may be less than about 400 mM. In another embodiment, the range of concentrations of freeze drying protective agent in the composition prior to freeze drying is less than about 100 mM. In other embodiments, the range of concentration of freeze-drying protective agent in the composition prior to freeze-drying may therefore range from about 0.5 mM to 400 mM or from about 2 mM to 200 mM or from about 30 mM to about 150 mM or from approximately 60-65 mM. In some embodiments, the freeze drying protective agent is also added in an amount to make the reconstituted composition isotonic.

The ratio of RAGE fusion protein to freeze drying protective agent in the preparation prior to freeze drying is selected for each combination of RAGE fusion protein and freeze drying protective agent. In an embodiment of an isotonically reconstituted preparation with a high concentration of RAGE fusion protein (e.g., greater than or equal to about 50 mg / ml), the molar ratio of freeze drying to RAGE fusion protein protective agent can be from about 50 to about 1500 moles of lyophilizing protective agent to 1 mole of RAGE fusion protein. In another embodiment, the molar ratio of freeze drying protective agent to RAGE fusion protein can be from about 150 to about 1000 moles of freeze drying protective agent to 1 mole fusion protein. In another embodiment, the molar ratio of freeze drying protective agent to RAGE fusion protein can be from about 150 to 300 mole freeze drying protective agent to 1 mole of RAGE fusion protein. These ranges may be suitable, for example, when the freeze drying protective agent is a non-reducing sugar such as sucrose, trehalose or mannitol.

In another embodiment of the invention, a surfactant can be added to the composition prior to freeze drying. Alternatively or additionally, the surfactant can be added to the freeze-dried preparation and / or the reconstituted preparation. Illustrative 90 surfactants include non-ionic surfactants such as polysorbates (e.g., polysorbates 20 or 80) (Tween 20 ™ or Tween 80 ™); poloxamers (e.g., poloxamer 188). The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particulate material after reconstitution. The surfactant may be present in the composition prior to lyophilization in, for example, an amount of about 0.001% to 0.5%. For example, in an embodiment wherein the surfactant comprises polysorbate 80, the surfactant may be present in the composition prior to freeze-drying in an amount of about 0.005% to 0.05% or about 0.008% to 0.012% or at about 0.01%. Alternatively, the surfactant may be present in the composition such that it comprises a final concentration ranging from 0.001 mg / ml to about 100 mg / ml, or about 0.01 mg / ml to about 10 mg / ml.

In certain embodiments of the invention, a mixture of the freeze drying protective agent (such as sucrose or histidine) and a volume-forming agent (e.g., mannitol or glycine) can be used in the preparation of the composition prior to freeze drying. The volume-forming agent can enable the production of a uniform freeze-dried cake without extensive cavities therein, and so on.

Other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Editor (1980) may be included in the preparation prior to freeze-drying (and / or the freeze-dried preparation and / or the reconstituted Preparation) provided that they do not adversely affect the desired characteristics of the preparation. Acceptable carriers, excipients, or stabilizing agents are non-toxic to recipients at the dosages and concentrations used and include additional buffering agents; preservatives; cosolvents; antioxidants including ascorbic acid and methionine; Chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers such as polyesters; and / or salt-forming counter ions such as sodium.

91

The RAGE fusion protein compositions of the present invention may also contain additional proteins as needed for the specific indication being treated. The additional proteins can be selected such that the proteins each have complementary activities that do not adversely affect each other or the RAGE fusion protein. Such proteins are suitably present in combination at amounts effective for the purpose intended.

The RAGE fusion protein compositions of the present invention can be sterile for in vivo administrations. This can be accomplished by filtration through sterile fllation membranes, prior to, or following freeze drying, and reconstitution.

After the RAGE fusion protein, freeze drying protective agent, and other optional ingredients have been mixed together, the composition can be freeze dried. Many different freeze dryers are available for this purpose such as Hull50 ™ (Huil, USA) or GT20 ™ (Leybold-Heraeus, Germany) freeze dryers. Freeze drying is accomplished by freezing the composition and subsequently subliming the ice from the frozen contents at a temperature suitable for primary drying. Under this condition, the temperature of the product is lower than the eutectic point or collapse temperature of the preparation. The temperature of storage for the primary drying will typically range from about -50 ° C to 25 ° C (provided that the product remains frozen during the primary drying) at a suitable pressure, which typically ranges from about 50 to 250 mTorr . In one embodiment, the pressure is about 100 mTorr and the sample can be freeze-dried between about -30 ° C and 25 ° C. The preparation, the size and type of the container containing the sample (for example a glass tube) and the volume of the liquid 25 can determine the time that may be required for drying, which can vary from a few hours to several days ( for example 40-60 hours). Conditions for freeze-drying can be varied depending on the preparation and the size of the tube.

In some cases, it may be desirable to perform lyophilization of the protein preparation in the container in which reconstitution of the protein is to be performed in order to prevent transfer steps. The container can in this case for example be a tube of 2, 3, 5, 10, 20, 50, 100 or 250 cc. In one embodiment, the container is any container suitable for preparing a reconstituted preparation that has a volume of less than or equal to 100 ml.

As a general issue, freeze drying will result in a freeze-dried preparation where its moisture content is less than about 5%. In an embodiment the moisture content of the freeze-dried preparation is lower than about 3%. In another embodiment, the moisture content of the freeze-dried preparation is less than about 1%.

Reconstitution of the freeze-dried preparation At the desired time, typically when it is time to administer the RAGE fusion protein to a patient or person, the freeze-dried preparation can be reconstituted with a diluent such that the concentration of the RAGE fusion protein in the reconstituted preparation is approximately higher than 10 mg / ml or higher than 20 mg / ml or higher than 50 mg / ml or approximately 30-50 mg / ml or approximately 50 mg / ml. In other embodiments, the concentration of the RAGE fusion protein in the reconstituted preparation may be at least 100 mg / ml, or 200 mg / ml or 400 mg / ml. In other embodiments, the concentration of the RAGE fusion protein in the reconstituted preparation may be, for example, in the range of from about 1 mg / ml to about 600 mg / ml or from about 1 mg / ml to about 500 mg / ml or from about 1 mg / ml to about 400 mg / ml or from about 1 mg / ml to about 200 mg / ml or from about 10 mg / ml to about 400 mg / ml or from about 10 mg / ml to about 200 mg / ml , or from about 40 mg / ml to about 400 mg / ml, or from about 40 mg / ml to about 200 mg / ml or from about 50 mg / ml to about 400 mg / ml, or from about 50 mg / ml to approximately 200 mg / ml. In other embodiments, the concentration of the RAGE fusion protein in the reconstituted preparation is from about 40 mg / ml to about 100 mg / ml or about 50 mg / ml to about 100 mg / ml, or about 40 mg / ml to approximately 50 mg / ml. Such concentrations of the RAGE fusion protein in the reconstituted preparation are considered to be particularly useful when considering subcutaneous delivery of the reconstituted preparation. However, for other routes of administration, such as intravenous administration, lower concentrations of the protein in the reconstituted preparation may be desired (e.g., from about 5-50 mg / ml, or from about 10-40 mg / ml RAGE fusion protein in the reconstituted preparation). 93 reconstituted preparation). Thus, in some embodiments, the concentration of the fusion protein in the reconstituted preparation may be the same or lower than 2 times the concentration of the fusion protein in the preparation prior to lyophilization.

In certain embodiments, the concentration of the fusion protein in the reconstituted preparation is considerably higher than that in the preparation prior to lyophilization. The concentration of the fusion protein in the reconstituted preparation may, for example, be in some embodiments about 2-40 or 2-10 or 3-8 times that of the preparation prior to lyophilization. In one embodiment, the concentration of the RAGE fusion protein in the reconstituted preparation may be about 3-6 times that of the preparation prior to lyophilization. In another embodiment where the concentration of the RAGE fusion protein in the preparation prior to freeze drying is about 15 mg / ml, the concentration of the RAGE fusion protein in the reconstituted preparation is greater than or equal to about 50 mg / ml ml (for example at least threefold or at least four times higher).

The delivery of a high protein concentration is often advantageous or necessary for subcutaneous administration due to volume restrictions (less than or equal to 1.5 ml) or dosage requirements (more than or equal to 100 mg). However, protein concentrations (more than or equal to 50 mg / ml) may be difficult to obtain in the preparation process because at high concentrations a protein may tend to aggregate during processing and become difficult to treat (e.g. pump) and sterile filtering. Alternatively, the freeze drying method can provide a method to allow concentration of a protein. For example, a RAGE fusion protein can be filled into tubes at a volume (Vf) and then freeze-dried. The freeze-dried RAGE fusion protein is then reconstructed with a smaller volume (Vr) of water or preservative (e.g. BWFI) than the original volume (e.g. Vr = 0.25 Vf) that is in a higher concentration of the RAGE fusion protein results in the reconstituted solution. This method also results in the concentration of the buffers and excipients. For subcutaneous administration, the solution is preferably isotonic.

