Truncated keratinocyte growth factor (KGF) having increased biological activity

Abstract

The present invention relates to a keratinocyte growth factor fragment, KGF des1-23 , or an analog thereof that is composed of a portion of an amino acid sequence of mature, full length keratinocyte growth factor, KGF 163 . The fragment exhibits at least a 2-fold increase in mitogenic activity as compared to a mature, recombinant keratinocyte growth factor, rKGF, but lacks a sequence comprising the first 23 amino acid residues; C-N-D-M-T-P-E-Q-M-A-T-N-V-N-C-S-S-P-E-R-H-T-R- (SEQ ID NO: 2) of the KGF 163 N-terminus. The present invention also relates to a DNA molecule encoding KGF des1-23 , an expression vector and a transformed host containing the DNA molecule, and a method of producing KGF des1-23 by culturing the transformed host. The present invention further relates to a conjugate of KGF des1-23 and a toxin molecule, and the use thereof for treatment of hyperproliferative disease of the epidermis. Moreover, the present invention relates to a therapeutic composition containing KGF des1-23 and a pharmaceutically acceptable carrier and the use thereof for wound healing purposes.

1 . An isolated polypeptide, wherein said polypeptide is a fragment of mature, full-length, human KGF as depicted in  FIG. 1  (amino acids 32-194 of SEQ ID NO:1) or an analog thereof, wherein said analog has the same number of amino acids as the fragment and has at least 80% sequence identity thereto and optionally further comprises a methionine residue at the N-terminus thereof, and further wherein said polypeptide exhibits an increase in bioactivity relative to said mature, full-length, human KGF as determined by the Balb/MK bioactivity assay. 
     
     
         2 . The polypeptide of  claim 1 , wherein said analog has at least 90% sequence identity to a fragment of the amino acid sequence of amino acids 1-163 of  FIG. 1  (amino acids 32-194 of SEQ ID NO:1). 
     
     
         3 . The polypeptide of  claim 1 , wherein said analog has at least 95% sequence identity to a fragment of the amino acid sequence of amino acids 1-163 of  FIG. 1  (amino acids 32-194 of SEQ ID NO:1). 
     
     
         4 . The polypeptide of  claim 1 , wherein said analog has at least 98% sequence identity to a fragment of the amino acid sequence of amino acids 1-163 of  FIG. 1  (amino acids 32-194 of SEQ ID NO:1). 
     
     
         5 . A composition comprising the polypeptide of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         6 . An isolated polypeptide, wherein said polypeptide is an analog of KGF des1-23  or a fragment thereof, wherein said analog has at least 80% sequence identity to the contiguous amino acid sequence of amino acids 24-163 of  FIG. 1  (amino acids 55-194 of SEQ ID NO:1) and optionally further comprises a methionine residue at the N-terminus thereof, and further wherein said polypeptide exhibits an increase in bioactivity relative to mature, full-length, human KGF as depicted in  FIG. 1  (amino acids 32-194 of SEQ ID NO:1) as determined by the Balb/MK bioactivity assay. 
     
     
         7 . The polypeptide of  claim 6 , wherein said polypeptide has at least 90% sequence identity to amino acids 24-163 of  FIG. 1  (amino acids 55-194 of SEQ ID NO:1). 
     
     
         8 . The polypeptide of  claim 6 , wherein said polypeptide has at least 95% sequence identity to amino acids 24-163 of  FIG. 1  (amino acids 55-194 of SEQ ID NO:1). 
     
     
         9 . A composition comprising the polypeptide of  claim 6 , and a pharmaceutically acceptable carrier. 
     
     
         10 . An isolated polypeptide, wherein said polypeptide is a fragment of mature, full-length, human KGF having an amino acid sequence comprising at least amino acids 24-163 of  FIG. 1  (amino acids 55-194 of SEQ ID NO:1) or an analog thereof, wherein said analog has the same number of amino acids as the fragment and has at least 80% sequence identity thereto and optionally further comprises a methionine residue at the N-terminus thereof, and further wherein said polypeptide exhibits an increase in bioactivity relative to mature, said full-length, human KGF as determined by the Balb/MK bioactivity assay and specifically stimulates epithelial cell proliferation. 
     
     
         11 . The polypeptide of  claim 10 , wherein said increase in bioactivity is at least about a 2-fold increase in bioactivity. 
     
     
         12 . The polypeptide of  claim 10 , wherein said increase in bioactivity is at least about a 2-fold to about a 10-fold increase in bioactivity. 
     
     
         13 . The polypeptide of  claim 10 , wherein said increase in bioactivity is at least about a 7-fold to about a 10-fold increase in bioactivity. 
     
     
         14 . The polypeptide of  claim 10 , wherein said increase in bioactivity is about a 10-fold increase in bioactivity. 
     
     
         15 . The polypeptide of  claim 10 , wherein said analog has at least 90% sequence identity to said fragment. 
     
     
         16 . The polypeptide of  claim 10 , wherein said analog has at least 95% sequence identity to said fragment. 
     
     
         17 . A composition comprising the polypeptide of  claim 10 , and a pharmaceutically acceptable carrier. 
     
