Chimeric fibroblast growth factor 19 proteins and methods of use

Abstract

The present invention relates to a chimeric protein that includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine fibroblast growth factor (“FGF”) and the C-terminus includes a C-terminal portion of an FGF19 molecule. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification. The present invention also relates to pharmaceutical compositions including chimeric proteins according to the present invention, methods for treating a subject suffering from diabetes, obesity, or metabolic syndrome, and methods of screening for compounds with enhanced binding affinity for the βKlotho-FGF receptor complex involving the use of chimeric proteins of the present invention.

1 . A chimeric protein comprising:
 an N-terminus coupled to a C-terminus, wherein the N-terminus comprises a portion of a paracrine fibroblast growth factor (“FGF”) and the C-terminus comprises a C-terminal portion of an FGF19 molecule and wherein the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification.   
     
     
         2 . The chimeric protein according to  claim 1 , wherein the paracrine FGF is FGF1 or FGF2. 
     
     
         3 . The chimeric protein according to  claim 1 , wherein the portion of the paracrine FGF comprises an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1. 
     
     
         4 . The chimeric protein according to  claim 1 , wherein the portion of the paracrine FGF comprises an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 151 to 155 of SEQ ID NO: 121. 
     
     
         5 . The chimeric protein according to  claim 3 , wherein the portion of the paracrine FGF comprises amino acid residues 1-150, 1-151, 1-152, 1-153, 1-154, 1-155, 2-150, 2-151, 2-152, 2-153, 2-154, 2-155, 3-150, 3-151, 3-152, 3-153, 3-154, 3-155, 4-150, 4-151, 4-152, 4-153, 4-154, 4-155, 5-150, 5-151, 5-152, 5-153, 5-154, 5-155, 6-150, 6-151, 6-152, 6-153, 6-154, 6-155, 7-150, 7-151, 7-152, 7-153, 7-154, 7-155, 8-150, 8-151, 8-152, 8-153, 8-154, 8-155, 9-150, 9-151, 9-152, 9-153, 9-154, 9-155, 10-150, 10-151, 10-152, 10-153, 10-154, 10-155, 11-150, 11-151, 11-152, 11-153, 11-154, 11-155, 12-150, 12-151, 12-152, 12-153, 12-154, 12-155, 13-150, 13-151, 13-152, 13-153, 13-154, 13-155, 14-150, 14-151, 14-152, 14-153, 14-154, 14-155, 15-150, 15-151, 15-152, 15-153, 15-154, 15-155, 16-150, 16-151, 16-152, 16-153, 16-154, 16-155, 17-150, 17-151, 17-152, 17-153, 17-154, 17-155, 18-150, 18-151, 18-152, 18-153, 18-154, 18-155, 19-150, 19-151, 19-152, 19-153, 19-154, 19-155, 20-150, 20-151, 20-152, 20-153, 20-154, 20-155, 21-150, 21-151, 21-152, 21-153, 21-154, 21-155, 22-150, 22-151, 22-152, 22-153, 22-154, 22-155, 23-150, 23-151, 23-152, 23-153, 23-154, 23-155, 24-150, 24-151, 24-152, 24-153, 24-154, 24-155, 25-150, 25-151, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 1. 
     
     
         6 . The chimeric protein according to  claim 4 , wherein the portion of the paracrine FGF comprises amino acid residues 1-151, 1-152, 1-153, 1-154, 1-155, 2-151, 2-152, 2-153, 2-154, 2-155, 3-151, 3-152, 3-153, 3-154, 3-155, 4-151, 4-152, 4-153, 4-154, 4-155, 5-151, 5-152, 5-153, 5-154, 5-155, 6-151, 6-152, 6-153, 6-154, 6-155, 7-151, 7-152, 7-153, 7-154, 7-155, 8-151, 8-152, 8-153, 8-154, 8-155, 9-151, 9-152, 9-153, 9-154, 9-155, 10-151, 10-152, 10-153, 10-154, 10-155, 11-151, 11-152, 11-153, 11-154, 11-155, 12-151, 12-152, 12-153, 12-154, 12-155, 13-151, 13-152, 13-153, 13-154, 13-155, 14-151, 14-152, 14-153, 14-154, 14-155, 15-151, 15-152, 15-153, 15-154, 15-155, 16-151, 16-152, 16-153, 16-154, 16-155, 17-151, 17-152, 17-153, 17-154, 17-155, 18-151, 18-152, 18-153, 18-154, 18-155, 19-151, 19-152, 19-153, 19-154, 19-155, 20-151, 20-152, 20-153, 20-154, 20-155, 21-151, 21-152, 21-153, 21-154, 21-155, 22-151, 22-152, 22-153, 22-154, 22-155, 23-151, 23-152, 23-153, 23-154, 23-155, 24-151, 24-152, 24-153, 24-154, 24-155, 25-151, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 121. 
     
