Rage Fusion Proteins And Methods Of Use

Abstract

Disclosed are RAGE fusion proteins comprising RAGE polypeptide sequences linked to a second, non-RAGE polypeptide. The RAGE fusion protein may utilize a RAGE polypeptide domain comprising a RAGE ligand binding site and an interdomain linker directly linked to an immunoglobulin 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 method of making a Receptor for Advanced Glycated Endproducts (RAGE) fusion protein comprising the step of covalently linking a RAGE polypeptide to a polypeptide comprising a C H 2 domain of an immunoglobulin or a portion of a C H 2 domain of an immunoglobulin, wherein the RAGE polypeptide comprises a RAGE ligand binding site, and wherein the fusion protein does not include an immunoglobulin Fc hinge region. 
     
     
         2 . The method of  claim 1 , wherein the RAGE fusion protein is encoded by a recombinant DNA construct. 
     
     
         3 . The method of  claim 2 , further comprising the step of incorporating the recombinant DNA construct into an expression vector. 
     
     
         4 . The method of  claim 3 , further comprising the step of inserting the expression vector into a host cell. 
     
     
         5 . The method of  claim 4 , further comprising the step of culturing the host cell under conditions such that the RAGE fusion protein is expressed. 
     
     
         6 . The method of  claim 2 , wherein the recombinant DNA construct comprises the nucleic acid sequence as set forth in SEQ ID NO: 30, or the nucleic acid sequence as set forth in SEQ ID NO: 31. 
     
     
         7 . The method of  claim 1 , wherein the RAGE ligand binding site comprises the amino acid sequence as set forth in SEQ ID NO: 9 or a sequence 90% identical thereto, or the amino acid sequence as set forth in SEQ ID NO: 10 or a sequence 90% identical thereto. 
     
     
         8 . The method of  claim 1 , wherein the RAGE polypeptide comprises a RAGE interdomain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-terminal amino acid of the RAGE interdomain linker, and the C-terminal amino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of the polypeptide comprising the C H 2 domain of the immunoglobulin or the portion of the C H 2 domain of the immunoglobulin. 
     
     
         9 . The method of  claim 1 , wherein the RAGE polypeptide comprises a first RAGE immunoglobulin domain and a first RAGE interdomain linker linked to a second RAGE immunoglobulin domain and a second RAGE interdomain linker, such that the N-terminal amino acid of the first RAGE interdomain linker is linked to the C-terminal amino acid of the first RAGE immunoglobulin domain, the N-terminal amino acid of the second RAGE immunoglobulin domain is linked to the C-terminal amino acid of the first RAGE interdomain linker, the N-terminal amino acid of the second RAGE interdomain linker is linked to the C-terminal amino acid of the second RAGE immunoglobulin domain, and the C-terminal amino acid of the second RAGE interdomain linker is directly linked to the N-terminal amino acid of the C H 2 domain of the immunoglobulin or the portion of the C H 2 domain of the immunoglobulin. 
     
     
         10 . The method of  claim 9 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34. 
     
     
         11 . The method of  claim 9 , wherein the second RAGE interdomain linker directly linked to the C H 2 domain of the immunoglobulin or the portion of the C H 2 domain of the immunoglobulin comprises the amino acid sequence as set forth in SEQ ID NO: 22 or a sequence 90% identical thereto. 
     
     
         12 . The method of  claim 1 , wherein the RAGE polypeptide comprises a single RAGE immunoglobulin domain linked via a RAGE interdomain linker to the N-terminal amino acid of the polypeptide comprising the C H 2 domain of the immunoglobulin or the portion of the C H 2 domain of the immunoglobulin. 
     
     
         13 . The method of  claim 12 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37. 
     
     
         14 . A method of making a Receptor for Advanced Glycated Endproducts (RAGE) fusion protein comprising making a recombinant DNA construct encoding a RAGE polypeptide that is directly linked to a polypeptide comprising a C H 2 domain of an immunoglobulin or a portion of a C H 2 domain of an immunoglobulin, wherein the RAGE polypeptide comprises a RAGE ligand binding site, and wherein the fusion protein does not include an immunoglobulin Fc hinge region. 
     
     
         15 . The method of  claim 14 , further comprising the step of incorporating the recombinant DNA construct into an expression vector. 
     
     
         16 . The method of  claim 15 , further comprising the step of inserting the expression vector into a host cell. 
     
     
         17 . The method of  claim 16 , further comprising the step of culturing the host cell under conditions such that the RAGE fusion protein is expressed. 
     
     
         18 . The method of  claim 14 , wherein the recombinant DNA construct comprises the nucleic acid sequence as set forth in SEQ ID NO: 30, or the nucleic acid sequence as set forth in SEQ ID NO: 31. 
     
     
         19 . The method of  claim 14 , wherein the RAGE ligand binding site comprises the amino acid sequence as set forth in SEQ ID NO: 9 or a sequence 90% identical thereto, or the amino acid sequence as set forth in SEQ ID NO: 10 or a sequence 90% identical thereto. 
     
     
         20 . The method of  claim 14 , wherein the RAGE polypeptide comprises a RAGE interdomain linker linked to a RAGE immunoglobulin domain such that the C-terminal amino acid of the RAGE immunoglobulin domain is linked to the N-terminal amino acid of the RAGE interdomain linker, and the C-terminal amino acid of the RAGE interdomain linker is directly linked to the N-terminal amino acid of the polypeptide comprising the C H 2 domain of the immunoglobulin or the portion of the C H 2 domain of the immunoglobulin. 
     
     
         21 . The method of  claim 14 , wherein the RAGE polypeptide comprises a first RAGE immunoglobulin domain and a first RAGE interdomain linker linked to a second RAGE immunoglobulin domain and a second RAGE interdomain linker, such that the N-terminal amino acid of the first RAGE interdomain linker is linked to the C-terminal amino acid of the first RAGE immunoglobulin domain, the N-terminal amino acid of the second RAGE immunoglobulin domain is linked to the C-terminal amino acid of the first RAGE interdomain linker, the N-terminal amino acid of the second RAGE interdomain linker is linked to the C-terminal amino acid of the second RAGE immunoglobulin domain, and the C-terminal amino acid of the second RAGE interdomain linker is directly linked to the N-terminal amino acid of the C H 2 domain of the immunoglobulin or the portion of the C H 2 domain of the immunoglobulin. 
     
     
         22 . The method of  claim 21 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34. 
     
     
         23 . The method of  claim 21 , wherein the second RAGE interdomain linker directly linked to the C H 2 domain of the immunoglobulin or the portion of the C H 2 domain of the immunoglobulin comprises the amino acid sequence as set forth in SEQ ID NO: 22 or a sequence 90% identical thereto. 
     
     
         24 . The method of  claim 14 , wherein the RAGE polypeptide comprises a single RAGE immunoglobulin domain linked via a RAGE interdomain linker to the N-terminal amino acid of the polypeptide comprising the C H 2 domain of the immunoglobulin or the portion of the C H 2 domain of the immunoglobulin. 
     
     
         25 . The method of  claim 24 , wherein the RAGE fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37.