Fibroblast growth factor mutants having improved functional half-life and methods of their use

Abstract

Mutant fibroblast growth factor (FGF) proteins having a polypeptide sequence with a high sequence identity to proteins encoded by members of the Fgf-1 subfamily of genes from a mammalian species, such as human, and with a specific amino acid substitution of an alanine at a position corresponding to amino acid position 66 of human FGF-1 with a cysteine and/or a specific amino acid substitution of a phenylalanine at a position corresponding to amino acid position 132 of human FGF-1 with a tryptophan (based on the 140 amino acid numbering scheme of human FGF-1) are provided. Other amino acid mutations or substitutions may be combined. Polynucleotide sequences encoding the mutant FGF proteins and host cells containing such polynucleotide sequences are provided. Methods of administering a mutant FGF protein to an individual to treat an ischemic condition or disease or a wound or tissue injury are also provided.

That which is claimed is: 
     
         1 . A mutant fibroblast growth factor (FGF) protein having a polypeptide sequence that is at least 90% identical to the polypeptide sequence of wild-type human FGF-2 protein (SEQ ID NO: 4), wherein the alanine (Ala) at an amino acid position of the mutant FGF protein corresponding to amino acid position 84 of wild-type human FGF-2 is replaced with cysteine (Cys). 
     
     
         2 . The mutant FGF protein of  claim 1 , wherein the mutant FGF protein has a polypeptide sequence that is at least 95% identical to the polypeptide sequence of wild-type human FGF-2 protein (SEQ ID NO: 4). 
     
     
         3 . The mutant FGF protein of  claim 1 , wherein the mutant FGF protein has the polypeptide sequence of SEQ ID NO: 6. 
     
     
         4 . The mutant FGF protein of  claim 1 , wherein the mutant FGF protein binds specifically to at least one FGF receptor (FGFR) arid triggers growth or proliferation of cultured fibroblast cells. 
     
     
         5 . A functional fragment of the mutant FGF protein of  claim 1 , wherein the functional fragment of the mutant FGF protein binds specifically to at least one FGF receptor (FGFR) and triggers growth or proliferation of fibroblasts, endothelial cells, chondrocytes, osteoblasts, myoblasts, smooth muscle cells, glial cells, or neuroblasts. 
     
     
         6 . A polynucleotide sequence encoding the mutant FGF protein of  claim 1 . 
     
     
         7 . A host cell containing the polynucleotide sequence of  claim 6 . 
     
     
         8 . The mutant FGF protein of  claim 1  in a pharmaceutical composition including a pharmaceutically acceptable carrier. 
     
     
         9 . A method, comprising the following steps:
 (a) identifying an individual having an ischemic condition or disease; and   (b) administering to the individual a composition comprising the mutant FGF protein of  claim 1 .   
     
     
         10 . The method of  claim 9 , wherein the ischemic condition or disease is coronary artery disease or peripheral vascular disease. 
     
     
         11 . The method of  claim 9 , wherein the composition is administered locally at or near the site of the ischemic condition or disease within the body of the individual. 
     
     
         12 . The method of  claim 11 , wherein the composition is administered locally at or near a site within the body of the individual causing the ischemic condition or disease. 
     
     
         13 . The method of  claim 12 , wherein the composition is administered locally at or near an occluded blood vessel. 
     
     
         14 . A method, comprising the following steps:
 (a) identifying an individual having a wound or tissue damage; and   (b) administering to the individual a composition comprising the mutant FGF protein of  claim 1 .   
     
     
         15 . The method of  claim 14 , wherein the wound or tissue damage is due to a traumatic injury or an immunologic disorder. 
     
     
         16 . The method of  claim 15 , wherein the wound or tissue damage is due to stroke, myocardial infarction, acute transverse myelitis (ATM), idiopathic transverse myelitis (ITM), brachial plexus injury, spinal cord injury, or peripheral nerve injury. 
     
     
         17 . The method of  claim 14 , wherein the composition is administered locally at or near the site of the wound or tissue damage within the body of the individual. 
     
     
         18 . A mutant fibroblast growth factor (FGF) protein having a polypeptide sequence that is at least 90% identical to the polypeptide sequence of wild-type human FGF-2 protein (SEQ ID NO: 4), wherein the phenylalanine (Phe) at an amino acid position of the mutant FGF protein corresponding to amino acid position 148 of wild-type human FGF-2 is replaced with tryptophan (Trp). 
     
     
         19 . The mutant FGF protein of  claim 18 , wherein the mutant FGF protein has a polypeptide sequence that is at least 95% identical to the polypeptide sequence of wild-type human FGF-2 protein (SEQ ID NO: 4). 
     
     
         20 . The mutant FGF protein of  claim 18 , wherein the mutant FGF protein has the polypeptide sequence of SEQ ID NO: 8. 
     
     
         21 . The mutant FGF protein of  claim 18 , wherein the mutant FGF protein binds specifically to a FGF receptor (FGFR) and triggers growth or proliferation of cultured fibroblast cells. 
     
     
         22 . A functional fragment of the mutant FGF protein of  claim 18 , wherein the functional fragment of the mutant FGF protein binds specifically to at least one FGF receptor (FGFR) and triggers growth or proliferation of fibroblasts, endothelial cells, chondrocytes, osteoblasts, myoblasts, smooth muscle cells, glial cells, or neuroblasts. 
     
     
         23 . A polynucleotide sequence encoding the mutant FGF protein of  claim 18 . 
     
     
         24 . A host cell containing the polynucleotide sequence of  claim 23 . 
     
     
         25 . The mutant FGF protein of  claim 18  in a pharmaceutical composition including a pharmaceutically acceptable carrier. 
     
     
         26 . The mutant FGF protein of  claim 18 , wherein the alanine (Ala) at an amino acid position of the mutant FGF protein corresponding to amino acid position 84 of wild-type human FGF-2 is replaced with cysteine (Cys). 
     
     
         27 . The mutant FGF protein of  claim 18 , wherein the mutant FGF protein has the polypeptide sequence of SEQ ID NO: 10. 
     
     
         28 . A method, comprising the following steps:
 (a) identifying an individual having an ischemic condition or disease; and   (b) administering to the individual a composition comprising the mutant FGF protein of  claim 18 .   
     
     
         29 . The method of  claim 28 , wherein the ischemic condition or disease is coronary artery disease or peripheral vascular disease. 
     
     
         30 . The method of  claim 28 , wherein the composition is administered locally at or near the site of the ischemic condition or disease within the body of the individual. 
     
     
         31 . The method of  claim 28 , wherein the composition is administered locally at or near a site within the body of the individual causing the ischemic condition or disease. 
     
     
         32 . The method of  claim 31 , wherein the composition is administered locally at or near an occluded blood vessel. 
     
     
         33 . A method, comprising the following steps:
 (a) identifying an individual having a wound or tissue damage; and   (b) administering to the individual a composition comprising the mutant FGF protein of  claim 18 .   
     
     
         34 . The method of  claim 33 , wherein the wound or tissue damage is due to a traumatic injury or an immunologic disorder. 
     
     
         35 . The method of  claim 34 , wherein the wound or tissue damage is due to stroke, myocardial infarction, acute transverse myelitis (ATM), idiopathic transverse myelitis (ITM), brachial plexus injury, spinal cord injury, or peripheral nerve injury. 
     
     
         36 . The method of  claim 33 , wherein the composition is administered locally at or near the site of the wound or tissue damage within the body of the individual.