Glycosaminoglycan-antagonising mcp-1 mutants and methods of using same

Abstract

Novel mutants of human monocyte chemoattractant protein 1 (MCP-1) with increased glycosaminoglycan (GAG) binding affinity and knocked-out or reduced GPCR activity compared to wild type MCP-1, and their use for therapeutic treatment of inflammatory diseases.

1 . An MCP1 mutant protein with increased GAG binding affinity and reduced GPCR activity compared to wild type MCP-1 protein, wherein the MCP-1 protein is modified in a structure-conserving way by insertion of at least one basic and/or electron donating amino acid or replacement of at least two amino acids by at least two basic and/or electron donating amino acids. 
     
     
         2 . The MCP-1 mutant protein of  claim 1 , wherein at least one amino acid of the first 10 amino acids of the N-terminal region of the wild type MCP-1 protein is modified by addition, deletion and/or replacement of at least one amino acid. 
     
     
         3 . The MCP-1 mutant protein of  claim 1 , wherein the amino acids that are replaced by the at least two basic and/or electron donating amino acids are non-basic amino acids. 
     
     
         4 . The MCP-1 mutant protein of  claim 1 , wherein the modification in a structure-conserving way is a deviation of the modified structure from wild type MCP1 structure of less than 30%, and preferably less than 20% as measured by far-UV CD spectroscopy. 
     
     
         5 . The MCP-1 mutant protein of  claim 1 , wherein the basic amino acids are selected from the group consisting of R, K, and H. 
     
     
         6 . The MCP-1 mutant protein of  claim 1 , wherein the electron donating amino acids are selected from the group consisting of N or Q. 
     
     
         7 . The MCP-1 mutant protein of  claim 1 , wherein at least two amino acids at positions 21, 23 and/or 47 are modified. 
     
     
         8 . The MCP-1 mutant protein of  claim 1 , wherein the Y at position 13 is substituted by an A. 
     
     
         9 . The MCP-1 mutant protein of  claim 1 , containing an N-terminal Met. 
     
     
         10 . The MCP-1 mutant protein of  claim 1 , wherein the N-terminal amino acid residues 2-8 are deleted. 
     
     
         11 . An MCP-1 mutant protein, it wherein the mutant protein comprises the amino acid sequence of the general formula: 
       
         
           
                 
                 
               
                   (M)nQ(PDAINA(Z1))mVTCC(X1)NFTN (Z2)(Z3)I(X2)V(X3)RLASYRRITSSKCP 
                     
                 
                     
                 
                   KEAVIFKTI(X4) AKEICADPKQ KWVQDSMDHL DKQTQTPKT 
                 
             
                
                
                
               
            
           
         
         wherein Z1 is selected from the group consisting of P, A, G, L, 
         wherein Z2 is selected from the group consisting of R and K, 
         wherein Z3 is selected from the group consisting of K and R, 
         wherein X1 is selected from the group consisting of Y and A, wherein X2 is selected from the group consisting of S, R, K, H, N and Q, 
         wherein X3 is selected from the group consisting of R, K, H, N and Q, 
         wherein X4 is selected from the group consisting of V, R, K, H, N and Q, and 
         wherein n and/or m can be either 0 or 1, and wherein at least two of the positions X2, X3 or X4 are modified. 
       
     
     
         12 . The MCP-1 mutant protein of  claim 1 , wherein the protein has it is an amino acid sequence selected from the group consisting of SEQ ID NO:5, and SEQ ID NO:6. 
     
     
         13 . An isolated polynucleic acid molecule which encodes the MCP-1 mutant protein of  claim 1 . 
     
     
         14 . The isolated polynucleic acid molecule of  claim 13 , wherein the nucleotide sequence is selected from the group consisting of SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9 and at least a part thereof. 
     
     
         15 . The isolated polynucleic acid molecule of  claim 13 , wherein the polynucleic acid molecule is in a vector. 
     
     
         16 . The isolated polynucleic acid of  claim 15 , wherein the vector is transfected into a recombinant cell. 
     
     
         17 . A pharmaceutical composition which comprises a protein according to  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         18 . A method for inhibiting or suppressing the biological activity of the wild type MCP-1 protein in vitro comprising using the MCP-1 mutant protein of  claim 1  in a diagnostic assay. 
     
     
         19 . A method for treating chronic or acute inflammatory disease or autoimmune conditions comprising administering the MCP-1 mutant protein of  claim 1  to a patient in need thereof. 
     
     
         20 . The method of  claim 19 , wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, uveitis, inflammatory bowel disease, myocardial infarction, congested heart failure and ischemia reperfusion injury.