RAGE Fusion Proteins with Improved Stability and Ligand Binding Affinity and Uses Thereof

Abstract

The present invention provides soluble RAGE-Fc fusion proteins with increased stability and extended half-life capable of binding endogenous RAGE ligands with high apparent affinity. The present invention also provides methods of making and using stable, soluble RAGE-Fc fusion proteins. These soluble RAGE-Fc fusion proteins are useful as therapeutics based on their ability to bind endogenous RAGE ligands.

1 . An isolated polypeptide comprising:
 (a) a first domain wherein said first domain has an amino acid sequence at least 97% identical to the sequence of SEQ ID NO:74; and   (b) a second domain comprising a fragment of a Fc region of an immunoglobulin,   wherein the carboxy terminus of said first domain is coupled to the amino terminus of said second domain by a peptide linkage.   
     
     
         2 . The isolated polypeptide of  claim 1 , wherein said polypeptide is resistant to cleavage by a disintegrin and metalloproteinase 10 (ADAM10). 
     
     
         3 . The isolated polypeptide of  claim 1 , wherein said polypeptide is at least 15% more resistant to cleavage by at least one of ADAM10, matrix metalloproteinase 9 (MMP9), and trypsin as compared to a polypeptide having the sequence of SEQ ID NO: 5. 
     
     
         4 . The isolated polypeptide of  claim 1 , wherein said polypeptide is at least 15% more resistant to degradation in human serum as compared to a polypeptide comprising the sequence of SEQ ID NO: 5, wherein the percent resistance equals the difference between the fraction of peptide that remains full length following incubation in human serum for a defined time period compared to a control peptide treated for the same time and under the same conditions. 
     
     
         5 . The isolated polypeptide of  claim 1 , wherein said polypeptide has increased thermal stability of at least 5° C. as compared to a polypeptide having the sequence of SEQ ID NO: 5. 
     
     
         6 . The isolated polypeptide of  claim 1 , wherein said polypeptide specifically binds an advanced glycation endproduct (AGE). 
     
     
         7 . The isolated polypeptide of  claim 1 , wherein said polypeptide specifically binds HMGB1 (Amphoterin). 
     
     
         8 . The isolated polypeptide of  claim 1 , wherein said polypeptide specifically binds at least one of the group consisting of: S100A1, S100A2, S100A4 (metastasin), S100A5, S100A6, S100A7 (psoriasin), S100A8/9, S100A11, S100A12, S100B, S100P, lipopolysaccharide (LPS), oxidized low-density lipoprotein (oxLDL), CD11b (MAC1), phosphatidyl serine, C3 a, S100P, S100G, S100Z, carbonylated proteins, malondialdehyde (MDA), laminin, type I Collagen, type IV Collagen, CAPZA1, CAPZA2, DDOST, LGALS3, MAPK1, MAPK3, PRKCSH, S100A4, S100A5, S100A6, S100A8, S100A9, S100P, and SAA1. 
     
     
         9 . The isolated polypeptide of  claim 1 , wherein said polypeptide specifically binds amyloid-beta. 
     
     
         10 . The isolated polypeptide of  claim 1 , wherein said polypeptide is a dimer of a polypeptide having the sequence of SEQ ID NO: 74. 
     
     
         11 . The isolated polypeptide of  claim 1 , wherein said first domain comprises at least one asparagine residue linked to a glycan. 
     
     
         12 . The isolated polypeptide of  claim 1 , wherein said first domain comprises an amino acid substitution at one or more of amino acid residues 3 or 59 of SEQ ID NO:74. 
     
     
         13 . The isolated polypeptide of  claim 12 , wherein said amino acid substitution is a substitution with glutamic acid or glutamine at amino acid residue 3. 
     
     
         14 . The isolated polypeptide of  claim 12 , wherein said amino acid substitution is a substitution with alanine, glutamic acid, or glutamine at amino acid residue 59. 
     
     
         15 . The isolated polypeptide of  claim 1 , wherein said first domain comprises an amino acid substitution at amino acid residue 60 of SEQ ID NO:74. 
     
     
         16 . The isolated polypeptide of  claim 1 , wherein said first domain has the sequence set forth in SEQ ID NO: 74. 
     
     
         17 . The isolated polypeptide of  claim 15 , wherein said amino acid substitution is a substitution with serine. 
     
     
         18 . The isolated polypeptide of  claim 1 , wherein said Fc fragment comprises CH2 and CH3 domains of a human IgG. 
     
     
         19 . An isolated polypeptide comprising a RAGE polypeptide coupled to a Fc region of an immunoglobulin, wherein the carboxy terminus of said RAGE polypeptide is coupled to the amino terminus of said immunoglobulin Fc region by a peptide linkage and wherein said RAGE polypeptide has the sequence of SEQ ID NO: 2. 
     
     
         20 . A pharmaceutical composition for treating a RAGE-mediated disorder comprising the isolated polypeptide of  claim 1 .