Modified FGF-21 polypeptides and their uses

• US 8,012,931 B2
• Filed: 03/19/2008
• Issued: 09/06/2011
• Est. Priority Date: 03/30/2007
• Status: Active Grant

• Current Assignee: Ambrx, Inc. (Johnson & Johnson)
• Original Assignee: Ambrx, Inc. (Johnson & Johnson)
• Inventors: Kraynov, Vadim, Mariani, Roberto, Cujec, Thomas P., Keefe, William M., Pinkstaff, Jason, Hays Putnam, Anna-Maria A., Ho, Lillian, Knudsen, Nick
• Primary Examiner(s): STOICA, ELLY GERALD
• Application Number:
  US12/051,830
• Publication Number:
  US 20080255045A1
• Time in Patent Office: 1,266 Days
• Field of Search: None
• US Class Current: 514/9.1
• 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 biologically active variant of FGF-21 comprising the amino acid sequence of SEQ ID NO:

1 and comprising one or more non-naturally encoded amino acids wherein at least one of the one or more non-naturally encoded amino acids occurs at position 108 in the FGF-21 variant, and wherein the non-naturally encoded amino acid has the structure;

2. The FGF-21 variant of claim 1, wherein the FGF-21 variant comprises one or more post-translational modifications.

3. The FGF-21 variant of claim 1, wherein the non-naturally occurring amino acid is linked to a linker, polymer, or biologically active molecule.

4. The FGF-21 variant of claim 3, wherein the variant is linked to a water soluble polymer.

5. The FGF-21 variant of claim 1, wherein the variant is linked to a bifunctional polymer, bifunctional linker, or at least one additional FGF-21 polypeptide.

6. The FGF-21 variant of claim 5, wherein the bifunctional linker or polymer is linked to a second polypeptide.

7. The FGF-21 variant of claim 6, wherein the second polypeptide is a FGF-21 polypeptide.

8. The FGF-21 variant of claim 4, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety.

9. The FGF-21 variant of claim 4, wherein said water soluble polymer is linked to a non-naturally encoded amino acid present in said FGF-21 variant.

10. The FGF-21 variant of claim 3, wherein the non-naturally encoded amino acid is a phenylalanine derivative.

11. The FGF-21 variant of claim 1, wherein the FGF-21 variant comprises one or more amino acid substitution, addition or deletion that modulates affinity of the FGF-21 variant for a FGF-21 receptor.

12. The FGF-21 variant of claim 1, wherein the FGF-21 variant comprises one or more amino acid substitution, addition or deletion that increases the stability or solubility of the FGF-21 variant.

13. The FGF-21 variant of claim 1, wherein the FGF-21 variant comprises one or more amino acid substitution, addition or deletion that increases the expression of the FGF-21 variant in a recombinant host cell or synthesized in vitro.

14. The FGF-21 variant of claim 1, wherein the FGF-21 variant comprises one or more amino acid substitution, addition or deletion that increases protease resistance of the FGF-21 variant.

15. The FGF-21 variant of claim 1, wherein the one or more non-naturally encoded amino acid(s) is reactive toward a linker, polymer, or biologically active molecule that is otherwise unreactive toward any of the 20 common amino acids in the polypeptide.

16. The FGF-21 variant of claim 1, wherein the one or more non-naturally encoded amino acid(s) comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.

17. The FGF-21 variant of claim 16, wherein the one or more non-naturally encoded amino acid(s) comprises a carbonyl group.

18. The FGF-21 variant of claim 4, wherein the water soluble polymer has a molecular weight of between about 0.1 kDa and about 100 kDa.

19. The FGF-21 variant of claim 18, wherein the water soluble polymer has a molecular weight of between about 0.1 kDa and about 50 kDa.

20. The FGF-21 variant of claim 4, which is made by reacting a FGF-21 polypeptide comprising a carbonyl-containing amino acid with a water soluble polymer comprising an aminooxy, hydrazine, hydrazide or semicarbazide group.

21. The FGF-21 variant of claim 20, wherein the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the water soluble polymer through an amide linkage.

22. The FGF-21 variant of claim 4, which is made by reacting a water soluble polymer comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid that comprises an aminooxy, a hydrazine, a hydrazide or a semicarbazide group.

