Methods of making FGF-21 mutants comprising non-naturally encoded phenylalanine derivatives

• US 8,383,365 B2
• Filed: 03/18/2011
• Issued: 02/26/2013
• Est. Priority Date: 03/30/2007
• Status: Active Grant

• Current Assignee: Ambrx, Inc. (Johnson & Johnson)
• Original Assignee: Ambrx, Inc. (Johnson & Johnson)
• Inventors: Cujec, Thomas P., Mariani, Roberto, Hays Putnam, Anna-Maria A., Keefe, William M., Knudsen, Nick, Pinkstaff, Jason, Kraynov, Vadim, Skidmore (Nee Ho), Lillian
• Primary Examiner(s): STOICA, ELLY GERALD
• Application Number:
  US13/051,953
• Publication Number:
  US 20110172401A1
• Time in Patent Office: 711 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 39/00 General protective or antinoxious agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/50 Fibroblast growth factor [FGF]
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Modified FGF-21 polypeptides and uses thereof are provided.

Claims

1. A method of making a modified human FGF-21 polypeptide comprising a non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is a phenylalanine derivative and

is at a position selected from the group consisting of residues 72, 77, 86, 87, 91, 108, 110, 126, 131, and 146 of SEQ ID NO:

1 or the corresponding amino acid position in another human FGF-21 polypeptide; and

wherein the non-naturally encoded amino acid is linked to a linker, polymer, or biologically active molecule, the method comprising ribosomally synthesizing a modified human FGF-21 polypeptide, which comprises a non-naturally encoded amino acid, and contacting the modified human FGF-21 polypeptide with a linker, polymer, or biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid of the modified human FGF-21 polypeptide.

2. The method of claim 1, wherein the polymer is a water soluble polymer.

3. The method of claim 1, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.

4. The method of claim 1, wherein the non-naturally encoded amino acid comprises a carbonyl moiety and the linker, polymer, or biologically active molecule comprises an aminooxy, a hydrazine, a hydrazide or a semicarbazide moiety.

5. The method of claim 4, wherein the aminooxy, hydrazine, hydrazide or semicarbazide moiety is linked to the linker, polymer, or biologically active molecule through an amide linkage.

6. The method of claim 1, wherein the non-naturally encoded amino acid comprises an alkyne moiety and the linker, polymer, or biologically active molecule comprises an azide moiety.

7. The method of claim 1, wherein the non-naturally encoded amino acid comprises an azide moiety and the linker, polymer, or biologically active molecule comprises an alkyne moiety.

8. The method of claim 3, wherein the azide or alkyne moiety is linked to a linker, polymer, or biologically active molecule through an amide linkage.

9. The method of claim 1, wherein the polymer comprises poly(ethylene glycol).

10. The method of claim 9, wherein the poly(ethylene glycol) moiety has an average molecular weight of between about 0.1kDa and about 100 kDa.

11. The method of claim 9, wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer.

12. The method of claim 2, wherein the polymer is an oligosaccharide.

13. The method of claim 1, wherein the modified human FGF-21 polypeptide comprising a non-naturally encoded amino acid exhibits an increased in vivo half-life over the corresponding modified human FGF-21 polypeptide without a non-naturally encoded amino acid.

14. The method of claim 13, wherein the half-life increased from about 10-fold to about 40-fold.

15. The method of claim 13, wherein the half-life increased from about 13-fold to about 30-fold.

16. The method of claim 13, wherein the half-life increased from about 13-fold to about 18-fold.

17. The method of claim 13, wherein the half-life increased from about 20-fold to about 30-fold.

18. The method of claim 9, wherein the modified human FGF-21 polypeptide comprising a non-naturally encoded amino acid exhibits an increased in vivo half-life over the corresponding modified human FGF-21 polypeptide without a non-naturally encoded amino acid.

19. The method of claim 18, wherein the half-life increased from about 10-fold to about 40-fold.

20. The method of claim 18, wherein the half-life increased from about 13-fold to about 30-fold.

21. The method of claim 18, wherein the half-life increased from about 13-fold to about 18-fold.

22. The method of claim 18, wherein the half-life increased from about 20-fold to about 30-fold.

23. The method of claim 1, wherein the modified human FGF-21 polypeptide comprising a non-naturally encoded amino acid exhibits an increased in vivo half-life over the corresponding modified human FGF-21 polypeptide with a non-naturally encoded amino acid.

24. The method of claim 23, wherein the half-life increased from about 10-fold to about 40-fold.

25. The method of claim 23, wherein the half-life increased from about 13-fold to about 30-fold.

26. The method of claim 23, wherein the half-life increased from about 13-fold to about 18-fold.

27. The method of claim 23, wherein the half-life increased from about 20-fold to about 30-fold.

28. The method of claim 9, wherein the modified human FGF-21 polypeptide comprising a non-naturally encoded amino acid exhibits an increased in vivo half-life over the corresponding modified human FGF-21 polypeptide with a non-naturally encoded amino acid.