Reconstitution generally takes place at a temperature of about 25 ° C to ensure complete hydration, although other temperatures may be used as desired. For example, the time required for reconstitution may depend on the type of diluent, the amount of excipient (s) and protein. Illustrative diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (for example, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In one embodiment, the diluent provides a reconstituted preparation suitable for injection. In another embodiment, wherein the diluent provides a reconstituted preparation suitable for injection, the diluent comprises water for injection (WFI). The diluent optionally contains a preservative. The amount of the preservative that is used can be determined by determining different concentrations of preservative for testing for compatibility with the protein and efficacy as a preservative.

In other embodiments, the reconstituted composition may have less than 8,000 or less than 6,000 or less than 4,000 or less than 2,000 or less than 1,000 or less than 600 or less than 400 or less than 200 or less than 100 or less than 50 particles that are equal in size or larger than 10 µm per 50 ml container. In other embodiments, the reconstituted composition may have less than 8,000 or less than 6,000 or less than 4,000 or less than 2,000 or less than 1,000 or less than 600 or less than 400 or less than 200 or less than 100 or less than 50 particles are equal in size or larger than 25 µm per 50 ml container.

Administration of the reconstituted preparation

The reconstituted preparation can be administered in accordance with known methods to a mammal in need of treatment with the RAGE-fiction protein, such as a human, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal , intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes.

In embodiments, the reconstituted composition may be administered to the mammal by subcutaneous (i.e., under the skin) administration. For such purposes, the reconstituted preparation can be injected by the use of a syringe. However, other devices for administering the reconstituted preparation 95 are available such as devices for injection (e.g., the Inject-ease ™ and Genject ™ devices); injection pens (such as the GenPen ™); needleless devices (e.g. MediJector ™ and Biojector ™); and subcutaneous delivery systems in the form of plasters.

The appropriate dosage or therapeutically effective amount of the RAGE fusion protein will depend, for example, on the condition to be treated, the severity and course of the condition, whether the RAGE fusion protein is administered for preventive or therapeutic purposes. , previous therapy, the medical history of the patient and the response to the RAGE fusion protein and the judgment of the attending physician. The RAGE fusion protein can be administered to the person (e.g., patient) at one time or during a series of treatments or can be administered at any time from diagnosis. The RAGE fusion protein can be administered as the sole treatment or in conjunction with other drugs or therapies useful for treating the condition of interest. In one embodiment, a dosage of about 0.1-20 mg / kg is an initial candidate dosage for administration to the subject or, for example, by one or more separate administrations. As described above, other dosage strategies may be useful.

20 Objects produced industrially

In another embodiment of the invention, an industrially produced article is provided which contains the freeze-dried preparation of the present invention and provides instructions for reconstitution and / or use thereof. The industrially produced article may comprise a container. Suitable containers include, for example, vials, tubes (for example, dual chamber tubes), syringes (such as dual chamber syringes) and test tubes. The container can be formed from a variety of materials such as glass or plastic. The container may comprise the freeze-dried preparation. In certain embodiments, a marker may be attached or associated with the holder. The label can indicate instructions for reconstitution and / or use. For example, in certain embodiments, the label may indicate that the freeze-dried preparation is reconstituted to protein concentrations as described above. The label may further indicate that the composition is useful or intended for subcutaneous administration.

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The container containing the preparation may be a multi-use tube allowing for repeated administrations (e.g., from 2-6, or 2-10, or 2-50 administrations) of the reconstituted preparation. The industrially produced article may further comprise a second container comprising a suitable diluent (e.g. WF1). By mixing the diluent and the freeze-dried preparation, the final concentration of the RAGE fusion protein in the reconstituted preparation will generally be at least 10 mg / ml. In one embodiment, the final concentration of the RAGE fusion protein in the reconstituted preparation is at least about 20 mg / ml. In another embodiment, the final concentration of the RAGE fusion protein in the reconstituted preparation is at least about 50 mg / ml. In other embodiments, the concentration of the RAGE fusion protein in the reconstituted preparation may be at least 100 mg / ml, or 200 mg / ml, or 400 mg / ml. In other embodiments, the final concentration of the RAGE fusion protein in the reconstituted preparation is between about 1-400 mg / ml, or 1-200 mg / ml, or 1-100 mg / ml, or 10-400 mg / ml ml, or 10-200 mg / ml, or 10-100 mg / ml, or from 40-400 mg / ml, or from 40-200 mg / ml, or from 40-100 mg / ml, or from 50-400 mg / ml, or from 50-200 mg / ml, or from 50-100 mg / ml, or from 40 mg / ml to 50 mg / ml. The industrially produced article may further comprise other materials that are desirable from a commercial standpoint and from a user standpoint, including other buffers, diluents, filters, needles, syringes, and package attachments with instructions for use.

EXAMPLES

Features and advantages of the inventive concept included in the present invention are further illustrated in the examples that follow. Example IA: Production of RAGE fusion proteins

Two plasmids were constructed to express RAGE-IgG fusion proteins. Both plasmids were constructed by ligating 30 different lengths of a 5 "cDNA sequence of human RAGE with the same 3" cDNA sequence of human IgG Fc (γΐ). These expression sequences (i.e., ligation products) were then inserted into expression vector pcDNA3.1 (Invitrogen, CA). The nucleic acid sequences encoding the coding region of the RAGE-97 fusion protein are shown in Figures 2 and 3. For the RAGE fusion protein TTP-4000, the nucleic acid sequence of 1 to 753 (marked in bold) encodes the N-terminal protein sequence of RAGE, while the nucleic acid sequence of 754 to 1386 encodes the Fc protein sequence of IgG without the hinge 5 (Figure 2). For TTP-3000, the nucleic acid sequence from 1 to 408 (marked in bold letters) encodes the N-terminal protein sequence of RAGE, while the nucleic acid sequence from 409 to 1041 encodes the Fc protein sequence of IgG without the hinge (Figure 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 transformants were selected for neomycin resistance conferred and cloned by the plasmid. High production clones, as detected by Westem blot analysis of supernatant, were expanded and the gene product was purified by affinity chromatography using Protein A columns. Expression was optimized such that cells produced recombinant TTP-4000 at levels of approximately 1.3 grams per liter.

Example 1B: Other production of four domain RAGE fusion proteins

A plasmid was constructed to express RAGE IgG

fusion proteins. The plasmid was constructed by ligating a 5 "cDNA sequence of human RAGE with a 3" cDNA sequence of human IgG (γΐ) without the hinge region. PCR was used to amplify the cDNA. At the 5 'end, the PCR primer further added a site for the Eco RI restriction enzyme for cloning and a Kozak consensus translation initiation sequence. At the 3 'end, the PCR primer added an Xho I restriction site just after the termination codon. At the 3 'end, the PCR primer also included two silent base changes that remove a cryptic RNA cleavage site in the immunoglobulin portion near the terminal codon. The codon encoding proline 30 (residue 459 based on the protein sequence numbering of SEQ ID NO: 32) was changed from CCG to CCC and the codon encoding glycine (residue 460 based on the protein sequence numbering of SEQ ID NO: 32) was changed from GGT to GGG. The PCR fragment was digested with Eco RI and Xho I and 98 then inserted into a retrovector plasmid (pCNS-newMCS-WPRE (new ori), available from Gala, Inc.) that had been digested with Mfe I (to form a compatible end with Eco RI) and digested with Xho I. Of the inserted part of the cloned plasmid and the transitions of cloning, the sequence 5 was analyzed to ensure that no mutations occurred during the cloning.

To produce the RAGE IgG fusion protein, the expression vector comprising the nucleic acid sequence SEQ ID NO: 54 was stably transfected into CHO cells.

The sequence of the isolated RAGE fusion protein TTP-4000, which was expressed by the transfected cells, was confirmed by various studies for characterization as either SEQ ID NO: 34 or SEQ ID NO: 56, or both SEQ ID NO: 34 and SEQ ID NO: 56. The signal sequence encoded by the first 23 amino acids of SEQ ID NO: 32 was thus split and the N-terminal residue was glutamine (Q) or pyroglutamic acid (pE) or a mixture thereof . Characterizing studies also showed glycosylation sites on N2 and N288 (based on the numbering of SEQ ID NO: 34 or SEQ ID NO: 56) and showed that the Ch3 region of the RAGE fusion protein may have removed the C-terminal residue by post-translational modification when expressed in this recombinant system.