     
         18 . An isolated polypeptide, wherein said polypeptide is (a) a fragment of mature, full-length, human KGF as depicted in  FIG. 1  (amino acids 32-194 of SEQ ID NO:1), wherein the amino acid sequence of said fragment has an N-terminus at a position between amino acid 9 and amino acid 26, inclusive, and extends to amino acid 163, of mature, full-length, human KGF, numbered relative to  FIG. 1 , or (b) an analog of the fragment of (a) that has the same number of amino acids as the fragment and has at least 80% sequence identity thereto and optionally further comprises a methionine residue at the N-terminus thereof, and further wherein said polypeptide exhibits an increase in bioactivity relative to said mature, full-length, human KGF as determined by the Balb/MK bioactivity assay and specifically stimulates epithelial cell proliferation. 
     
     
         19 . The polypeptide of  claim 18 , wherein said increase in bioactivity is at least about a 2-fold increase in bioactivity. 
     
     
         20 . The polypeptide of  claim 18 , wherein said increase in bioactivity is about 2-fold to about a 10-fold increase in bioactivity. 
     
     
         21 . The polypeptide of  claim 18 , wherein said increase in bioactivity is about a 7-fold to about a 10-fold increase in bioactivity. 
     
     
         22 . The polypeptide of  claim 18 , wherein said increase in bioactivity is about a 10-fold increase in bioactivity. 
     
     
         23 . The polypeptide of  claim 18 , wherein said analog has at least 90% sequence identity to said fragment. 
     
     
         24 . The polypeptide of  claim 18 , wherein said analog has at least 95% sequence identity to said fragment. 
     
     
         25 . The polypeptide of  claim 18 , wherein said polypeptide is a fragment of said mature, full-length, human KGF, wherein the amino acid sequence of said fragment has an N-terminus at a position between amino acid 9 and amino acid 26, inclusive, and extends to amino acid 163, of mature, full-length, human KGF, numbered relative to  FIG. 1 . 
     
     
         26 . A composition comprising the polypeptide of  claim 18 , and a pharmaceutically acceptable carrier. 
     
     
         27 . A method of treating a gastrointestinal disorder comprising administering the composition of  claim 5 , to a subject in need thereof. 
     
     
         28 . A method of treating a gastrointestinal disorder comprising administering the composition of  claim 9 , to a subject in need thereof. 
     
     
         29 . A method of treating a gastrointestinal disorder comprising administering the composition of  claim 17 , to a subject in need thereof. 
     
     
         30 . A method of treating a gastrointestinal disorder comprising administering the composition of  claim 26 , to a subject in need thereof. 
     
     
         31 . A method for wound healing comprising administering the composition of  claim 5 , to a subject in need thereof. 
     
     
         32 . A method for wound healing comprising administering the composition of  claim 9 , to a subject in need thereof. 
     
     
         33 . A method for wound healing comprising administering the composition of  claim 17 , to a subject in need thereof. 
     
     
         34 . A method for wound healing comprising administering the composition of  claim 26 , to a subject in need thereof. 
     
     
         35 . An antibody selective for the polypeptide of  claim 1 . 
     
     
         36 . An antibody selective for the polypeptide of  claim 6 . 
     
     
         37 . An antibody selective for the polypeptide of  claim 10 . 
     
     
         38 . An antibody selective for the polypeptide of  claim 18 . 
     
     
         39 . An isolated polynucleotide comprising a coding sequence encoding the polypeptide of  claim 1 . 
     
     
         40 . An isolated polynucleotide comprising a coding sequence encoding the polypeptide of  claim 6 . 
     
     
         41 . An isolated polynucleotide comprising a coding sequence encoding the polypeptide of  claim 10 . 
     
     
         42 . An isolated polynucleotide comprising a coding sequence encoding the polypeptide of  claim 18 . 
     
     
         43 . A non-human host cell that expresses a polypeptide according to  claim 1 . 
     
     
         44 . A non-human host cell that expresses a polypeptide according to  claim 6 . 
     
     
         45 . A non-human host cell that expresses a polypeptide according to  claim 10 . 
     
     
         46 . A non-human host cell that expresses a polypeptide according to  claim 18 . 
     
     
         47 . The host cell of  claim 43 , wherein the host cell is a bacterial cell. 
     
     
         48 . The host cell of  claim 44 , wherein the host cell is a bacterial cell. 
     
     
         49 . The host cell of  claim 45 , wherein the host cell is a bacterial cell. 
     
     
         50 . The host cell of  claim 46 , wherein the host cell is a bacterial cell. 
     
     
         51 . The host cell of  claim 47 , wherein the host cell is  E. coli.    
     
     
         52 . The host cell of  claim 48 , wherein the host cell is  E. coli.    
     
     
         53 . The host cell of  claim 49 , wherein the host cell is  E. coli.    
     
     
         54 . The host cell of  claim 50 , wherein the host cell is  E. coli.    
     
     
         55 . A method of producing a recombinant polypeptide comprising culturing the host cell of  claim 43 , under conditions whereby the polypeptide is expressed. 
     
     
         56 . A method of producing a recombinant polypeptide comprising culturing the host cell of  claim 44 , under conditions whereby the polypeptide is expressed. 
     
     
         57 . A method of producing a recombinant polypeptide comprising culturing the host cell of  claim 45 , under conditions whereby the polypeptide is expressed. 
     
     
         58 . A method of producing a recombinant polypeptide comprising culturing the host cell of  claim 46 , under conditions whereby the polypeptide is expressed.