Modified FGF-21 Polypeptides and Their Uses

• US 20080255045A1
• Filed: 03/19/2008
• Published: 10/16/2008
• Est. Priority Date: 03/30/2007
• Status: Active Grant

• Current Assignee: Ambrx, Inc. (Johnson & Johnson)
• Original Assignee: Ambrx, Inc. (Johnson & Johnson)
• Inventors: HAYS PUTNAM, Anna-Maria A., MARIANI, Roberto, CUJEC, Thomas P., PINKSTAFF, Jason, KNUDSEN, Nick, KRAYNOV, Vadim, KEEFE, William M., HO, Lillian
• Granted Patent: US 8,012,931 B2
• Time in Patent Office: Days
• US Class Current: 514/12
• 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 FGF-21 polypeptide comprising one or more non-naturally encoded amino acids.

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

3. The FGF-21 polypeptide of claim 1, wherein the polypeptide is linked to a linker, polymer, or biologically active molecule.

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

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

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

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

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

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

10. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminus of the protein), and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

11. The FGF-21 polypeptide of claim 10, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 10, 52, 117, 126, 131, 162, 87, 77, 83, 72, 69, 79, 91, 96, 108, 110, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

12. The FGF-21 polypeptide of claim 10, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 10, 52, 77, 117, 126, 131, 162, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids of SEQ ID NOs;

2-7).

13. The FGF-21 polypeptide of claim 10, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 87, 77, 83, 72, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids of SEQ ID NOs;

2-7).

14. The FGF-21 polypeptide of claim 10, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 69, 79, 91, 96, 108, 110, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids of SEQ ID NOs;

2-7).

15. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues in the leader or signal sequence of SEQ ID NOs:

3, 4, 6, or 7.

16. The FGF-21 polypeptide of claim 4, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminus of the protein), and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

17. The FGF-21 polypeptide of claim 16, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 10, 52, 117, 126, 131, 162, 87, 77, 83, 72, 69, 79, 91, 96, 108, 110, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

18. The FGF-21 polypeptide of claim 16, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 10, 52, 77, 117, 126, 131, 162, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids of SEQ ID NOs;

2-7).

19. The FGF-21 polypeptide of claim 16, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 87, 77, 83, 72, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids of SEQ ID NOs;

2-7).

20. The FGF-21 polypeptide of claim 16, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 69, 79, 91, 96, 108, 110, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids of SEQ ID NOs;

2-7).

21. The FGF-21 polypeptide of claim 4, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues in the leader or signal sequence of SEQ ID NOs:

3, 4, 6, or 7.

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

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

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

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

26. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid 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.

27. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.

28. The FGF-21 polypeptide of claim 27, wherein the non-naturally encoded amino acid comprises a carbonyl group.

29. The FGF-21 polypeptide of claim 28, wherein the non-naturally encoded amino acid has the structure:

30. The FGF-21 polypeptide of claim 27, wherein the non-naturally encoded amino acid comprises an aminooxy group.

31. The FGF-21 polypeptide of claim 27, wherein the non-naturally encoded amino acid comprises a hydrazide group.

32. The FGF-21 polypeptide of claim 27, wherein the non-naturally encoded amino acid comprises a hydrazine group.

33. The FGF-21 polypeptide of claim 27, wherein the non-naturally encoded amino acid residue comprises a semicarbazide group.

34. The FGF-21 polypeptide of claim 27, wherein the non-naturally encoded amino acid residue comprises an azide group.

35. The FGF-21 polypeptide of claim 34, wherein the non-naturally encoded amino acid has the structure:

36. The FGF-21 polypeptide of claim 27, wherein the non-naturally encoded amino acid comprises an alkyne group.

37. The FGF-21 polypeptide of claim 36, wherein the non-naturally encoded amino acid has the structure:

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

39. The FGF-21 polypeptide of claim 38, wherein the water soluble polymer has a molecular weight of between about 0.1 kDa and about 50 kDa.

40. The FGF-21 polypeptide 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.

41. The FGF-21 polypeptide of claim 40, wherein the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the water soluble polymer through an amide linkage.

42. The FGF-21 polypeptide 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.

43. The FGF-21 polypeptide of claim 4, which is made by reacting a FGF-21 polypeptide comprising an alkyne-containing amino acid with a water soluble polymer comprising an azide moiety.