Example 1C: Other production of three domain RAGE fusion proteins

A plasmid can be constructed to express three-domain RAGE-IgG fusion proteins (e.g., one RAGE domain and two IgG domains) such as TTP-3000 in the manner described above for TTP-4000. The plasmid was constructed by ligating a 5 "cDNA sequence of human RAGE encoding amino acids 1-136 of human RAGE with a 3" cDNA sequence of human IgG Fc (γ1) without the hinge region. PCR can be used to amplify the cDNA. At the 5 'end, a PCR primer 30 may add a restriction site (e.g., an site for an Eco RI restriction enzyme as used for TTP-4000) for cloning and a Kozak consensus translation initiation sequence. At the 3 'end, the PCR primer can also add a restriction site (for example, an Xho 1 restriction site) just after the 99 termination codon. The PCR primer also includes silent base changes as may be needed to remove any cryptic RNA cleavage site, such as the cryptic RNA cleavage sites located at the 3 'end of the C ^ domain of the immunoglobulin, as in Example 1B. To remove these cryptic cleavage sites, the codon encoding proline 344 of SEQ 1D NO: 35 (ie residues 1030-1032 based on the DNA sequence numbering of SEQ ID NO: 31) can be changed from CCG to CCC and the codon encoding glycine 345 of SEQ ID NO: 35 (residues 1033-1035 based on the DNA sequence numbering of SEQ ID NO: 31) can be changed from GGT to GGG. The PCR fragment can then be digested with the appropriate restriction enzymes (e.g., Eco RI and Xho I) and inserted into the retrovector plasmid pCNS-newMCS-WPRE (new ori; available from Gala, Ine.). The vector can be digested with Mfel to form a compatible RI with Eco RI and also digested with Xho I. From the inserted part of the cloned plasmid and the transitions from cloning, the sequence can be analyzed to ensure that no mutations have occurred during the cloning.

To produce the RAGE IgG fusion protein, the expression vector comprising the nucleic acid sequence SEQ ID NO: 55 (ie which includes the alteration of the DNA sequence to remove cryptic cleavage sites) can be stably converted into CHO- cells are transfected as described in Examples 1A and 1B.

The sequence of the isolated RAGE fusion protein TTP-3000, which is expressed by the transfected cells, can be either SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 57 or a combination of SEQ ID NO: 36, SEQ ID NO: 37 and / or SEQ ID NO: 57. The signal sequence encoded by the first 22 and / or 23 amino acids of SEQ ID NO: 35 can thus be cleaved and the N-terminal residue can be glutamine (Q) or pyroglutamic acid (pE) or a mixture thereof. Glycosylation can occur at sites N2 and NI74 (based on the numbering of SEQ ID NO: 37 or SEQ ID NO: 57) and / or other glycosylation sites that may be present. The CH3 region of the RAGE fusion protein may have removed its C-terminal residue from it by post-translational modification when expressed in this recombinant system.

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Example 2: Method for testing activity of a RAGE-IgG1 fusion protein A. Binding of ligand in vitro Known RAGE ligands were coated on the surface of Maxisorb plates at a concentration of 5 micrograms per well. Plates were incubated overnight at 4 ° C. Following the incubation with the ligand, the plates were aspirated and a 1% BSA blocking buffer in 50 mM imidizole buffer (pH 7.2) was added to the plates for 1 hour at room temperature. The plates were then filtered off with suction and / or washed with wash buffer (20 mM imidizole, 150 mM NaCl, 0.05% Tween-20, 5 mM CaCb and 5 mM MgCb, 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 pg / ml were prepared. The RAGE fusion protein was added with increasing dilutions of the initial sample. The RAGE fusion protein was allowed to incubate with the immobilized ligand for one hour at 37 ° C after which the plate was washed and tested for binding of the RAGE fusion protein. Binding was detected by the addition of an immunodetection complex containing a monoclonal mouse anti-human IgG1 diluted 1: 11,000 to a final test concentration (FAC) of 21 ng / 100 μΐ, a biotinylated goat anti-mouse IgG 1 : 500 is diluted to an FAC of 500 ng / μΐ and contains an avidin-linked alkaline phosphatase. The complex was incubated with the immobilized RAGE fusion protein for one hour at room temperature, after which the plate was washed and the substrate for alkaline phosphatase, para-nitrophenyl phosphate (PNPP) was added. Binding of the complex to the immobilized RAGE fusion protein was quantified by measuring the conversion of PNPP to para-xophenol (PNP) which was measured spectrophotometrically at 405 nm.

As illustrated in Figure 7, the RAGE fusion proteins TTP-4000 (TT4) and TTP-3000 (TT3) exhibit specific interaction with known RAGE 30 amyloid-beta ligands (Abeta), SI00b (SI00) and amphoterin (Ampho) . In the absence of ligand, i.e. coating with BSA alone (BSA or BSA + wax), there was no increase in absorbance above levels due to non-specific binding of the immunodetection complex. When amyloid beta is used as the labeling 101 ligand, it may be necessary to incubate the amyloid beta prior to the test. Prior incubation may allow the amyloid beta itself to aggregate in the form of a pleated sheet, since amyloid beta RAGE preferably binds in the form of a pleated sheet.

Additional evidence for a specific interaction between RAGE fiction proteins TTP-4000 and TTP-3000 with RAGE ligands is illustrated in studies showing that a RAGE ligand is able to compete effectively with a known RAGE ligand for binding to the RAGE fusion proteins. In these studies, amyloid beta (A beta) was immobilized on a Maxisorb plate and RAGE fusion protein was added as described above. In addition, a ligand of RAGE was added to some of the wells at the same time as the RAGE fusion protein.

It was found that the ligand of RAGE could block the binding of TTP-4000 (TT4) by about 25% to 30% when TTP-4000 was present at 123 pg / ml (1: 3 dilution, Figure 8). When the initial solution of TTP-4000 was diluted by a factor of 10 or 30 (1:10 or 1:30), the binding of the RAGE fusion protein to the immobilized ligand was completely inhibited by the ligand of RAGE. Similarly, the RAGE ligand blocked binding of TTP-3000 (TT3) by about 50% when TTP-3000 was present at 360 pg / ml (1: 3 dilution, Figure 9). When the initial solution of TTP-3000 was diluted by a factor of 10 (1:10), the binding of the RAGE fusion protein to the immobilized ligand was completely inhibited by the ligand of RAGE. Specificity of binding of the RAGE fusion protein to the RAGE ligand was thus dose dependent. In Figures 8 and 9 it is also shown that essentially no binding was detected in the absence of RAGE fusion protein, i.e. with the use of only the immunodetection complex ("Complex only").

B. Effect of RAGE fusion protein in cell-based assays

Previous work has shown that the myeloid THP-1 cells can secrete TNF-α in response to RAGE ligands. In this test, THP-1 cells were grown in RPMI-1640 media supplemented with 10% FBS using a protocol provided by ATCC. The cells were induced to secrete TNF-α by stimulation of RAGE with 0.1 mg / ml SI00b both in the absence of 102 and in the presence of RAGE fiction proteins TTP-3000 (TT3) or TTP-4000 (TT4) (10 pg), sRAGE (10 pg) and a human IgG (10 μβ) (ie as a negative control). The amount of TNF-α secreted by the THP-1 cells was measured 24 hours after the addition of the proteins to the cell culture using a commercially available ELISA kit for TNF-α (R&D Systems, Minneapolis) , MN). The results in Figure 10 show that the RAGE-fiction proteins inhibited the S100b / RAGE-induced production of TNF-α in these cells. As shown in Figure 10, by adding 10 µg of the RAGE-fiction proteins, TTP-3000 or TTP-4000 induction of TNF-α by SI00b (0.1 mg / ml FAC) was increased by approximately 45% to 10 70, respectively % reduced. TTP-4000 fusion protein can be just as effective in blocking TNF-α induction by SI00b as sRAGE (Figure 10). Specificity of the inhibition for TTP-4000 and TTP-3000 RAGE sequences has been shown by the experiment in which IgG was only added to SI00b stimulated cells. Addition of IgG and SI00b to the test shows the same levels of TNF-α as SI00b alone. Specificity of the inhibition of TNF-α induction by TTP-4000 and TTP-3000 for sequences of the RAGE fusion protein has been shown by an experiment in which IgG was only added to SlOOb-stimulated cells. It can be seen that the addition of IgG, i.e. human IgG without the sequence of RAGE (Human IgG from Sigma added at 10 pg / well) and SI00b to the test shows the same levels of TNF-20α as S100b alone.

In another cell-based assay, the ability of TTP-4000 to prevent the RAGE, HMGB1, ligand from interacting with RAGE and other HMGB1 receptors was assessed. Unlike anti-RAGE antibodies that bind to RAGE and prevent the interaction of a RAGE ligand with RAGE, TTP-4000 can block the interaction of a RAGE ligand with RAGE by binding to the RAGE ligand. HMGB1 is reported to be a ligand for RAGE and Toll-like receptors 2 and 4 (Parit et al., J. Biol. Chem., 2004; 279 (9): 7370-7). All of these three receptors (RAGE, Toll-like receptor 2 and Toll-like receptor 4) are expressed on THP-1 cells (Parker, et al., J. Immunol., 2004, 172 (8): 4977- 86). In this experiment, THP-1 cells were stimulated to produce TNF-α by HMGB1 (50 mg / ml) in the presence or absence of either TTP4000 or anti-RAGE antibodies. Under the conditions used in the test, HMGB1 should be the only inducer of TNFa. The amount of TNFα secreted by the 103 THP-1 cells was measured 24 hours after the addition of the proteins to the cell culture using a commercially available ELISA kit for TNF-α (R&D Systems, Minneapolis, MN) . The results in Figure 11 show that the anti-RAGE antibody and RAGE fusion protein block TTP-4000 HMGB1 to respond with RAGE expressed on TFIP-1 cells, but that TTP-4000 HMGB1-induced inhibits TNF-α production to a greater extent than the anti-RAGE antibody does. The data thus indicate that TTP-4000 can inhibit HMGB1 activity to a greater extent than the anti-RAGE antibody by inhibiting HMGB1 to interact with Toll-like receptors 2 and 4 as well as the RAGE that is on THP-1 cells is present.