     
         7 . The chimeric protein according to  claim 3 , wherein the modification comprises one or more substitutions located at one or more amino acid residues of SEQ ID NO: 1 selected from the group consisting of N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof. 
     
     
         8 . The chimeric protein according to  claim 7 , wherein the one or more substitutions are selected from the group consisting of N33T, K127D, K128Q, N129T, K133V, R134L, R137H, Q142M, K143T/L/I, and combinations thereof. 
     
     
         9 . The chimeric protein according to  claim 4 , wherein the modification comprises one or more substitutions located at one or more amino acid residues of SEQ ID NO: 121 selected from the group consisting of N36, K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof. 
     
     
         10 . The chimeric protein according to  claim 9 , wherein the one or more substitutions are selected from the group consisting of N36T, K128D, R129Q, K134V, K138H, Q143M, K144T/L/I, C78S, C96S, and combinations thereof. 
     
     
         11 . The chimeric protein according to  claim 1 , wherein the C-terminal portion comprises a β-Klotho co-receptor binding domain. 
     
     
         12 . The chimeric protein according to  claim 1 , wherein the C-terminal portion from FGF19 begins at a residue corresponding to any one of amino acid residues 169, 197, or 204 of SEQ ID NO: 233. 
     
     
         13 . The chimeric protein according to  claim 1 , wherein the C-terminal portion from FGF19 comprises an amino acid sequence selected from the group consisting of amino acid residues 204 to 216, amino acid residues 197 to 216, and amino acid residues 169 to 216 of SEQ ID NO: 233. 
     
     
         14 . The chimeric protein according to  claim 1 , wherein the C-terminal portion from FGF19 comprises an amino acid sequence selected from the group consisting of TGLEAV(R/N)SPSFEK (SEQ ID NO: 281); MDPFGLVTGLEAV(R/N)SPSFEK (SEQ ID NO: 282); and LP(M/I)(V/A)PEEPEDLR(G/R)HLESD(M/V)FS SPLETDSMDPFGLVTGLEAV(R/N)SPSFEK (SEQ ID NO: 283). 
     
     
         15 . The chimeric protein according to  claim 11 , wherein the C-terminal portion from FGF19 further comprises one or more substitutions, additions, or deletions while retaining the ability to bind β-Klotho. 
     
     
         16 . The chimeric protein according to  claim 11 , wherein the C-terminal portion from FGF19 further comprises one or more substitutions, additions, or deletions to enhance binding affinity for β-Klotho. 
     
     
         17 . A pharmaceutical composition comprising the chimeric protein according to  claim 1  and a pharmaceutically-acceptable carrier. 
     
     
         18 . The pharmaceutical composition according to  claim 17  further comprising one or more agents selected from the group consisting of an anti-inflammatory agent, an antifibrotic agent, an antihypertensive agent, an antidiabetic agent, a triglyceride-lowering agent, and a cholesterol-lowering agent. 
     
     
         19 . The pharmaceutical composition according to  claim 18  further comprising an organotropic targeting agent. 
     
     
         20 . A method for treating a subject suffering from a disorder, the method comprising:
 selecting a subject suffering from the disorder;   providing a chimeric fibroblast growth factor (“FGF”) protein, wherein the chimeric FGF protein comprises an N-terminus coupled to a C-terminus, wherein the N-terminus comprises a portion of a paracrine FGF and the C-terminus comprises a C-terminal portion of FGF19, and wherein the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification; and   administering a therapeutically effective amount of the chimeric FGF protein to the selected subject under conditions effective to treat the disorder.   
     
     
         21 . The method according to  claim 20 , wherein the paracrine FGF is FGF1 or FGF2. 
     
     
         22 . The method according to  claim 20 , wherein the portion of the paracrine FGF comprises an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1. 
     
     
         23 . The method according to  claim 20 , wherein the portion of the paracrine FGF comprises an amino acid sequence beginning at any one of residues 1 to 25 and ending at any one of residues 151 to 155 of SEQ ID NO: 121. 
     
