RAGE fusion proteins and methods of use
Disclosed are RAGE fusion proteins comprising RAGE polypeptide sequences linked to a second, non-RAGE polypeptide. The RAGE fusion protein may utilize a RAGE polypeptide domain comprising a RAGE ligand binding site and an interdomain linker directly linked to an immunoglobulin C H 2 domain. Such fusion proteins may provide specific, high affinity binding to RAGE ligands. Also disclosed is the use of the RAGE fusion proteins as therapeutics for RAGE-mediated pathologies.
1 . A fusion protein comprising a RAGE polypeptide linked to a second, non-RAGE polypeptide wherein the RAGE polypeptide comprises a RAGE ligand binding site.
2 . The fusion protein of claim 1 , wherein the RAGE polypeptide is linked to a polypeptide comprising an immunoglobulin domain or a portion of an immunoglobulin domain.
3 . The fusion protein of claim 2 , wherein the polypeptide comprising an immunoglobulin domain comprises at least a portion of at least one of the C H 2 or the C H 3 domains of a human IgG.
4 . The fusion protein of claim 1 , wherein the RAGE ligand binding site comprises SEQ ID NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90% identical thereto.
5 . The fusion protein of claim 1 , wherein the RAGE polypeptide comprises the amino acid sequence SEQ ID NO: 8 corresponding to amino acids 24-116 of human RAGE.
6 . The fusion protein of claim 1 , wherein the RAGE polypeptide comprises the amino acid sequence SEQ ID NO: 14 corresponding to amino acids 24-123 of human RAGE.
7 . The fusion protein of claim 1 , wherein the RAGE polypeptide comprises the amino acid sequence SEQ ID NO: 18 corresponding to amino acids 24-226 of human RAGE.
8 . The fusion protein of claim 1 , wherein the RAGE polypeptide comprises the amino acid sequence SEQ ID NO: 5 corresponding to amino acids 24-339 of human RAGE or sRAGE.
9 . An isolated nucleic acid sequence encoding a RAGE polypeptide linked to a second, non-RAGE polypeptide wherein the RAGE polypeptide comprises a RAGE ligand binding site.
10 . The isolated nucleic acid sequence of claim 9 , wherein the RAGE polypeptide is linked to a polypeptide comprising an immunoglobulin domain or a portion of an immunoglobulin domain.
11 . The isolated nucleic acid sequence of claim 10 , wherein the polypeptide comprising an immunoglobulin domain comprises at least a portion of at least one of the C H 2 or the C H 3 domains of a human IgG.
12 . The isolated nucleic acid sequence of claim 9 , wherein the RAGE ligand binding site comprises SEQ ID NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90% identical thereto.
13 . The isolated nucleic acid sequence of claim 9 , comprising SEQ ID NO: 25 or a fragment thereof.
14 . The isolated nucleic acid sequence of claim 9 , comprising SEQ ID NO: 26 or a fragment thereof.
15 . The isolated nucleic acid sequence of claim 9 , comprising SEQ ID NO: 28 or a fragment thereof.
16 . A composition comprising a therapeutically effective amount of a RAGE fusion protein in a pharmaceutically acceptable carrier, wherein the RAGE fusion protein comprises a RAGE polypeptide linked to a second, non-RAGE polypeptide wherein the RAGE polypeptide comprises a RAGE ligand binding site.
17 . The composition of claim 16 , wherein the RAGE polypeptide is linked to a polypeptide comprising an immunoglobulin domain or a portion of an immunoglobulin domain.
18 . The composition of claim 17 , wherein the polypeptide comprising an immunoglobulin domain comprises at least a portion of at least one of the C H 2 or the C H 3 domains of a human IgG.
19 . The composition of claim 16 , wherein the RAGE ligand binding site comprises SEQ ID NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90% identical thereto.
20 . The composition of claim 16 , wherein the RAGE polypeptide comprises the amino acid sequence SEQ ID NO: 8 corresponding to amino acids 24-116 of human RAGE.
21 . The composition of claim 16 , wherein the RAGE fusion protein is formulated as an injectable solution.
