Modified FGF-21 polypeptides comprising non-naturally occurring amino acids

• US 9,079,971 B2
• Filed: 01/02/2013
• Issued: 07/14/2015
• 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, Ho, Lillian, Pinkstaff, Jason, Kraynov, Vadim
• Primary Examiner(s): STOICA, ELLY GERALD
• Application Number:
  US13/732,522
• Publication Number:
  US 20130150564A1
• Time in Patent Office: 923 Days
• US Class Current: 1/1
• 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 modified FGF-21 polypeptide comprising a non-naturally encoded amino acid, wherein:

(a) 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;

(b) the non-naturally encoded amino acid has the structure;

2. The modified polypeptide of claim 1, wherein the non-naturally encoded amino acid is a phenylalanine derivative.

3. The modified polypeptide of claim 1, wherein the non-naturally encoded amino acid comprises a first functional group and the linker, polymer, or biologically active molecule comprises a second functional group, wherein the first functional group and second functional group are not identical and each comprise a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.

4. The modified polypeptide of claim 3, wherein the first functional group on the non-naturally encoded amino acid is a carbonyl moiety and the second functional group on the linker, polymer, or biologically active molecule is an aminooxy moiety, and the resultant covalent linkage created by the reaction of the first and second functional groups is an oxime linkage.

5. The modified polypeptide of claim 3, wherein the first functional group on the non-naturally encoded amino acid is an azide moiety and the second functional group on the linker, polymer, or biologically active molecule is an alkyne moiety.

6. The modified polypeptide of claim 1, wherein the non-naturally encoded amino acid is linked to a polymer.

7. The modified polypeptide of claim 6, wherein the polymer comprises poly(ethylene glycol).

8. The modified polypeptide of claim 7, wherein the poly(ethylene glycol) has an average molecular weight of between about 0.1 kDa and about 100 kDa.

9. The modified polypeptide of claim 7, wherein the poly(ethylene glycol) has an average molecular weight of about 30 kDa.

10. The modified polypeptide of claim 6, wherein the polymer is an oligosaccharide.

11. The modified polypeptide of claim 1, wherein the polypeptide has an in vivo half-life at least five-fold greater than the human FGF-21 polypeptide in SEQ ID NO:

1.

12. The modified polypeptide of claim 1, wherein the polypeptide has an in vivo half-life about 10-fold to about 40-fold greater than the human FGF-21 polypeptide in SEQ ID NO:

1.

13. The modified polypeptide of claim 1, wherein the non-naturally encoded amino acid is at a position corresponding to residue 108.

14. The modified polypeptide of claim 1, wherein the non-naturally encoded amino acid is selected from a para-substituted, ortho-substituted, or meta-substituted phenylalanine comprising a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.

15. The modified polypeptide of claim 14, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

16. The modified polypeptide of claim 1, wherein the 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.

17. The modified polypeptide of claim 16, wherein

(a) the modified polypeptide comprises SEQ ID NO:

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

(b) the phenylalanine derivative is linked to a polymer, and

(c) the polymer comprises poly(ethylene glycol) having an average molecular weight of about 30 kDa.

18. The modified polypeptide of claim 1, wherein the polypeptide comprises a sequence at least 95% identical to SEQ ID NO:

1.

19. A modified FGF-21 polypeptide having biological activity associated with native human FGF-21, wherein:

(a) the polypeptide comprises a non-naturally encoded amino acid that is linked to a linker, polymer, or another biologically active molecule; and

(b) the non-naturally encoded amino acid is a phenylalanine derivative at a position corresponding to residue 72, 77, 86, 104, 108, 110, 126, 131, or 146 of SEQ ID NO;

1.

20. A modified FGF-21 polypeptide comprising the polypeptide of SEQ ID NO:

1 except that an amino acid in the polypeptide is substituted by a non-naturally encoded amino acid, wherein;

(a) said non-naturally encoded amino acid is at a position corresponding to residue 108 of SEQ ID NO;

1;

(b) said non-naturally encoded amino acid comprises para-acetyl phenylalanine linked to a polymer comprising a poly(ethylene glycol), wherein said poly(ethylene glycol) has an average molecular weight of about 30 kDa; and

(c) said non-naturally encoded amino acid is linked to said polymer through an oxime linkage.

21. The modified polypeptide of claim 20, wherein said polymer comprises an aminooxy group, and said non-naturally encoded amino acid is linked to said polymer by said oxime linkage formed by a reaction of said para-acetyl phenylalanine with said aminooxy group.

22. A modified FGF-21 polypeptide comprising the polypeptide of SEQ ID NO:

1 except that the amino acid at position 108 of SEQ ID NO;

1 is substituted by a non-naturally encoded amino acid, wherein;

(a) said non-naturally encoded amino acid comprises para-acetyl phenylalanine, and

(b) said non-naturally encoded amino acid is linked to a polymer comprising a poly(ethylene glycol) having an average molecular weight of about 30 kDa.

23. A modified FGF-21 polypeptide comprising a non-naturally encoded amino acid, wherein:

(a) 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;

(b) the non-naturally encoded amino acid has the structure;

24. The modified polypeptide of claim 23, wherein the non-naturally encoded amino acid is a phenylalanine derivative.

25. The modified polypeptide of claim 23, wherein the non-naturally encoded amino acid comprises a first functional group and the linker, polymer, or biologically active molecule comprises a second functional group, wherein the first functional group and second functional group are not identical and each comprise a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.

26. The modified polypeptide of claim 25, wherein the first functional group on the non-naturally encoded amino acid is a carbonyl moiety and the second functional group on the linker, polymer, or biologically active molecule is an aminooxy moiety, and the resultant covalent linkage created by the reaction of the first and second functional groups is an oxime linkage.

27. The modified polypeptide of claim 25, wherein the first functional group on the non-naturally encoded amino acid is an azide moiety and the second functional group on the linker, polymer, or biologically active molecule is an alkyne moiety.

28. The modified polypeptide of claim 23, wherein the non-naturally encoded amino acid is linked to a polymer.

29. The modified polypeptide of claim 28, wherein the polymer comprises poly(ethylene glycol).

30. The modified polypeptide of claim 29, wherein the poly(ethylene glycol) has an average molecular weight of between about 0.1 kDa and about 100 kDa.

31. The modified polypeptide of claim 29, wherein the poly(ethylene glycol) has an average molecular weight of about 30 kDa.

32. The modified polypeptide of claim 28, wherein the polymer is an oligosaccharide.

33. The modified polypeptide of claim 23, wherein the polypeptide has an in vivo half-life at least five-fold greater than the human FGF-21 polypeptide in SEQ ID NO:

1.

34. The modified polypeptide of claim 23, wherein the polypeptide has an in vivo half-life about 10-fold to about 40-fold greater than the human FGF-21 polypeptide in SEQ ID NO:

1.

35. The modified polypeptide of claim 23, wherein the non-naturally encoded amino acid is at a position corresponding to residue 108.

36. The modified polypeptide of claim 23, wherein the non-naturally encoded amino acid is selected from a para-substituted, ortho-substituted, or meta-substituted phenylalanine comprising a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.

37. The modified polypeptide of claim 36, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

38. The modified polypeptide of claim 23, wherein the 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.

39. The modified polypeptide of claim 38, wherein

(a) the modified polypeptide comprises SEQ ID NO:

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

(b) the phenylalanine derivative is linked to a polymer, and

(c) the polymer comprises poly(ethylene glycol) having an average molecular weight of about 30 kDa.