     
         24 . The method according to  claim 22 , wherein the portion of the paracrine FGF comprises amino acid residues 1-150, 1-151, 1-152, 1-153, 1-154, 1-155, 2-150, 2-151, 2-152, 2-153, 2-154, 2-155, 3-150, 3-151, 3-152, 3-153, 3-154, 3-155, 4-150, 4-151, 4-152, 4-153, 4-154, 4-155, 5-150, 5-151, 5-152, 5-153, 5-154, 5-155, 6-150, 6-151, 6-152, 6-153, 6-154, 6-155, 7-150, 7-151, 7-152, 7-153, 7-154, 7-155, 8-150, 8-151, 8-152, 8-153, 8-154, 8-155, 9-150, 9-151, 9-152, 9-153, 9-154, 9-155, 10-150, 10-151, 10-152, 10-153, 10-154, 10-155, 11-150, 11-151, 11-152, 11-153, 11-154, 11-155, 12-150, 12-151, 12-152, 12-153, 12-154, 12-155, 13-150, 13-151, 13-152, 13-153, 13-154, 13-155, 14-150, 14-151, 14-152, 14-153, 14-154, 14-155, 15-150, 15-151, 15-152, 15-153, 15-154, 15-155, 16-150, 16-151, 16-152, 16-153, 16-154, 16-155, 17-150, 17-151, 17-152, 17-153, 17-154, 17-155, 18-150, 18-151, 18-152, 18-153, 18-154, 18-155, 19-150, 19-151, 19-152, 19-153, 19-154, 19-155, 20-150, 20-151, 20-152, 20-153, 20-154, 20-155, 21-150, 21-151, 21-152, 21-153, 21-154, 21-155, 22-150, 22-151, 22-152, 22-153, 22-154, 22-155, 23-150, 23-151, 23-152, 23-153, 23-154, 23-155, 24-150, 24-151, 24-152, 24-153, 24-154, 24-155, 25-150, 25-151, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 1. 
     
     
         25 . The method according to  claim 23 , wherein the portion of the paracrine FGF comprises amino acid residues 1-151, 1-152, 1-153, 1-154, 1-155, 2-151, 2-152, 2-153, 2-154, 2-155, 3-151, 3-152, 3-153, 3-154, 3-155, 4-151, 4-152, 4-153, 4-154, 4-155, 5-151, 5-152, 5-153, 5-154, 5-155, 6-151, 6-152, 6-153, 6-154, 6-155, 7-151, 7-152, 7-153, 7-154, 7-155, 8-151, 8-152, 8-153, 8-154, 8-155, 9-151, 9-152, 9-153, 9-154, 9-155, 10-151, 10-152, 10-153, 10-154, 10-155, 11-151, 11-152, 11-153, 11-154, 11-155, 12-151, 12-152, 12-153, 12-154, 12-155, 13-151, 13-152, 13-153, 13-154, 13-155, 14-151, 14-152, 14-153, 14-154, 14-155, 15-151, 15-152, 15-153, 15-154, 15-155, 16-151, 16-152, 16-153, 16-154, 16-155, 17-151, 17-152, 17-153, 17-154, 17-155, 18-151, 18-152, 18-153, 18-154, 18-155, 19-151, 19-/152, 19-153, 19-154, 19-155, 20-151, 20-152, 20-153, 20-154, 24-155, 21-151, 21-152, 21-153, 21-154, 21-155, 22-151, 22-152, 22-153, 22-154, 22-155, 23-151, 23-152, 23-153, 23-154, 23-155, 24-151, 24-152, 24-153, 24-154, 24-155, 25-151, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 121. 
     
     
         26 . The method according to  claim 22 , wherein the modification comprises one or more substitutions located at one or more amino acid residues of SEQ ID NO: 1 selected from the group consisting of N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof. 
     
     
         27 . The method according to  claim 26 , wherein the one or more substitutions are selected from the group consisting of N33T, K127D, K128Q, N129T, K133V, R134L, R137H, Q142M, K143T/L/I, and combinations thereof. 
     
     
         28 . The method according to  claim 23 , wherein the modification comprises one or more substitutions located at one or more amino acid residues of SEQ ID NO: 121 selected from the group consisting of N36, K128, R129, K134, K138, Q143, K144, C78, C96, and combinations thereof. 
     
     
         29 . The method according to  claim 28 , wherein the one or more substitutions are selected from the group consisting of N36T, K128D, R129Q, K134V, K138H, Q143M, K144T/L/I, C78S, C96S, and combinations thereof. 
     
     
         30 . The method according to  claim 20 , wherein the C-terminal portion comprises a β-Klotho co-receptor binding domain. 
     
     
         31 . The method according to  claim 20 , wherein the C-terminal portion from FGF19 begins at a residue corresponding to any one of amino acid residues 169, 197, or 204 of SEQ ID NO: 233. 
     
