MODIFIED ANIMAL ERYTHROPOIETIN POLYPEPTIDES AND THEIR USES

• US 20100093608A1
• Filed: 09/25/2009
• Published: 04/15/2010
• Est. Priority Date: 09/26/2008
• Status: Active Grant

• Current Assignee: Elanco US Inc. (Elanco Animal Health, Inc.), Ambrx, Inc. (Johnson & Johnson)
• Original Assignee: Eli Lilly and Company, Ambrx, Inc. (Johnson & Johnson)
• Inventors: CHU, Stephanie, SILADI, Marc E., ATKINSON, Kyle, SONG, Frank, LEE, Darin, BARNETT, Richard S., HAYS PUTNAM, Anna-Maria A., TIAN, Feng, SHEFFER, Joseph, CANNING, Peter C.
• Granted Patent: US 8,278,418 B2
• Time in Patent Office: Days
• US Class Current: 514/1.1
• 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 38/1816 Erythropoietin [EPO]
• 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 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids

Abstract

Modified animal erythropoietin polypeptides and uses thereof are provide.

Claims

1. A feline erythropoietin (fEPO) polypeptide comprising a non-naturally encoded amino acid.

2. The fEPO polypeptide of claim 1, wherein the fEPO polypeptide is linked to at least one additional fEPO polypeptide.

3. The fEPO polypeptide of claim 1, wherein the non-naturally encoded amino acid is linked to a water soluble polymer.

4. The fEPO polypeptide of claim 3, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety.

5. The fEPO polypeptide of claim 4, wherein the poly(ethylene glycol) molecule is a bifunctional polymer.

6. The fEPO polypeptide of claim 5, wherein the bifunctional polymer is linked to a second polypeptide.

7. The fEPO polypeptide of claim 6, wherein the second polypeptide is a non-fEPO polypeptide.

8. The fEPO polypeptide of claim 4, comprising at least two amino acids linked to a water soluble polymer comprising a poly(ethylene glycol) moiety.

9. The fEPO polypeptide of claim 8, wherein at least one amino acid linked to said water soluble polymer is a non-naturally encoded amino acid.

10. The fEPO polypeptide of claim 4, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 1-7, 27-54, 84-89, 114-137, 162-166 from SEQ ID NO:

2.

11. The fEPO polypeptide of claim 4, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 1, 2, 3, 4, 5, 6, 8, 9, 17, 21, 24, 25, 26, 27, 28, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 43, 45, 47, 50, 51, 52, 53, 54, 55, 56, 57, 58, 65, 68, 72, 76, 77, 78, 79, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 107, 110, 111, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 154, 157, 158, 159, 160, 162, 163, 164, 165 and 166 from SEQ ID NO:

2.

12. The fEPO polypeptide of claim 11, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 2, 4, 17, 21, 24, 27, 28, 30, 31, 32, 34, 36, 37, 38, 40, 50, 53, 55, 58, 65, 68, 72, 76, 80, 82, 83, 85, 86, 87, 89, 113, 115, 116, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 134, 136, and 162, and a combination thereof from SEQ ID NO:

2.

13. The fEPO polypeptide of claim 11, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 21, 24, 28, 30, 31, 36, 37, 38, 55, 72, 83, 85, 86, 87, 89, 113, 116, 119, 120, 121, 123, 124, 125, 126, 127, 128, 129, 130, and 162, and a combination thereof from SEQ ID NO:

2.

14. The fEPO polypeptide of claim 11, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 21, 24, 38, 83, 85, 86, 89, 116, 119, 121, 124, 125, 126, 127, and 128, and a combination thereof from SEQ ID NO:

2.

15. The fEPO polypeptide of claim 4, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of 24, 36, 38, 58, 65, 83, 86, 113, 115, 126, and a combination thereof, from SEQ ID NO:

2.

16. The fEPO polypeptide of claim 1, wherein the fEPO polypeptide comprises a substitution, addition or deletion that increases affinity of the fEPO polypeptide for an erythropoietin receptor.

17. The fEPO polypeptide of claim 1, wherein the fEPO polypeptide comprises an amino acid substitution, addition or deletion that increases the stability or solubility of the fEPO polypeptide.

18. The fEPO polypeptide of claim 16, comprising an amino acid substitution selected from the group consisting of but not limited to, S9A, F48S, Y49S, A50S, Q59A, A73G, G101A, T106A, L108A, T132A, R139A, K140A, R143A, S146A, N147A, R150A, and K154A and combination thereof in SEQ ID NO:

2.

19. The fEPO polypeptide of claim 1, wherein the non-naturally encoded amino acid is reactive toward a water soluble polymer that is otherwise unreactive toward any of the 20 common amino acids.

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

21. The fEPO polypeptide of claim 20, wherein the non-naturally encoded amino acid comprises a carbonyl group.

22. The fEPO polypeptide of claim 21, wherein the non-naturally encoded amino acid has the structure:

23. The fEPO polypeptide of claim 20, wherein the non-naturally encoded amino acid comprises an aminooxy group.

24. The fEPO polypeptide of claim 20, wherein the non-naturally encoded amino acid comprises a hydrazide group.

25. The fEPO polypeptide of claim 20, wherein the non-naturally encoded amino acid comprises a hydrazine group.

26. The fEPO polypeptide of claim 20, wherein the non-naturally encoded amino acid residue comprises a semicarbazide group.