29. The method of claim 28, wherein the half-life increased from about 10-fold to about 40-fold.

30. The method of claim 28, wherein the half-life increased from about 13-fold to about 30-fold.

31. The method of claim 28, wherein the half-life increased from about 13-fold to about 18-fold.

32. The method of claim 28, wherein the half-life increased from about 20-fold to about 30-fold.

33. The method of claim 1, wherein the modified human FGF-21 polypeptide comprises SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7 containing a substitution of an amino acid with a non-naturally encoded amino acid.

34. The method of claim 1, wherein the modified human FGF-21 polypeptide comprises SEQ ID NO:

1 containing a substitution of an amino acid with a non-naturally encoded amino acid.

35. The method of claim 1, wherein the non-naturally encoded amino acid is substituted at residue 108.

36. The method of claim 1, wherein the non-naturally encoded amino acid is selected from the group consisting of a para-substituted phenylalanine, an ortho-substituted phenylalanine, and a meta-substituted phenylalanine.

37. The method of claim 36, wherein the para-substituted, ortho-substituted, or meta-substituted phenylalanine comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.

38. The method of claim 1, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

39. The method of claim 1, wherein the non-naturally encoded amino acid is selected from the group consisting of a para-substituted phenylalanine, an ortho-substituted phenylalanine, and a meta-substituted phenylalanine and wherein the polymer is poly(ethylene glycol).

40. The method of claim 1, wherein the polymer is poly(ethylene glycol) having an average molecular weight of about 30 kDa, wherein the modified human FGF-21 polypeptide comprises SEQ ID NO:

1 containing para-acetyl-L-phenylalanine at residue 108.

41. The method of claim 1, which results in a biologically active FGF-21 polypeptide having increased in vivo circulating half-life compared to an unmodified FGF-21 polypeptide having the sequence of SEQ ID NO:

1.

42. The method of claim 9, wherein the poly(ethylene glycol) moiety has an average molecular weight of about 30 kDa.

60. The method of claim 42, wherein the polypeptide has a serum half-life that is at least five times the serum half-life of FGF-21 polypeptide consisting of SEQ ID NO:

1.

43. A method of making a polypeptide comprising a non-naturally encoded amino acid, wherein the polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7;

wherein the polypeptide contains a substitution of an amino acid with the non-naturally encoded amino acid at a position selected from the group consisting of the corresponding residues 72, 77, 86, 87, 91, 108, 110, 126, 131, and 146 of SEQ ID NO;

1 or the corresponding amino acid position in SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7;

wherein the polypeptide maintains the biological activity of human FGF-21 polypeptides; and

wherein the non-naturally encoded amino acid is a phenylalanine derivative and is linked to a linker, polymer, or biologically active molecule, the method comprising ribosomally synthesizing a polypeptide, which comprises a non-naturally encoded amino acid, and contacting the polypeptide with a linker, polymer, or biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid of the polypeptide.

44. The method of claim 43, wherein the polymer comprises poly(ethylene glycol).

45. The method of claim 44, wherein the poly(ethylene glycol) moiety has an average molecular weight of between about 0.1 kDa and about 100 kDa.

46. The method of claim 44, wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer.

47. The method of claim 44, wherein the poly(ethylene glycol) moiety has an average molecular weight of about 30 kDa.

48. The method of claim 43, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

49. The method of claim 43, wherein the non-naturally encoded amino acid is substituted at residue 108.

50. The method of claim 43, wherein the polymer is poly(ethylene glycol) having an average molecular weight of about 30 kDa, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine substituted at residue 108.

51. The method of claim 43, wherein the polypeptide has a serum half-life that is at least five times the serum half-life of FGF-21 polypeptide consisting of SEQ ID NO:

1.

52. The method of claim 43, wherein the polypeptide comprises a sequence at least 95% identical to SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, or SEQ ID NO;

7.

53. The method of claim 52 wherein the polymer comprises poly(ethylene glycol).

54. The method of claim 53, wherein the poly(ethylene glycol) moiety has an average molecular weight of between about 0.1 kDa and about 100 kDa.

55. The method of claim 53, wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer.

56. The method of claim 53, wherein the poly(ethylene glycol) moiety has an average molecular weight of about 30 kDa.

57. The method of claim 52, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

58. The method of claim 52, wherein the non-naturally encoded amino acid is substituted at residue 108.

59. The method of claim 52, wherein the polymer is poly(ethylene glycol) having an average molecular weight of about 30 kDa, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine substituted at residue 108.