44. The FGF-21 polypeptide of claim 4, which is made by reacting a FGF-21 polypeptide comprising an azide-containing amino acid with a water soluble polymer comprising an alkyne moiety.

45. The FGF-21 polypeptide of claim 27, wherein the azide or alkyne group is linked to a water soluble polymer through an amide linkage.

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

47. The FGF-21 polypeptide of claim 46, wherein each branch of the water soluble polymer has a molecular weight of between about 1 kDa and about 100 kDa.

48. The FGF-21 polypeptide of claim 1, wherein the polypeptide is a FGF-21 antagonist.

49. The FGF-21 polypeptide of claim 48, wherein the polypeptide comprises one or more post-translational modification, linker, polymer, or biologically active molecule.

50. The FGF-21 polypeptide of claim 49, wherein the polymer comprises a moiety selected from a group consisting of a water soluble polymer and poly(ethylene glycol).

51. The FGF-21 polypeptide according to claim 48, wherein the polypeptide prevents activation of the FGF-21 receptor.

52. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid comprises a saccharide moiety.

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

56. A method of making the FGF-21 polypeptide of claim 3, the method comprising contacting an isolated FGF-21 polypeptide comprising a non-naturally encoded amino acid with a linker, polymer, or biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid.

57. The method of claim 56, wherein the polymer comprises a moiety selected from a group consisting of a water soluble polymer and poly(ethylene glycol).

58. The method of claim 56, 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.

59. The method of claim 56, 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.

60. The method of claim 59, wherein the aminooxy, hydrazine, hydrazide or semicarbazide moiety is linked to the linker, polymer, or biologically active molecule through an amide linkage.

61. The method of claim 56, wherein the non-naturally encoded amino acid comprises an alkyne moiety and the linker, polymer, or biologically active molecule comprises an azide moiety.

62. The method of claim 56, wherein the non-naturally encoded amino acid comprises an azide moiety and the linker, polymer, or biologically active molecule comprises an alkyne moiety.

63. The method of claim 58, wherein the azide or alkyne moiety is linked to a linker, polymer, or biologically active molecule through an amide linkage.

64. The method of claim 57, wherein the poly(ethylene glycol) moiety has an average molecular weight of between about 0.1 kDa and about 100 kDa.

65. The method of claim 57, wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer.

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

67. The composition of claim 66, wherein the non-naturally encoded amino acid is linked to a water soluble polymer.

68. A method of treating a patient having a disorder modulated by FGF-21 comprising administering to the patient a therapeutically-effective amount of the composition of claim 66.

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

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

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

100. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid comprises a ketone group.

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

102. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 56, 59, 69, and 122, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

103. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 42, 54, 77, 81, 86, 88, 122, 125, 126, 130, 131, 139, 145, 146, 152, 154, 156, 161, 163, 170, 172, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

104. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 25, 38, 58, 59, 60, 69, 79, 87, 91, 101, 102, 114, 116, 122, 126, 130, 133, 140, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

105. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 31, 33, 35, 37, 41, 42, 43, 50, 54, 58, 62, 66, 67, 69, 72, 73, 76, 77, 79, 80, 81, 82, 84, 85, 90, 92, 94, 95, 100, 102, 104, 107, 109, 110, 115, 117, 118, 119, 121, 122, 123, 124, 125, 126, 127, 129, 130, 132, 134, 135, 137, 138, 139, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

106. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 59 and 122 (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

107. The FGF-21 polypeptide of claim 1, wherein the polypeptide also has following substitutions Leu118Cys-Ala134Cys (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

108. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 19, 21, 26, 28, 29, 30, 36, 39, 42, 50, 56, 61, 64, 65, 68, 70, 71, 77, 81, 85, 86, 90, 92, 94, 98, 107, 108, 112, 113, 123, 124, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

109. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 24, 27, 37, 40, 44, 46, 49, 57, 88, 89, 106, 110, 111, 115, 120, 139, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

110. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 18, 45, 47, 48, 78, 83, 99, 103, 125, 128, 131, 132, 138, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

111. The FGF-21 polypeptide of claim 1, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 25, 38, 58, 59, 60, 69, 79, 87, 91, 101, 102, 114, 116, 122, 126, 130, 133, 140, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

54. An isolated nucleic acid comprising a polynucleotide that hybridizes under stringent conditions to SEQ ID NO:

8, 9, 10, 11, 12, 13, or 14, wherein the polynucleotide comprises at least one selector codon.

