Gag binding proteins

Abstract

A method is provided for introducing a GAG binding site into a protein comprising the steps: identifying a region in a protein which is not essential for structure maintenance introducing at least one basic amino acid into said site and/or deleting at least one bulky and/or acidic amino acid in said site, whereby said GAG binding site has a GAG binding affinity of Kd≦10 μM, preferably ≦1 μM, still preferred ≦0.1 μM, as well as modified GAG binding proteins.

1 - 20 . (canceled) 
     
     
         21 . A method of making a modified GAG binding protein by modifying a GAG binding site of the GAG binding protein, wherein the GAG binding protein is a C-terminal α-helix of a chemokine, and wherein the GAG binding site is modified by a method comprising the steps of:
 (a) introducing at least one basic amino acid into the C-terminal α-helix; and/or 
 (b) deleting at least one bulky and/or acidic amino acid in the C-terminal α-helix; 
 wherein the GAG binding region has a GAG binding affinity of Kd≦10 μM. 
 
     
     
         22 . The method according to  claim 21 , wherein the GAG binding site has a GAG binding affinity of ≦1 μM. 
     
     
         23 . The method according to  claim 21 , wherein the GAG binding site has a GAG binding affinity of ≦0.1 μM. 
     
     
         24 . The method according to  claim 21 , wherein the GAG binding affinity is higher by a factor of minimum 5 compared with wild-type GAG binding protein. 
     
     
         25 . A modified chemokine, wherein GAG binding site is a C-terminal α-helix in the chemokine, and wherein the GAG binding site is modified by a method comprising the steps of:
 (a) substituting and/or inserting of at least one amino acid selected from the group consisting of Arg, Lys and His; and/or 
 (b) deleting of at least one amino acid in order to increase the relative amount of basic amino acids in the C-terminal helix; and/or 
 (c) reducing the amount of bulky and/or acidic amino acids in the C-terminal α-helix, thereby increasing the GAG binding affinity of the modified chemokine compared to the GAG binding affinity of a respective wild-type chemokine. 
 
     
     
         26 . The modified chemokine according to  claim 25 , wherein the chemokine is RANTES or MCP-1. 
     
     
         27 . The modified chemokine according to  claim 25 , wherein the chemokine is SDF-1α, MGSA/GROα, MIP2α/GROβ, NAP-2, PF-4, MCP-2, MCP-3, MIP-1α, MIP-1β, MPIF-1, or MIP-5/HCC-1. 
     
     
         28 . The modified chemokine according to  claim 25 , wherein the increased GAG binding affinity is an increased binding affinity to heparan sulphate and/or heparin. 
     
     
         29 . The modified chemokine according to  claim 25 , wherein a further biologically active region is modified thereby inhibiting or down-regulating a further biological activity of the chemokine. 
     
     
         30 . The modified chemokine according to  claim 29 , wherein the further biologically active region is modified by deletion, insertion, and/or substitution with alanine, a sterically and/or electrostatically similar residue. 
     
     
         31 . The modified chemokine according to  claim 30 , wherein the further biological activity is leukocyte activation. 
     
     
         32 . An isolated polynucleic acid molecule that codes for a chemokine according to  claim 25 . 
     
     
         33 . A vector that comprises an isolated DNA molecule according to  claim 32 . 
     
     
         34 . A recombinant cell that comprises an isolated DNA molecule according to  claim 32 , wherein the recombinant cell is not of human origin. 
     
     
         35 . A pharmaceutical composition that comprises a chemokine according to  claim 25 . 
     
     
         36 . A pharmaceutical composition that comprises a polynucleic acid according to  claim 32 . 
     
     
         37 . A pharmaceutical composition that comprises a vector according to  claim 33 . 
     
     
         38 . A pharmaceutical composition according to  claim 32 , wherein the composition comprises a pharmaceutically acceptable carrier. 
     
     
         39 . A method of treating an inflammatory condition comprising a Chemokine according to  claim 32 , wherein the chemokine is selected from the group consisting of MGSA/GRO, MIP2alpha, GRO, NAP-2, PF-4, SDF-1, RANTES, MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1β, MPIF-1, and MIP-5. 
     
     
         40 . The methods according to  claims 39 , wherein the inflammatory condition is selected from the group consisting of rheumatoid arthritis, psoriasis, osteoarthritis, asthma, Alzheimer's disease, and multiple sclerosis.