Example 3: Pharmacokinetic profile of TTP-4000

To determine whether TTP-4000 would have a superior pharmacokinetic profile compared to human sRAGE, rats and non-human primates received an intravenous (IV) injection of TTP-4000 (5 mg / kg) and then plasma was tested for presence of TTP-4000. In these experiments, two naive male monkeys received a single IV bolus dose of TTP-4000 (5 mg / ml / kg) in a peripheral vein followed by a saline flush of approximately 1.0 milliliter (ml). Blood samples (approximately 1.0 ml) were taken prior to the dose (ie prior to injection of the TTP-4000) or after 0.083, 0.25, 0.5, 2, 4, 8, 12, 24, 48, 72, 96, 120, 168, 240, 288 and 336 hours after the dose were collected in vials containing lithium heparin. Following collection, the tubes were placed on wet ice (up to 30 minutes) until centrifugation with cooling (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 it was tested for RAGE polypeptide using an ELISA at various times following the injection as described in Example 6.

The kinetic profile shown in Figure 12 revealed that once TTP-4000 is saturated with its ligands, as shown by the reasonable 30 steep slopes of the alpha phase in 2 animals, it retains a terminal half-life of more than 300 hour. This half-life is considerably higher than the half-life of human sRAGE in plasma (generally about 2 hours) and provides a possibility for single injections for acute and semi-chronic indications. In Figure 104, each curve represents a different animal under the same experimental conditions.

Example 4: TTP-4000 Fc Activation Experiments were performed to measure the activation of the Fc receptor by RAGE-fiction protein TTP-4000 compared to human IgG. Activation of the Fc receptor was measured by measuring secretion of TNF-α from THP-1 cells expressing the Fc receptor. In these experiments, a 96-well plate was coated with 10 µg / well of TTP-4000 or human IgG. Fc stimulation results in excretion of TNF-α. The amount of TNF-α was measured by an enzyme-linked immune-absorbing test (ELISA).

In this test, the myeloid cell line THP-1 (ATTC # TIB-202) was maintained in RPMI-1640 media supplemented with 10% fetal bovine serum according to ATCC instructions. Typically, 40,000-80,000 cells per well were induced to secrete 15 TNF-alpha by stimulation of the Fc receptor by pre-coating the wells with 10 pg / well of either heat-aggregated (63 ° C for 30 minutes) TTP-4000 or human IgG1. The amount of TNF-alpha secreted by the THP-1 cells was measured in supernatants collected from 24-hour cultures in the treated wells using a commercially available TNF ELISA kit (R&D Systems) , Minneapolis, MN # DTA00C) according to instructions. Results are shown in Figure 13 where it can be seen that TTP-4000 generated less than 2 ng / well of TNF and IgG generated more than 40 ng / well.

Example 5: In vivo activity of TTP-4000

The activity of TTP-4000 was compared with sRAGE in various in vivo models of human disease.

A. TTP-4000 in an animal model of restenosis The RAGE-fiction protein TTP-4000 was assessed in a model of restenosis of a diabetic rat that involved measuring smooth muscle proliferation and intima extension 21 days following vascular damage . In these experiments, damage to the left general carotid artery was performed by a balloon at 105

Zucker diabetic and non-diabetic rats by using a standard method. A loading dose (3 mg / rat) of IgG, TTP-4000 or phosphate buffered saline (PBS) was administered intraperitoneally (IP) one day prior to injury. A maintenance dose was delivered every other day until day 7 after damage 5 (i.e. on days 1, 3, 5 and 7 after damage). The maintenance dose was high = 1 mg / animal for one group or low = 0.3 mg / animal for the second group. To measure the proliferation of vascular smooth muscle cells (VSMC), animals were sacrificed 4 days and 21 days after the injury.

For the measurement of cell proliferation, animals received an intraperitoneal injection of bromodoxyuridine (BrDdU) 50 mg / kg after 4 days at 18, 12 and 2 hours prior to euthanasia. After the sacrifice, the entire left and right carotid arteries were harvested. Samples were stored in Histochoice for at least 24 hours before the samples were embedded. Determination of proliferation of VSMC was performed using mouse anti-BrdU monoclonal antibody. A fluorescently labeled goat anti-mouse secondary antibody was administered. The number of BrdU-positive nuclei per coupe was counted by two observers who were not aware of the treatment strategies.

The remaining rats were sacrificed after 21 days for morphometric analysis. Morphometric analyzes were performed by an observer who was not aware of the groups being studied, using computerized digital microscopic planimetry software Image-Pro Plus on successive sections, (5 mm distance) carotid arteries stained with Van Gieson staining. All data were expressed as mean ± SD. Statistical analysis was performed with the use of SPSS software. Continuous variables were compared using unpaired t tests. A value of P <0.05 was considered to be statistically significant.

As seen in Figures 14A and 14B, reduced treatment with TTP-4000 significantly reduced intima / media ratios and proliferation of vascular smooth muscle cells in a dose-responsive manner. In Figure 14B, the y-axis represents the number of BrdU proliferating cells.

B. TTP4000 in an animal model of AD

106

Experiments were performed to assess whether TTP-4000 could influence amyloid formation and cognitive dysfunction in a mouse AD model. The experiments used 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 (Αβ). It has been shown above that treatment with sRAGE for 3 months reduced both the formation of amyloid plaques in the brain and the associated increase of markers of inflammation in this model.

The APP mice (male) used in this experiment were made by micro-injection of the human APP gene (with the Swedish and London mutations) into mouse eggs under the control of the promoter of the platelet-derived gene growth factor B (PDGF-B) chain. The mice were generated on a C57BL / 6 background and were developed by Molecular Therapeutics Inc. Animals were fed ad libitum and held by crossings between brothers and sisters. The mice generated from this construct developed amyloid deposits starting at 6 months of age. Animals were bred up to the age of 6 months and then held for 90 days and sacrificed for quantification of amyloid.

APP transgenic mice were given adjuvant or TTP4000 [qod (i.p.)] every other day for 90 days starting at 6 months of age. At the end of the experiment, animals were sacrificed and examined for the presence of Αβ plaques in the brain (i.e., number of plaques). A 6-month control APP group was used to determine the baseline of amyloid deposition. In addition, the animals were subjected to behavioral analysis at the end of the study (Morris water maze (maze)). The researchers were not informed of the compounds that were being investigated. Samples were given to the mice at 0.25 ml / mouse / every other day. In addition, one group of mice received 200 pg / day of human sRAGE.

1. Deposition of amyloid beta

For histological examination, the animals were anesthetized with an intraperitoneal injection (IP) of sodium pentobarbital (50 mg / kg). The animals were transcardially perfused with phosphate buffered saline (PBS) at 4 ° C, followed by 107 4% paraformaldehyde. The brains were removed and placed in 4% paraformaldehyde overnight. The brain was worked up to paraffin and embedded. Ten consecutive 30 µm thickness sections through the brain were obtained. Sections were subjected to primary antibody (Αβ 5 peptide antibody) overnight at 4 ° C to detect amyloid deposits in the brains 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 temperature. After washing, sections were incubated as instructed in the Vector ABC 10 Elite kit (Vector Laboratories) and stained with diaminobenzoic acid (DAB). The reactions were stopped in water and covered with a coverslip after treatment with xylene. The amyloid area in each coupe was determined with a computer-assisted image analysis system, which consists of a Power Macintosh computer equipped with a Quick Capture frame grabber card, Hitachi CCD camera mounted on an Olympus microscope and a camera standard. NIH Image Analysis Software, v. 1.55 was used. The images were recorded and the total area of amyloid was determined over the ten sections. A single investigator who was not aware of any status of treatment performed all measurements. Addition of the amyloid volumes of the sections and dividing by the total number of sections was performed to calculate the amyloid volume.

For quantitative analysis, an enzyme-linked immune-absorbing test (ELISA) was used to measure the levels of human total Αβ, Αβίο ^ ι and Αβι.42 in the brains of APP transgenic mice (Biosource International, Camarillo, CA). Αβίοΐωΐ and Αβι-42 were extracted from the brain of mouse by guanidine hydrochloride and quantified as described by the manufacturer. This test extracts the total Αβ peptide from the brain (both soluble and aggregated).