55. The isolated nucleic acid of claim 54, wherein the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, and a four-base codon.

69. A cell comprising the nucleic acid of claim 54.

70. The cell of claim 69, wherein the cell comprises an orthogonal tRNA synthetase or an orthogonal tRNA.

71. A method of making a FGF-21 polypeptide comprising a non-naturally encoded amino acid, the method comprising, culturing cells comprising a polynucleotide or polynucleotides encoding a FGF-21 polypeptide comprising a selector codon, an orthogonal RNA synthetase and an orthogonal tRNA under conditions to permit expression of the FGF-21 polypeptide comprising a non-naturally encoded amino acid;

and purifying the FGF-21 polypeptide.

72. A method of modulating serum half-life or circulation time of a FGF-21 polypeptide, the method comprising substituting one or more non-naturally encoded amino acids for any one or more naturally occurring amino acids in the FGF-21 polypeptide.

73. A FGF-21 polypeptide 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 polypeptide comprises at least one non-naturally encoded amino acid.

74. The FGF-21 polypeptide of claim 73, wherein the non-naturally encoded amino acid is linked to a linker, polymer, water soluble polymer, or biologically active molecule.

75. The FGF-21 polypeptide of claim 74, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety.

76. The FGF-21 polypeptide of claim 73, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminus of the protein), and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

77. The FGF-21 polypeptide of claim 76, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 10, 52, 117, 126, 131, 162, 87, 77, 83, 72, 69, 79, 91, 96, 108, 110, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NOs;

2-7).

78. The FGF-21 polypeptide of claim 76, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 10, 52, 77, 117, 126, 131, 162, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids of SEQ ID NOs;

2-7).

79. The FGF-21 polypeptide of claim 76, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 87, 77, 83, 72, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids of SEQ ID NOs;

2-7).

80. The FGF-21 polypeptide of claim 76, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 69, 79, 91, 96, 108, 110, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids of SEQ ID NOs;

2-7).

81. The FGF-21 polypeptide of claim 73, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues in the leader or signal sequence of SEQ ID NOs:

3, 4, 6, or 7.

82. The FGF-21 polypeptide of claim 73, 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.

83. The FGF-21 polypeptide of claim 75, wherein the poly(ethylene glycol) moiety has a molecular weight of between about 0.1 kDa and about 100 kDa.

84. The FGF-21 polypeptide of claim 75, wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer.

85. The FGF-21 polypeptide of claim 84, wherein the poly(ethylene glycol) moiety has a molecular weight of between about 1 kDa and about 100 kDa.

86. A composition comprising the FGF-21 polypeptide of claim 73 and a pharmaceutically acceptable carrier.

87. A FGF-21 polypeptide comprising one or more amino acid substitution, addition or deletion that increases the expression of the FGF-21 polypeptide in a recombinant host cell.

88. A FGF-21 polypeptide comprising a water soluble polymer linked by a covalent bond to the FGF-21 polypeptide at a single amino acid.

89. The FGF-21 polypeptide of claim 88, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety.

90. The FGF-21 polypeptide of claim 88, wherein the amino acid covalently linked to the water soluble polymer is a non-naturally encoded amino acid.

92. A FGF-21 polypeptide comprising at least one linker, polymer, or biologically active molecule, wherein said linker, polymer, or biologically active molecule is attached to the polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into the polypeptide.

93. The FGF-21 polypeptide of claim 92, wherein said FGF-21 polypeptide is monoPEGylated.

94. A FGF-21 polypeptide comprising a linker, polymer or biologically active molecule that is attached to one or more non-naturally encoded amino acids wherein said non-naturally encoded amino acid is ribosomally incorporated into the polypeptide at pre-selected sites.

95. The FGF-21 polypeptide of claim 94, wherein the FGF-21 polypeptide comprises one said linker, polymer, or biologically active molecule.

98. A method of modulating immunogenicity of a FGF-21 polypeptide, the method comprising substituting one or more non-naturally encoded amino acids for any one or more naturally occurring amino acids in the FGF-21 polypeptide.

99. A FGF-21 polypeptide having the amino acid sequence shown as SEQ ID NO:

1.