22 . The composition of claim 16 , wherein the RAGE fusion protein is formulated as a sterile lyophilized powder.
23 . A method of making a RAGE fusion protein comprising the step of covalently linking a RAGE polypeptide linked to a second, non-RAGE polypeptide wherein the RAGE polypeptide comprises a RAGE ligand binding site.
24 . The method of claim 23 , where the linked RAGE polypeptide and the second, non-RAGE polypeptide are encoded by a recombinant DNA construct.
25 . The method of claim 24 , further comprising the step of incorporating the DNA construct into an expression vector.
26 . The method of claim 24 , further comprising inserting the expression vector into a host cell.
27 . A method for the detection of RAGE modulators comprising: (a) providing a fusion protein comprising a RAGE polypeptide comprising a RAGE ligand binding site linked to a second, non-RAGE polypeptide; (b) mixing a compound of interest and a ligand having a known binding affinity for RAGE with the fusion protein; and (c) measuring binding of the known RAGE ligand to the RAGE fusion protein in the presence of the compound of interest.
28 . A kit for the detection of RAGE modulators comprising: (a) compound having known binding affinity to RAGE as a positive control; (b) a RAGE fusion protein comprising a RAGE polypeptide comprising a RAGE ligand binding site linked to a second, non-RAGE polypeptide; and (c) instructions for use.
29 . A method of treating a RAGE-mediated disorder in a subject comprising administering to a subject a polypeptide comprising a RAGE polypeptide comprising a RAGE ligand binding site linked to a second, non-RAGE polypeptide.
30 . The method of claim 29 , wherein the RAGE polypeptide is linked to a polypeptide comprising an immunoglobulin domain or a portion of an immunoglobulin domain.
31 . The method of claim 30 , wherein the polypeptide comprising an immunoglobulin domain comprises at least a portion of at least one of the C H 2 or the C H 3 domains of a human IgG.
32 . The method of claim 29 , wherein the RAGE ligand binding site comprises SEQ ID NO: 9 or a sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90% identical thereto.
33 . The method of claim 29 , wherein the RAGE polypeptide comprises the amino acid sequence SEQ ID NO: 8 corresponding to amino acids 24-116 of human RAGE.
34 . The method of claim 29 , comprising intravenous administration of the RAGE fusion protein to the subject.
35 . The method of claim 29 , comprising intraperitoneal administration of the RAGE fusion protein to the subject.
36 . The method of claim 29 , comprising subcutaneous administration of the RAGE fusion protein to the subject.
37 . The method of claim 29 , wherein the fusion protein is used to treat a symptom of diabetes or a symptom of diabetic late complications.
38 . The method of claim 37 , wherein the symptom of diabetes or diabetic late complications comprises diabetic nephropathy.
39 . The method of claim 37 , wherein the symptom of diabetes or diabetic late complications comprises diabetic retinopathy.
40 . The method of claim 37 , wherein the symptom of diabetes or diabetic late complications comprises a diabetic foot ulcer.
41 . The method of claim 37 , wherein the symptom of diabetes or diabetic late complications comprises a cardiovascular complication.
42 . The method of claim 37 , wherein the symptom of diabetes or diabetic late complications comprises diabetic neuropathy.
43 . The method of claim 29 , wherein the fusion protein is used to treat amyloidosis.
44 . The method of claim 29 , wherein the fusion protein is used to treat Alzheimer's disease.
45 . The method of claim 29 , wherein the fusion protein is used to treat cancer.
46 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with autoimmunity.
47 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with inflammatory bowel disease.
48 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with rheumatoid arthritis.
49 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with psoriasis.
50 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with multiple sclerosis.
51 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with hypoxia.
52 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with stroke.
53 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with heart attack.
54 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with hemorraghic shock.
55 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with sepsis.
56 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with organ transplantation.
57 . The method of claim 29 , wherein the fusion protein is used to treat inflammation associated with impaired wound healing.
58 . The method of claim 29 , wherein the fusion protein is used to treat kidney failure.