2. Cognitive function

Testing with the Morris water maze (maze) was performed as follows: All 30 mice were tested once in the Morris water maze (maze) test at the end of the experiment. Mice were trained in a water maze with an open field of 1.2 m. The bath was filled with water to a depth of 30 cm and the water was kept at a temperature of 25 ° C. The escape platform (10 square cm) was placed 108 cm under the water surface. The platform was removed from the bath during the tests. The cued (with clues) testing was performed in the bath that was surrounded with white curtains to hide all extra cues (clues) for the maze. All animals underwent non-spatial pre-training ("non-spatial pre-training"; NSP) for three consecutive days. These tests are for preparing the animals for the final behavioral test for determining the retention of memory for finding the platform. These tests were not recorded, but were for training purposes only. For the studies of training and learning, the 10 curtains for extra cues (clues) were removed from the maze (this made it possible to identify animals with damage to the ability of swimming). On day 1, the mice were placed on the hidden platform for 20 seconds (test 1), during tests 2-3 the animals were released at a distance of 10 cm from the cued (with directions) platform or hidden platform (test 4) allowed to swim to the platform. On the second day of the experiments, the hidden platform was randomly moved between the center of the bath or the center of each quadrant. The animals were released into the bath, head-to-wall randomly, and allowed to reach the platform for 60 seconds (3 experiments). At the 20th third trial, the animals received three trials, two with a hidden platform and one with a cued (with clues) platform. Two days after the NSP, animals were subjected to the final behavioral tests (Morris water maze (maze) test). For these experiments (3 per animal), the platform was placed in the middle of one quadrant of the bath and the animals were released with the head facing the wall in a random manner. The animal was allowed to find the platform or swim for 60 seconds (latency period, the time it takes to find the platform). All animals were tested within 4-6 hours of dosing and randomly selected for testing by a researcher who was not aware of the test groups.

30 The results are expressed as the mean ± standard deviations (SD). The significance of differences in the studies of amyloid and the behavioral studies were analyzed using a t-test. Comparisons were made between the APP control group of 6 month old mice and the TTP-4000 treated 109 animals as well as an APP treated group and the 9 month old TTP-4000 treated animals. Differences of less than 0.05 were considered significant. Percentage changes in amyloid and behavior were determined by summing the data from each group and dividing by the comparison (i.e., 1.5 i.p./6 months of control =% change).

Figures 15A and 15B show that mice treated with either TTP-4000 or mouse sRAGE for 3 months have fewer Αβ-plaques and less cognitive dysphuction than animals treated with vehicle and human IgG1 (IgG1) that serves as a negative control. These data indicate that TTP-4000 is effective in lowering pathology of AD in a transgenic mouse model. It was also found that just like sRAGE TTP-4000 can lower the inflammatory cytokines IL-1 and TNF-α (data not shown).

C. Efficacy of TTP-4000 in an animal model of stroke TTP-4000 was also compared with sRAGE in a disease-relevant animal model for stroke. In this model, the middle carotid artery of a mouse was ligated for 1 hour followed by reperfusion for 23 hours, at which point the mice were sacrificed and the area of brain infarction determined. Mice were treated with sRAGE or TTP-4000 or control immunoglobulin immediately prior to reperfusion.

In these experiments, male C57BL / 6 were injected with excipient at 250 μΐ / mouse or TTP test articles (TTP-3000, TTP-4000 at 250 μΐ / mouse). Mice were injected intraperitoneally, 1 hour after the initiation of ischemia. Mice were subjected to one hour of cerebral ischemia followed by reperfusion for 24 hours. To induce ischemia, each mouse was anesthetized and body temperature was maintained at 36-37 ° C by external heating. The left general carotid artery (CCA) was exposed by a midline incision in the neck. A microsurgical clamp was placed around the origin of the internal carotid artery (ICA). The distal end of the ECA was ligated with silk and cut transversely. A 6-0 silk was knotted loosely around the ECA stump. The flame-polished end of a nylon suture was carefully inserted into the ECA stub. The loop of the 6-0 side was tightened around the stump and the nylon suture was further inserted into and through the internal carotid artery 110 (ICA) until it was placed in the anterior cerebral artery thereby occluding the anterior communicating and middle cerebral artery . After the nylon suture was inserted for 1 hour, the animal was again anesthetized, the rectal temperature was recorded, and the suture was removed and the incision closed.

The volume of the infarction was determined by anesthetizing the animals with an intraperitoneal injection of sodium pentobarbital (50 mg / kg) and then removing the brain. The brains were then cut into four 2 mm sections through the infarction region and placed in 2% triphenyl tetrazolium chloride (TTC) for 30 minutes. The sections were then placed in 4% paraformaldehyde overnight. The area of the infarction in each coupe 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 a camera standard. NIH Image Analysis Software, 15 v. 1.55 was used. The images were recorded and the total area of the infarction was determined over the sections. A single researcher who was not aware of the status of treatment conducted all experiments. The total volume of the infarction was calculated by adding up the volumes of the infarction of the sections. The results are expressed as the mean ± standard deviation (SD). The significance of the difference of the infarction volume data was analyzed using a t-test.

As illustrated by the data in Table 2, TTP-4000 was more effective than sRAGE in limiting the infarction area in these animals, suggesting that TTP-4000, due to its better half-life in plasma, was able to better protection in these mice.

Example 6: Detection of RAGE fusion protein by ELISA

First, 50 μΐ of the RAGE-specific 1HB1011 monoclonal antibody was coated onto plates by overnight incubation at 10 µg / ml in IX PBS pH 7.3. When ready for use, the plates were washed three times with 300 μΐ of IX imidazole-Tween wash buffer and blocked with 1% BSA. The samples (diluted) and standard dilutions of known dilutions of TTP-4000 are added at a final volume of 100 μΐ. The monsters

They are allowed to incubate for one hour at room temperature. After incubation, the plates are washed three times. An anti-human IgG1 1 (Sigma A3312) AP conjugate of goat in 1XPBS with 1% BSA is added and allowed to incubate for 1 hour at room temperature. The plates are washed three times. Color 5 is developed with paranitrophenyl phosphate.

Example 7: Quantification of binding of RAGE ligand to RAGE fusion protein

Figure 16 shows the TTP-4000 saturation binding curves to several immobilized known RAGE ligands. The ligands are immobilized on a microtiter plate and incubated in the presence of increasing concentrations of RAGE fusion protein from 0 to 360 nM. The interaction of RAGE fusion protein ligand is detected by the use of a polyclonal antibody conjugated to alkaline phosphatase specific for the IgG portion of the fusion chimera. Relative Kds were calculated using Graphpad Prizm software and correspond to the values for RAGE ligand values of RAGE set in the literature. HMG1B = Amphoterin, CML = Carboxymethyllysine, A beta = Amyloid beta 1-40.

Example 8: Use of RAGE fusion protein to prevent allogeneic transplant rejection

Blockage of RAGE can be expected to block allogeneic graft rejection. These experiments investigated whether blockage of interactions of ligand-RAGE through the use of a RAGE fusion protein of the invention would weaken rejection of islet cells transplanted from a healthy donor to a diabetic animal as measured by the time period during which the transplanted animals can maintain a blood glucose level below a target concentration. As discussed herein, it was found that administration of a RAGE fusion protein (e.g., TTP-4000) to diabetic animals that had received islet cell transplants significantly delayed the return of hyperglycemia and thus rejection of islet transplanted cells at two (allogeneic and syngeneic) models of transplant animals.

112 A. Allogeneic island transplantation in mice

The first set of experiments tested whether administration of a RAGE fusion protein (TTP-4000) the allogeneic rejection of transplanted islet cells and the return of diabetes in a model of diabetes of a C57BL / 6J (B6) mouse 5 could modulate.

Animal model of diabetes C57BL / 6J (6-8 weeks old) (B6) mice were made diabetic by a single intravenous injection of streptozotocin (STZ) (Sigma Chemical Co., St. Louis, 10 MO) at 200 mg / kg. BALB / cJ (6-8 weeks old) (BALB) mice served as donors for islet transplantation thus providing an allogeneic mismatch for islet transplantation.

Islet isolation 15 Mice (BALB / c) were anesthetized with ketamine HCl / xylazine HCl solution (Sigma, St. Louis MO). After intraductal injection of 3 ml of cold Hank's balanced saline solution (HBSS, Gibco, Grand Island, NY) containing 1.5 mg / ml of collagenase P (Roche Diagnostics, Branchburg, NJ), pancreas were surgically obtained for 20 minutes at 37 ° C digested. The islets were washed with HBSS and purified by discontinuous gradient centrifugation using Polysucrose 400 (Cellgro, Hemdon VA) which has four different densities (26%, 23%, 20% and 11%). The tissue fragments at the interface of the 20% and 23% layers were collected, washed and resuspended in HBSS. Individual islets that are free from connected acinar, vascular and ductal tissues were carefully selected under an inverted microscope to provide highly purified islets for transplantation.

Transplantation of islets

Streptozotocin-induced diabetic C57BL / 6 (B6) mice received islet grafts within 2 days of diagnosis. BALB / cJ (6-8 weeks old) (BALB) mice served as donors for allogeneic islet transplantation. For transplantation, 500-600 freshly isolated islets (i.e., approximately 113 550 islet equivalents) from donor mice were taken with an infusion set and transplanted into the right-hand kidney subcapsular space of a recipient.

Treatment with test compounds Test compounds were administered at the time the islets were transplanted; administration was continued for approximately 60 days depending on how the control animal behaves. Mice were injected with 0.25 ml of either phosphate buffered saline (PBS), TTP-4000 in PBS or IgG in PBS according to the strategy below (Table 3).

