Chimeric fibroblast growth factor 21 proteins and methods of use

Patent metadata

Field	Value
Publication number	US-9926355-B2
Application number	US-201615289447-A
Country	US
Kind code	B2
Filing date	Oct 10, 2016
Priority date	Jun 7, 2012
Publication date	Mar 27, 2018
Grant date	Mar 27, 2018

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 FGF21 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.

First claim

What is claimed:

1. A chimeric protein comprising:
an N-terminus coupled to a C-terminus,
wherein the N-terminus comprises an FGF1 portion beginning at any one of residues 1 to 25 and ending at any one of residues 150 to 155 of SEQ ID NO: 1,
wherein the FGF1 amino acid positions corresponding to those selected from the group consisting of N33, K127, K128, N129, K133, R134, R137, Q142, K143, and combinations thereof are substituted to decrease binding affinity for heparin and/or heparan sulfate compared to FGF1 without the substitution, and
wherein the C-terminus comprises a C-terminal portion of an FGF21 comprising amino acid residues 168 to 209 of SEQ ID NO: 233.

2. The chimeric protein according to claim 1 , wherein the FGF1 portion is amino acid residues 1-150 of SEQ ID NO: 1.

3. The chimeric protein according to claim 1 , wherein the FGF1 portion is amino acid residues 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, 25-151, 25-152, 25-153, 25-154, or 25-155 of SEQ ID NO: 1.

4. The chimeric protein according to claim 1 , wherein the one or more substitutions are selected from the group consisting of N33T; K127D; K128Q; N129T; K133V; R134L; R137H; Q142M; K143T, K143L, or K1431; and combinations thereof.

5. The chimeric protein according to claim 1 , wherein the FGF1 portion is amino acid residues 25-150 of SEQ ID NO: 1.

6. The chimeric protein according to claim 1 , wherein the chimeric protein comprises the amino acid sequence of SEQ ID NO: 339 or SEQ ID NO: 340.

7. A pharmaceutical composition comprising the chimeric protein of claim 1 and a pharmaceutically-acceptable carrier.

8. The pharmaceutical composition according to claim 7 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.

9. The chimeric protein according to claim 1 , wherein the one or more amino acid substitutions comprises a substitution at amino acid residue N33.

10. The chimeric protein according to claim 9 , wherein the substitution is N33T.

11. The chimeric protein according to claim 1 , wherein the one or more amino acid substitutions comprises a substitution at amino acid residue K127.

12. The chimeric protein according to claim 11 , wherein the substitution is K127D.

13. The chimeric protein according to claim 1 , wherein the one or more amino acid substitutions comprises a substitution at amino acid residue K128.

14. The chimeric protein according to claim 13 , wherein the substitution is K128Q.

15. The chimeric protein according to claim 1 , wherein the one or more amino acid substitutions comprises a substitution at amino acid residue N129.

16. The chimeric protein according to claim 15 , wherein the substitution is N129T.

17. The chimeric protein according to claim 1 , wherein the one or more amino acid substitutions comprises a substitution at amino acid residue K133.

18. The chimeric protein according to claim 17 , wherein the substitution is K133V.

19. The chimeric protein according to claim 1 , wherein the one or more amino acid substitutions comprises a substitution at amino acid residue R134.

20. The chimeric protein according to claim 19 , wherein the substitution is R134L.

21. The chimeric protein according to claim 1 , wherein the one or more amino acid substitutions comprises a substitution at amino acid residue R137.

22. The chimeric protein according to claim 21 , wherein the substitution is R137H.

23. The chimeric protein according to claim 1 , wherein the one or more amino acid substitutions comprises a substitution at amino acid residue Q142.

24. The chimeric protein according to claim 23 , wherein the substitution is Q142M.

25. The chimeric protein according to claim 1 , wherein the one or more amino acid substitutions comprises a substitution at amino acid residue K143.

26. The chimeric protein according to claim 25 , wherein the substitution is K143T.

27. The chimeric protein according to claim 25 , wherein the substitution is K143L.

28. The chimeric protein according to claim 25 , wherein the substitution is K1431.

29. The chimeric protein according to claim 1 , wherein the one or more amino acid substitutions comprises substitutions at amino acid residues K127, K128, and K133.

30. The chimeric protein according to claim 29 , wherein the substitutions are K127D, K128Q, and K133V.

31. An isolated FGF1 peptide comprising at least 90% sequence identity to amino acid residues 25-155 of SEQ ID NO:1 with K127D, K128Q, and K133V substitutions (FGF1 ΔNTΔHBS ), wherein the isolated FGF1 peptide comprises K127D, K128Q, and K133V substitutions.

32. The isolated FGF1 peptide according to claim 31 , wherein the isolated FGF1 peptide comprises at least 95% sequence identity to amino acid residues 25-155 of SEQ ID NO:1 with K127D, K128Q, and K133V substitutions (FGF1 ΔNTΔHBS ).

33. The isolated FGF1 peptide according to claim 31 , wherein the isolated FGF1 peptide comprises at least 99% sequence identity to amino acid residues 25-155 of SEQ ID NO:1 with K127D, K128Q, and K133V substitutions (FGF1 ΔNTΔHBS ).

34. The isolated FGF1 peptide according to claim 31 , wherein the isolated FGF1 peptide comprises amino acid residues 25-155 of SEQ ID NO:1 with K127D, K128Q, and K133V substitutions (FGF1 ΔNTΔHBS ).

35. The isolated FGF1 peptide according to claim 31 , wherein the isolated FGF1 peptide consists of amino acid residues 25-155 of SEQ ID NO:1 with K127D, K128Q, and K133V substitutions (FGF1 ΔNTΔHBS ).

36. An isolated FGF1 peptide comprising at least 90% sequence identity to amino acid residues 1-155 of SEQ ID NO:1 with K127D, K128Q, and K133V substitutions (FGF1 ΔHBS ), wherein the isolated FGF1 peptide comprises K127D, K128Q, and K133V substitutions.

37. The isolated FGF1 peptide according to claim 36 , wherein the isolated FGF1 peptide comprises at least 95% sequence identity to amino acid residues 1-155 of SEQ ID NO:1 with K127D, K128Q, and K133V substitutions (FGF1 ΔHBS ).

38. The isolated FGF1 peptide according to claim 36 , wherein the isolated FGF1 peptide compr

Assignees

• Univ New York

Inventors

• Mohammadi Moosa
• Goetz Regina

Classifications

• A61P3/10

  for hyperglycaemia, e.g. antidiabetics · CPC title

• G01N2800/042

  Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism · CPC title

• G01N2500/02

  Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor) · CPC title

• G01N2333/50

  Fibroblast growth factors [FGF] · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title