     
         32 . The method according to  claim 20 , wherein the C-terminal portion from FGF19 comprises an amino acid sequence selected from the group consisting of amino acid residues 204 to 216, amino acid residues 197 to 216, and amino acid residues 169 to 216 of SEQ ID NO: 233. 
     
     
         33 . The method according to  claim 20 , wherein the C-terminal portion from FGF19 comprises an amino acid sequence selected from the group consisting of TGLEAV(R/N)SPSFEK (SEQ ID NO: 281); MDPFGLVTGLEAV(R/N)SPSFEK (SEQ ID NO: 282); and LP(M/I)(V/A)PEEPEDLR(G/R)HLESD(M/V)FSSPLETDSMDPFGLVTGLEAV(R/N)SPSFEK (SEQ ID NO: 283). 
     
     
         34 . The method according to  claim 32 , wherein the C-terminal portion from FGF19 further comprises one or more substitutions, additions, or deletions while retaining the ability to bind β-Klotho. 
     
     
         35 . The method according to  claim 32 , wherein the C-terminal portion from FGF19 further comprises one or more substitutions, additions, or deletions to enhance binding affinity for β-Klotho. 
     
     
         36 . The method according to  claim 20 , wherein the disorder is associated with diabetes, obesity, or metabolic syndrome. 
     
     
         37 . The method according to  claim 36 , wherein the disorder is type II diabetes, gestational diabetes, or drug-induced diabetes. 
     
     
         38 . The method according to  claim 36 , wherein the disorder is type I diabetes. 
     
     
         39 . The method according to  claim 36 , wherein the disorder is obesity. 
     
     
         40 . The method according to  claim 37 , wherein the disorder is metabolic syndrome. 
     
     
         41 . The method according to  claim 20 , wherein the administering is performed subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. 
     
     
         42 . The method according to  claim 20 , wherein the chimeric protein is administered with a pharmaceutically-acceptable carrier. 
     
     
         43 . The method according to  claim 20 , wherein the selected subject is a mammal. 
     
     
         44 . The method according to  claim 20 , wherein the selected subject is a human. 
     
     
         45 . The method according to  claim 20 , wherein the chimeric FGF is co-administered with one or more agents selected from the group consisting of an anti-inflammatory agent, an antifibrotic agent, an antihypertensive agent, an antidiabetic agent, a triglyceride-lowering agent, and a cholesterol-lowering agent. 
     
     
         46 . A method of making a chimeric fibroblast growth factor (“FGF”) protein possessing enhanced endocrine activity, the method comprising:
 introducing one or more modifications to a FGF protein, wherein the modification decreases the affinity of the FGF protein for heparin and/or heparan sulfate; and 
 coupling a C-terminal portion of FGF19 comprising a β-Klotho co-receptor binding domain to the modified FGF protein's C-terminus, whereby a chimeric FGF protein possessing enhanced endocrine activity is made. 
 
     
     
         47 .- 60 . (canceled) 
     
     
         61 . A method of facilitating fibroblast growth factor receptor (“FGFR”)-βKlotho complex formation, the method comprising:
 providing a cell comprising βKlotho co-receptor and an FGFR; 
 providing a chimeric fibroblast growth factor (“FGF”) protein comprising a C-terminal portion of FGF19 and a portion of a paracrine FGF, wherein the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification; and 
 contacting the cell with the chimeric FGF protein under conditions effective to cause FGFR-Klotho co-receptor complex formation. 
 
     
     
         62 .- 77 . (canceled) 
     
     
         78 . A method of screening for agents capable of facilitating fibroblast growth factor receptor (“FGFR”)—βKlotho co-receptor complex formation in the treatment of a disorder, the method comprising:
 providing a chimeric fibroblast growth factor (“FGF”) comprising an N-terminus coupled to a C-terminus, wherein the N-terminus comprises a portion of a paracrine FGF and the C-terminus comprises a C-terminal portion of FGF19, and wherein the portion of the paracrine FGF is modified to decrease binding affinity for heparin and/or heparan sulfate compared to the portion without the modification; 
 providing binary βKlotho-FGFR complex; 
 providing one or more candidate agents; 
 combining the chimeric FGF, the binary βKlotho-FGFR complex, and the one or more candidate agents under conditions permitting the formation of a ternary complex between the chimeric FGF and the binary βKlotho-FGFR complex in the absence of the one or more candidate agents; and 
 identifying the one or more candidate agents that decrease ternary complex formation between the chimeric FGF and the binary βKlotho-FGFR complex compared to the ternary complex formation in the absence of the one or more candidate agents as suitable for treating the disorder. 
 
     
     
         79 .- 86 . (canceled)