Table 3

Administration of test compounds and / or excipient Test group Number of mice Loading dose Maintenance dose Strategy

Untreated 8 control

Control with 8 0.25 ml / dose / mouse 0.25 ml / dose / mouse Once every other day

excipient on day 1 starting on day 2 (QOD) x 60 days; IP

(PBS) liÖ 8 (300 pg) (100 Mg) (100 pg)

0.25 ml / dose / mouse 0.25 ml / dose / mouse Once every other day on day 1 starting on day 2 (QOD) x 60 days; IP

TTP-4000 8 I (300 pg) (100 pg) (100 pg)

0.25 ml / dose / mouse 0.25 ml / dose / mouse Once every other day on day 1 starting on day 2 (QOD) x 60 days; IP

TTP-4000 δ (300 µg) (3 µg δ (3 µggj

0.25 ml / dose / mouse 0.25 ml / dose / mouse Once every other day on day 1 starting on day 2 (QOD) x 60 days; IP

Tracking the function of the islet graft

The functioning of the islet graft was followed by daily sequential measurement of blood glucose during the first 2 weeks after islet transplantation, followed by measuring every other day. Diabetes reversal was defined if the blood had a glucose level of less than 200 mg / dl in two consecutive measurements. Loss of the graft was determined when blood glucose exceeds 250 mg / dl in two consecutive measurements. The results are shown in Table 4.

114

Table 4

Effects of TTP-4000 on allogeneic islet transplantation of islets *

TTP-4000 TTP-4000 µgG

300 pg LD + 300 pg + 30 300 pg LD + Non-100 pg qod ip PBS μg qod ip 100gqipip treated (Group 1) (Group 2) (Group 3) (Group 4) control 14 9 Ï3 8 9 16 8 14 9 8 Ï3 TÖ Ï2 ÏÖ 9 13 8 12 8 TÖ 12 Π Π 8 9 16 8 Π 8 8 15 8 8 9 Π 14 8 8 Π 9 7 9 8 9

Average 25J25 8/75 17/25 8/575 8.833333 ~ SD 1.457738 1.164965 2.141212 1.129999 1.029857 ~ n 8 8 8 8 12 * Values in Table 4 reflect the day of graft loss for each animal as defined by return of elevated levels of glucose in the blood

The effects of TTP-4000 administration on BALB / c islet allogeneic graft rejection in B6 mice are shown as a Kaplan-10 Meier Cumulative survival graph in Figure 17. It can be seen that there is an increase in time before graft failure is detected for animals treated with TTP-4000 (Groups 1 and 3) as opposed to animals not treated at all (Control) or animals treated with the adjuvant (PBS) or human IgG1 . By using a variety of statistical analyzes (Mantel-Cox Logrank, Breslow-Gehan-Wilcoxon; Tarone-Ware, Peto-Peto-Wilcoxin; and Harrington-Fleming) the differences between the Control and TTP-4000 (Groups 1 and 3) significant (Table 5).

115

Table 5

Statistical method Control versus Group 1 Control versus Group 3 (TTP-4000) (TTP-4000)

Chi-square DF * P-value Chi-square DF P-value Logrank (Mantel-Cox) 18.777 ï <0.0001 7.6262 ï 0.0056

Breslow-Gehan-Wilcoxone 15,092 1 0,0001 4,904 1 0.0268

Tarone-Ware 16.830 ≤ 0.0001 6.212 ≤ 0.0127

Peto-Peto-Wilcoxon 14.359 0.0002 4.315 0.0378

Harrington Fleming (rho = 0.5) 16.830 1 <0.0001 6.212 1 0.0127 * Freedom degrees 5 B. Islet transplantation in NOD mice as a model of autoimmune disease

In the second set of experiments, it was tested whether administration of RAGE fusion protein (i.e., TTP-4000 or TTP-3000) would modulate the course of recurrent diabetes in NOD mice by using a synogenic NOD model of 10 transplant.

Animal models of diabetes

Spontaneous autoimmune non-obese diabetic mice (NOD / LU) (12-25 weeks old) served as islet cells, while young pre-15 diabetic NOD / LtJ mice (6-7 weeks old) served as donors to syngeneic transplantation of islets. Islets for transplantation were isolated as described above in Part A (Allogeneic islet transplantation).

Islet transplantation: Diabetic NOD / LU mice received islet grafts within 2 days of diabetes diagnosis. 500-600 freshly isolated islets (approximately 550 islet equivalents) from donor mice were taken with an infusion set and transplanted into the subcapsular space of the right kidney.

Treatment with test compounds 116

Test compounds were administered at the time the islets were transplanted and administration was continued for approximately 8 weeks. Mice were injected with 0.25 ml of either PBS, TTP-4000 in PBS or TTP-3000 in PBS according to the strategy outlined below (Table 6).

5

Table 6

Group Number of mice Volume Volume Strategy loading dose maintenance dose TTP-4000 8 (300 pg) (100 pg) (100 pg)

0.25 ml / dose / mouse 0.25 ml / dose / mouse Once every other day on day 1 starting on day 2 (QOD) x 8 weeks; IP

TTP-3000 8 (300 pg) (100 pg) (100 pg)

0.25 ml / dose / mouse 0.25 ml / dose / mouse Once every other day on day 1 starting on day 2 (QOD) x 8 weeks; IP

PBS 8 0.25 ml / dose / mouse 0.25 ml / dose / mouse Once every other day

on day 1 starting on day 2 (QOD) x 8 weeks; IP

Tracking the function of the islet graft

The functioning of the islet graft was followed by successively measuring daily glucose of the blood during the first 2 weeks after the islet transplantation, followed by measuring every other day. Diabetes reversal was defined if the blood had a glucose level of less than 200 mg / dl in two consecutive measurements. Loss of the graft was determined when blood glucose exceeds 250 mg / dl in two consecutive measurements. The results are shown in Table 7.

Table 7

Effects of TTP-4000 and TTP-3000 on diabetes return in islet syngeneic transplantation in NOD mice * TTP-4000 TTP-3000 Control 300 pg LD + 100 pg 300 pg + qod ip (Group 1) 100 pg qod ip ( Group 2) 35 44 23 38 46 25 40 42 26 43 41 22 36 34 22 117 45 32 24 44 30 21 38 2Ö _ _ 24

Average 39 ^ 875 38.42857 22.727273 ~ SD 3.758324 6.32079 1.8488326 ~ n 8 7 Π * Values reflect the day of transplant loss for each animal as defined by return of elevated levels of glucose in the blood

The effects of TTP-4000 administration on rejection of syngeneic transplanted islets in diabetic NOD mice are shown as a Kaplan-Meier Cumulative survival graph in Figure 18. As shown in the data in Table 7, there was an increase in time before graft failure is detected for animals treated with TTP-4000 (Group 1) and TTP-3000 (Group 2) as opposed to animals that have not been treated at all (Control). Figure 18 shows the increase in time prior to detection of graft failure for animals treated with TTP-4000 (Group 1) and animals not treated at all. By using a variety of statistical analyzes (Mantel-Cox Logrank, Breslow-Gehan-Wilcoxon; Tarone-Ware, Peto-Peto-Wilcoxin; Harrington-Fleming) it was shown that the differences between the Control and TTP-4000 (Group 1) and the Control and TTP-3000 (Group 2) are significant (Table 8).

Table 8

Statistical method Control versus Group 1 Control versus Group 2 (TTP-4000) (TTP-3000)

Chi-square DF * P-value Chi-square DF P-value

Logrank (Mantel-Cox) 18.410 1 <0.0001 16.480 1 <0.0001

Breslow-Gehan-Wilcoxon 14.690 1 0.0001 12.927 1 0.0001

Tarone-Ware 16,529 ï 0.0001 14.686 ï 0.0001

Peto-Peto-Wilcoxon 14.812 1 0.0001 13.027 1 0.0003

Harrington-Fleming (rho = 0.5) 16.529 1 <0.0001 14.686 1 0.0001 * Freedom degrees 118

Example 9 - Lyophilized preparation of RAGE fusion protein

In the development of a freeze-dried preparation using the RAGE fusion protein TTP-4000, freeze-drying and buffering protective agents were first screened by measuring the stability of the protein after freeze-drying and reconstitution. The freeze-dried protein in each preparation was also subjected to accelerated stability studies to determine the possible stability of the protein during its shelf life.

In initial screening studies, experiments were designed to assess the conditions of preparation that could provide appropriate solubility and stability for TTP-4000 that is prepared as a frozen amount and that provide reconstituted compositions that contain concentrations of the RAGE fusion protein at about 50 mg / ml or higher. Preparations containing sodium acetate, sodium citrate, sodium phosphate, sodium succinate, histidine and sodium chloride were tested together with sucrose and mannitol. Various pHs between 5.5 and 7.0 were also assessed.

The biophysical and chemical stability of TTP-4000 was assessed using size exclusion chromatography (SEC), SDS-PAGE, Fourier-Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), peptide mapping and ultraviolet - visible absorption (UV-Vis).

Based on the solubility of the RAGE fusion protein in preparations containing one or more of sodium citrate, histidine, sucrose, mannitol and Tween 80 at a pH of 7.0 or lower, preparations containing one or more of these buffers contain freeze drying or surfactant protective agents chosen for further study.