23. The FGF-21 variant of claim 4, wherein the water soluble polymer is a branched or multiarmed polymer.

24. The FGF-21 variant of claim 23, wherein each branch of the water soluble polymer has a molecular weight of between about 1 kDa and about 100 kDa.

25. The FGF-21 variant of claim 1, wherein the variant is a FGF-21 antagonist.

26. The FGF-21 variant of claim 25, wherein the variant comprises one or more post-translational modification, linker, polymer, or biologically active molecule.

27. The FGF-21 variant of claim 26, wherein the polymer comprises a moiety selected from a group consisting of a water soluble polymer and poly(ethylene glycol).

28. The FGF-21 variant according to claim 25, wherein the polypeptide prevents activation of the FGF-21 receptor.

29. The FGF-21 variant of claim 1, wherein the one or more non-naturally encoded amino acid(s) comprises a saccharide moiety.

30. The FGF-21 variant of claim 3, wherein the linker, polymer, or biologically active molecule is linked to the polypeptide via a saccharide moiety.

31. A composition comprising the FGF-21 variant of claim 1 and a pharmaceutically acceptable carrier.

32. The composition of claim 31, wherein the one or more non-naturally encoded amino acid(s) is linked to a water soluble polymer.

45. The FGF-21 variant of claim 10 wherein said non-naturally encoded amino acid is linked to a poly(ethylene glycol) molecule.

46. The FGF-21 polypeptide of claim 45, wherein said FGF-21 polypeptide is monoPEGylated.

49. The FGF-21 variant of claim 1, wherein the FGF-21 variant comprises one or more amino acid substitution, addition, or deletion that modulates immunogenicity of the FGF-21 polypeptide.

50. The FGF-21 variant of claim 1, wherein the FGF-21 variant comprises one or more amino acid substitution, addition, or deletion that modulates serum half-life or circulation time of the FGF-21 variant.

51. The FGF-21 variant of claim 1, wherein the one or more non-naturally encoded amino acid(s) comprises a ketone group.

52. The FGF-21 variant of claim 1, wherein the polypeptide also comprises a naturally encoded amino acid substitution.

33. An FGF-21 variant encoded by a polynucleotide having a sequence shown in SEQ ID NO:

8, 9, 10, 11, 12, 13, or 14, wherein said polynucleotide comprises a selector codon, and wherein said variant comprises at least one non-naturally encoded amino acid substituted at a corresponding residue position 108 in SEQ ID NO;

1 or the corresponding amino acids of SEQ ID NOs;

2-7, wherein the non-naturally occurring amino acid has the structure;

34. The FGF-21 variant of claim 33, wherein the non-naturally encoded amino acid is linked to a linker, polymer, water soluble polymer, or biologically active molecule.

35. The FGF-21 variant of claim 34, wherein the water soluble polymer comprises a polyethylene glycol) moiety.

36. The FGF-21 variant of claim 33, wherein the non-naturally encoded amino acid is a phenylalanine derivative.

37. The FGF-21 variant of claim 33, 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.

38. The FGF-21 variant of claim 35, wherein the poly(ethylene glycol) moiety has a molecular weight of between about 0.1 kDa and about 100 kDa.

39. The FGF-21 variant of claim 35, wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer.

40. The FGF-21 variant of claim 39, wherein the poly(ethylene glycol) moiety has a molecular weight of between about 1 kDa and about 100 kDa.

41. A composition comprising the FGF-21 variant of claim 33 and a pharmaceutically acceptable carrier.

43. The FGF-21 variant of claim 35, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety.

44. The FGF-21 variant of claim 35, wherein the amino acid covalently linked to the water soluble polymer is a non-naturally encoded amino acid.

42. A FGF-21 variant comprising one or more amino acid substitution, addition or deletion wherein at least one of the one or more amino acid substitution, addition, or deletions occurs at amino acid position 108 of SEQ ID NO.:

1 that increases the expression of the FGF-21 variant in a recombinant host cell.

47. A biologically functional FGF-21 variant comprising a non-naturally encoded amino acid at position 108 of SEQ ID NO.:

1, and a linker, polymer or biologically active molecule that is attached to the non-naturally encoded amino acid wherein said non-naturally encoded amino acid is ribosomally incorporated into the polypeptide.

48. The FGF-21 variant of claim 47, wherein the FGF-21 variant comprises one said linker, polymer, or biologically active molecule.