25

Example 10 - Lyophilized preparation of RAGE fusion protein.

Based on the information collected in Example 9, additional preparations for TTP-4000 were investigated. The studies were aimed at identifying the freeze drying buffers and / or protective agents useful for maintaining a stable product by the freeze drying method and possibly obtaining a high concentration of TTP-4000 during reconstitution (ie of approximately or higher than 50 mg / ml). Six preparations were examined during an accelerated stability study of 2 weeks. The compositions are 119 summarized in Table 9. Studies also focused on developing a freeze drying cycle for higher doses of TTP-4000 (250 mg) as described below.

Following lyophilization, the sample tubes for development of the preparation were sealed by melting and placed in a 40 ° C chamber for 2 weeks. The samples for developing upscaling were stored at 2-8 ° C until testing was performed.

Methods for analysis of the sample The concentration of TTP-4000 in samples was measured by UV-Vis spectrophotometry. An Aglient UV-Vis was used to obtain protein spectra as well as spectra of buffer as a blank. Once obtained, the values of absorption were corrected for any scattering of light that can occur as a result of any protein aggregation.

Remaining moisture was analyzed using a Karl Fisher titration method. Freeze-dried samples for developing a preparation were reconstituted with the correct amount of WFI. The time of reconstitution was considered to be the time from the time the water was added to the time that no visible solids were present. The pH of each sample after reconstitution was measured using a properly calibrated semi-micro probe.

Size exclusion (SEC) chromatography was performed using a TSKgel Super SW2000 column to analyze or monitor the physical stability (degradation and formation of soluble aggregates) of TTP-4000 during lyophilization and storage under accelerating conditions. Samples were injected into an Aglient 1100 series LC equipped with two TSKgel Super SW2000, 4.6 x 300 mm, 4 µm columns (Tosoh Bioscience, 18674).

To measure the particle count, a 1 ml sample was diluted 20-fold in 20 ml. The sample was degassed by sonication for about 30 seconds and by gently pivoting manually without introducing bubbles. Three amounts, each with a volume of 5 ml, were taken in the light-darkened counter sensor. When the device was set in a cumulative manner 120, particles were collected at settings greater than or equal to 10 μιη and greater or equal to 25 μιη.

Results: Based on the preliminary preparation studies of Example 9, two buffers were initially investigated: sodium citrate and L-histidine. The concentrated compositions 1-6 (Table 9) were prepared by centrifuge concentrators. The final protein concentration in the compositions 1-6 in Table 9 was in a range of between about 4-15 mg / ml.

10

Table 9. TTP-4000 preparations

Sodium citrate Histidine Sucrose Mannitol Tween 80 pH

(mM) (mM) (mM) (raM) (%)

1 ÏÖ 6Ö M

~ 2 ÏÖ 3Ö 5Ö M

~ 3 1Ö 6Ö Ö ^ ÏÏ M

~ 4 60 60 M

1 30 30 6 6 0

~ 6 ÏÖ 6Ö m

By using preparations 1-6 in Table 9, tubes (2 ml) were used with volumes for filling 0.7 ml. The space above the liquid ("headspace") in the tube was filled with air. Prior to drying, samples were frozen at a storage temperature of between -50 ° C to -20 ° C for approximately 12 hours. The samples were dried at a reduced pressure of 100 mTorr and a storage temperature of -20 ° C and -10 ° C for approximately 36 hours, followed by a storage temperature of 20 ° C for approximately 12 hours. Following freeze drying, lids were placed on the tubes and the top was melted. Solid cakes were produced from each of the compositions 1-6.

The freeze-dried products were subjected to accelerated stability studies to determine the chemical stability of the compositions. The freeze-dried products were placed for 2 weeks on storage at 40 ° C and 75% relative humidity.

121

The freeze-dried products were reconstituted with 0.206 ml of WFI. All freeze-dried products were reconstituted within 20 seconds or less. The pH remained constant with all reconstituted preparations during the 2 week storage period. Residual moisture values determined for the freeze-dried products at time 0 and 2 weeks were between 3.0% and 0.8% and showed that freeze-drying cycle was able to sufficiently dry the pre-freeze-drying preparations . The osmolarity of all reconstituted compositions was within a desired isotonic range between 250 mOsm / kg and 400 mOsm / kg (see Table 10). The viscosity of each reconstituted preparation was lower than 3.7 cP (centiPoise).

Table 10. Reconstituted preparations of TTP-4000 and osmolarity

Sodium citrate Histidine Sucrose Mannitol Tween 80 Osmolarity (mM) (mM) (mM) (mM) (%) (mOsm / kg) 1 ÏÖ 60 322 ~ 2 ÏÖ 30 50 385 1 ÏÖ 60 Öjïl 324 1 TÖ 60 264 1 ÏÖ 3Ö 50 336 "6 6 6 Ö analyse 26 262 SEC analysis was performed on samples of preparation 5 in Table 10 taken at various steps during the lyophilization process and indicate that in these preparations TTP-4000 was not particularly sensitive to any of the steps of the freeze drying process based on the constant low levels of aggregated material and material that has degraded.

SEC analysis was also performed on the compositions prior to freeze-drying before performing freeze-drying as well as on the reconstituted compositions at time zero and after an accelerated stability study of 2 weeks. The amount of impurities (aggregate or degraded product) was constantly low (i.e., less than 4%) (Table 11).

Table 11. Reconstituted preparations of TTP-4000 and% intact protein

Sodium Histidine Sucrose Mannitol Tween 80% intact% intact protein 122 citrate (mM) (mM) (mM) (%) protein (T = 0) (T = 2 wk) 1 ÏÖ 6Ö 963 973 ~ 2 ÏÖ 3Ö 5Ö 96 ^ 8 973 1 ÏÖ 6Ö ÖÏÖÏ 963 973 1 ÏÖ 6Ö 973 973 1 ÏÖ 3Ö 50 973 963 ~ 6 ÏÖ 6Ö ÖiÖÏ 97/7 96Ï9

Peptide mapping revealed no oxidation or deamination of TTP-4000 by lyophilization or storage under accelerating conditions.

SDS-PAGE was performed on compositions 1, 3, 4 and 6 and showed that TTP-4000 retained physical stability during the freeze drying process and storage in these compositions.

To scale up the freeze drying method for use with 50 ml freeze drying tubes and a dose of 250 mg TTP-4000, samples were prepared by concentrating the solution to 15 mg / ml TTP-4000 using ultrafiltration and then diafilter the solution against 10 mM histidine and 65 mM sucrose at pH 6.0. Tween 80 was added to a final amount of 0.01% (vol / vol).

The preparation prior to freeze drying (16.67 ml) was added to each 50 ml tube. The samples were exposed to a freeze drying cycle in which the samples were cooled for 30 minutes at a storage temperature of between 5 ° C and -5 ° C, followed by cooling at a storage temperature of -50 ° C for approximately 3 hours. The samples were dried at a reduced pressure of 100 mTorr and a storage temperature of between -20 ° C and -10 ° C for approximately 34 hours, followed by a storage temperature of between 5 ° C and 20 ° C for approximately 11 hours. Following freeze drying, a lid was placed on the tubes and the top was melted shut. A pharmaceutical excellent white cake was produced. The cake appeared firm and did not lose its structure during handling and storage.

The concentration of the reconstituted sample, as determined by absorbance at 280 nm, was 40.5 mg / ml TTP-4000. In additional studies, under similar conditions, the concentration of TTP-4000 in the reconstituted sample was constantly about 50 mg / ml.

123

The reconstituted sample was measured for particle content after 0, 2 and 6 hours of storage at room temperature. The results in Table 12 show that the reconstituted sample had a low amount of particulate material.

5

Table 12. Number of particles detected in the reconstituted sample during storage

Size of particles Time = 0 Time = 2 hours Time = 6 hours Particles per ml> 10 pm 562 368 948> 25 pm 8 16 20

Particles per container> 10 pm 2753 1803 4645> 25 pm 39 78 98

In summary, TTP-4000 was prepared in citrate and histidine buffers containing one or more of sucrose, mannitol and Tween 80. Tests showed that compositions containing sodium citrate or histidine had similar characteristics of execution and that TTP-4000 may be more soluble in compositions containing histidine.

Further scaling studies focused on preparations containing histidine. Following a freeze drying cycle, the chemical and physical stability of TTP-4000 was assessed and no significant differences between the histidine preparations were detected. Mannitol was also excluded from the preparation. The final preparation selected from the study of developing a preparation contained 10 mM histidine, 60 mM sucrose and 0.01% Tween 80 at about pH 6.0. This preparation showed a superior ability to keep the TTP-4000 stable during lyophilization and storage and also provided the highest concentration of TTP-4000 during the study. In the scaling study, the level of sucrose was increased to 65 mM to adjust the osmolarity of the preparation closer to isotonicity. During the scaling study, it was also found that maintaining the pH of the TTP-4000 preparation prior to lyophilization and reconstituted preparation at or near pH 6.0 and below 6.7 was a useful pH for lowering of precipitation or aggregation of the protein.

The foregoing is only considered illustrative of the principle of the invention. Since numerous modifications and 124 changes can be readily apparent to those skilled in the art, it is not intended to limit the invention to the precise embodiments as they have been shown and described and all suitable modifications and equivalents that are within the scope of protection. of the appended claims are considered to fall within the concept of the present invention.

Claims (55)

A composition comprising a freeze-dried mixture of a freeze-drying protective agent, a RAGE fusion protein and a buffer, wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ 1D NO: 34, without the C- terminal lysine. 2. A composition comprising a freeze-dried mixture of a freeze-drying protective agent, a RAGE fusion protein and a buffer, wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 34. The composition of claim 1 or 2, wherein the freeze drying protective agent comprises a non-reducing sugar. The composition of claim 3, wherein the non-reducing sugar comprises at least one of sucrose, mannitol or trehalose. The composition of claim 1 or 2, wherein the buffer comprises histidine. The composition of claim 1 or 2, further comprising at least one of a surfactant, a chelating agent or a volume-forming agent. A reconstituted preparation comprising a freeze-dried RAGE fusion protein reconstituted in a diluent, the concentration of the A reconstituted preparation comprising a freeze-dried RAGE fusion protein reconstituted in a diluent, wherein the concentration of the RAGE fusion protein in the reconstituted preparation is within the range of about 1 mg / ml to about 400 mg / ml, and wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 34. A reconstituted preparation according to claim 7 or 8, wherein the diluent comprises water for injection (WFT). A reconstituted preparation according to claim 7 or 8, wherein the reconstituted preparation is suitable for subcutaneous or intramuscular administration. The reconstituted preparation of claim 7 or 8, wherein the reconstituted preparation is isotonic. The reconstituted composition of claim 7 or 8, wherein the composition is about 1-400 mg / ml of the RAGE fusion protein; about 1 mM to about 100 15 mM histidine buffer; about 60 mM to about 65 mM sucrose; and about 0.001% to about 0.05% Tween 80; and has a pH of about 6.0 to 6.5. A reconstituted preparation according to claim 7 or 8, wherein the preparation is about 40-50 mg / ml of the RAGE fusion protein; about 10 mM histidine; and about 65 mM sucrose; about 0.01% Tween 80; and has a pH of about 6.0. The reconstituted composition of claim 7 or 8, wherein the composition exhibits less than 5% degradation after 1 week at 40 degrees Celsius. 25 The reconstituted composition of claim 7 or 8, wherein less than about 10% of the RAGE fusion protein is present as an aggregate in the composition after reconstitution. A reconstituted preparation according to claim 7 or 8, wherein the freeze-dried preparation of the RAGE fusion protein comprises a protective agent for freeze-drying. The reconstructed preparation of claim 16, wherein the freeze drying protective agent comprises a non-reducing sugar. A reconstituted preparation according to claim 17, wherein the non-reducing sugar comprises at least one of sucrose, mannitol or trehalose. A reconstituted preparation according to claim 7 or 8, wherein the freeze-dried preparation of the RAGE fusion protein comprises at least one of a buffer, a surfactant, a chelating agent or a volume-forming agent. 10 An article comprising a container containing a freeze-dried RAGE fusion protein and instructions for reconstituting the freeze-dried preparation with a diluent and wherein the RAGE fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 34, without the C-terminal lysine. 15 21. Object comprising a container containing a freeze-dried RAGE fusion protein and instructions for reconstituting the freeze-dried preparation with a diluent and wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 34. 20 The article of claim 20 or 21, wherein the diluent comprises water for injection (WFI). 23. Article according to claim 20 or 21, wherein the reconstituted preparation is suitable for subcutaneous or intramuscular administration. The article of claim 20 or 21, wherein the reconstituted preparation is isotonic. The article of claim 20 or 21, wherein by reconstitution according to the instructions, the composition comprises about 1-400 mg / ml of the RAGE fusion protein; about 1 mM to about 100 mM histidine buffer; about 60 mM to about 65 mM sucrose; and about 0.001% to about 0.05% Tween 80; and has a pH of about 6.0 to 6.5. RAGE fusion protein in the reconstituted preparation is within the range of about 1 mg / ml to about 400 mg / ml, and wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 34, without the C-terminal lysine. An article according to claim 20 or 21, wherein by reconstitution the preparation is about 40-50 mg / ml of the RAGE fusion protein; about 10 mM histidine; about 65 mM sucrose; and about 0.01% Tween 80; and has a pH of about 6.0. 27. The article of claim 20 or 21, wherein the instructions for reconstituting the freeze-dried preparation are such that the concentration of the RAGE fusion protein in the reconstituted preparation is in the range of about 40 mg / ml to about 100. mg / ml. The article of claim 20 or 21, wherein the freeze-dried preparation 15 is sterile. An article according to claim 20 or 21, further comprising a second container containing a diluent for reconstituting the freeze-dried composition, wherein the diluent is water for injection (WFI). 20 The article of claim 20 or 21, wherein by reconstitution according to the instructions, the composition exhibits less than 5% degradation after 1 week at 40 degrees Celsius. The article of claim 20 or 21, wherein after reconstitution, less than about 10% of the RAGE iusion protein is present in the composition as an aggregate. The article of claim 20 or 21, wherein the freeze-dried material of the RAGE fusion protein comprises a protective agent for freeze-drying. The article of claim 32, wherein the freeze drying protective agent comprises a non-reducing sugar. The article of claim 32, wherein the non-reducing sugar comprises at least one of sucrose, mannitol or trehalose. The article of claim 20 or 21, wherein the freeze-dried RAGE fusion protein comprises at least one of a buffer, a surfactant, a chelating agent, or a volume-forming agent. 36. A method for preparing a stable reconstituted preparation of a RAGE fusion protein comprising reconstituting a freeze-dried mixture of the RAGE fusion protein and a protective agent for freeze-drying in a diluent such that the concentration of the RAGE fusion protein in the reconstituted preparation is in a range of from about 1 mg / ml to about 400 mg / ml and wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 34, without the C-terminal lysine. 37. Method for preparing a stable reconstituted preparation of a RAGE fusion protein, comprising reconstituting a freeze-dried mixture of the RAGE fusion protein and a protective agent for freeze-drying in a diluent such that the concentration of the RAGE fusion protein in the reconstituted preparation is in a range of from about 1 mg / ml to about 400 mg / ml and wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 34. The method of claim 36 or 37, wherein the freeze drying protective agent comprises a non-reducing sugar. The method of claim 38, wherein the freeze drying protective agent comprises at least one of sucrose, mannitol or trehalose. 30 The method of claim 36 or 37, wherein the freeze-dried mixture further comprises at least one of a buffer, a surfactant, a chelating agent, or a volume-forming agent. The method of claim 36 or 37, wherein the reconstituted composition is about 40-100 mg / ml of the RAGE fusion protein; about 2 mM to about 60 mM histidine; about 60 mM to about 65 mM sucrose; and about 5 0.001% to about 0.05% Tween 80; and has a pH of about 6.0 to 6.5. 42. The method according to claim 36 or 37, further comprising preparing the freeze-dried RAGE fusion protein by freeze-drying a mixture comprising the RAGE-fiction protein and a freeze-drying amount of a freeze-drying protective agent. The composition of any one of claims 1 to 19 for the treatment of a RAGE-mediated disorder in a subject. The composition of claim 43, wherein the composition is for at least one of intravenous, intraperitoneal or subcutaneous administration to the subject. 45. A composition according to claim 43 for use in treating a symptom of diabetes or a symptom of late complications of diabetes. 46. A composition according to claim 45, wherein the symptom of diabetes or late complications of diabetes comprises at least one of diabetic nephropathy, diabetic retinopathy, a diabetic foot ulcer, a cardiovascular complication, or diabetic neuropathy. 47. The composition of claim 43 for use in treating at least one of amyloidoses, Alzheimer's disease, cancer, renal failure or inflammation associated with autoimmunity, inflammatory bowel disease, rheumatoid arthritis, psoriasis, multiple sclerosis, hypoxia, stroke, heart attack , haemorrhagic shock, sepsis, organ transplantation or weakened wound healing. The composition of claim 43 for use in treating osteoporosis. The composition of claim 48, wherein the treatment of osteoporosis comprises increasing the bone density of the person or lowering the rate of decrease of the bone density of a person. The composition of claim 47, wherein the autoimmunity rejection of at least one of skin cells, pancreas cells, nerve cells, muscle cells, endothelial cells, heart cells, liver cells, kidney cells, a heart, bone marrow cells, bone, blood cells, arterial cells, venous cells , cartilage cells, thyroid cells or stem cells. The composition of claim 43 for use in treating renal failure. 52. A composition according to claim 43 for use in treating inflammation and / or rejection associated with transplantation of at least one of an organ, a tissue or a variety of cells from a first location to a second location. The composition of claim 52, wherein the first and second sites are with different people. The composition of claim 52, wherein the first and second sites are with the same person. 55. A composition according to claim 52, wherein the transplanted cells, tissue or organ is a cell, tissue or organ of a pancreas, skin, liver, kidney, heart, bone marrow, blood, bone, muscle, artery, vein, cartilage, thyroid gland, nervous system or stem cells.