27. The fEPO polypeptide of claim 20, wherein the non-naturally encoded amino acid residue comprises an azide group.

28. The fEPO polypeptide of claim 27, wherein the non-naturally encoded amino acid has the structure:

29. The fEPO polypeptide of claim 20, wherein the non-naturally encoded amino acid comprises an alkyne group.

30. The fEPO polypeptide of claim 29, wherein the non-naturally encoded amino acid has the structure:

31. The fEPO polypeptide of claim 4, wherein the poly(ethylene glycol) molecule has a molecular weight of between about 1 and about 100 kDa.

32. The fEPO polypeptide of claim 31, wherein the poly(ethylene glycol) molecule has a molecular weight of between 1 kDa and 50 kDa.

33. The fEPO polypeptide of claim 4, which is made by reacting a fEPO polypeptide comprising a carbonyl-containing amino acid with a poly(ethylene glycol) molecule comprising an aminooxy, a hydroxylamine, hydrazine, hydrazide or semicarbazide group.

34. The fEPO polypeptide of claim 33, wherein the aminooxy, hydroxylamine, hydrazine, hydrazide or semicarbazide group is linked to the poly(ethylene glycol) molecule through an amide linkage.

35. The fEPO polypeptide of claim 4, which is made by reacting a poly(ethylene glycol) molecule comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid that comprises an aminooxy, a hydroxylamine, hydrazide or semicarbazide group.

36. The fEPO polypeptide of claim 4, which is made by reacting a fEPO polypeptide comprising an alkyne-containing amino acid with a poly(ethylene glycol) molecule comprising an azide moiety.

37. The fEPO polypeptide of claim 4, which is made by reacting a fEPO polypeptide comprising an azide-containing amino acid with a poly(ethylene glycol) molecule comprising an alkyne moiety.

38. The fEPO polypeptide of claim 4, wherein the functional group of the non-naturally encoded amino acid is linked to the poly(ethylene glycol) molecule through an amide linkage.

39. The fEPO polypeptide of claim 4, wherein the poly(ethylene glycol) molecule is a branched or multiarmed polymer.

40. The fEPO polypeptide of claim 39, wherein each branch of the poly(ethylene glycol) branched polymer has a molecular weight of between 5 kDa and 30 kDa.

41. The fEPO polypeptide of claim 1, wherein the polypeptide is an erythropoietin antagonist.

42. The fEPO polypeptide of claim 41, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues including, but not limited to, V11, R14, Y15, D96, K97, S100, R103, S104, T107, L108, and R110, and a combination thereof from SEQ ID NO:

2.

43. The fEPO polypeptide of claim 41, wherein the non-naturally encoded amino acid is linked to a water soluble polymer.

44. The fEPO polypeptide of claim 41, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety.

45. The fEPO polypeptide according to claim 41, wherein the non-naturally encoded amino acid linked to a water soluble polymer is present within the Site II region of the fEPO polypeptide.

46. The fEPO polypeptide according to claim 41, wherein the non-naturally encoded amino acid linked to a water soluble polymer prevents dimerization of the fEPO receptor by preventing the fEPO antagonist from binding to a second fEPO receptor.

47. The fEPO polypeptide according to claim 41, wherein an amino acid other than leucine is substituted for L108 in SEQ ID NO:

2.

48. The fEPO polypeptide according to claim 47, wherein arginine is substituted for L108 in SEQ ID NO:

2.

49. The fEPO polypeptide of claim 1, wherein the non-naturally encoded amino acid comprises a saccharide moiety.

50. The fEPO polypeptide of claim 3, wherein the water soluble polymer is linked to the polypeptide via a saccharide moiety.

53. A method of making the fEPO polypeptide of claim 4, the method comprising contacting an isolated fEPO polypeptide comprising a non-naturally encoded amino acid with a water soluble polymer comprising a moiety that reacts with the non-naturally encoded amino acid.

54. The method of claim 53, wherein the water soluble polymer comprises a polyethylene glycol moiety.

55. The method of claim 53, wherein the non-naturally encoded amino acid residue comprises a carbonyl group, an aminooxy group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.

56. The method of claim 55, wherein the non-naturally encoded amino acid residue comprises a carbonyl moiety and the water soluble polymer comprises an aminooxy, a hydroxylamine, hydrazide or semicarbazide moiety.

57. The method of claim 55, wherein the non-naturally encoded amino acid residue comprises an alkyne moiety and the water soluble polymer comprises an azide moiety.

58. The method of claim 55, wherein the non-naturally encoded amino acid residue comprises an azide moiety and the water soluble polymer comprises an alkyne moiety.

59. The method of claim 54, wherein the polyethylene glycol moiety has an average molecular weight of between about 1 and about 100 kDa.

60. The method of claim 58, wherein the polyethylene glycol moiety is a branched or multiarmed polymer.

61. A composition comprising the fEPO polypeptide of claim 1 and a pharmaceutically acceptable carrier.

62. The composition of claim 61, wherein the non-naturally encoded amino acid is linked to a water soluble polymer.

63. A method of treating a patient having a disorder modulated by fEPO comprising administering to the patient a therapeutically-effective amount of the pharmaceutical composition of claim 61.

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

24, SEQ ID NO;

25, SEQ ID NO;

26, or SEQ ID NO;

27 wherein the polynucleotide comprises at least one selector codon.

52. The isolated nucleic acid of claim 51, 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.

64. A cell comprising the nucleic acid of claim 51.

65. The cell of claim 64, wherein the cell comprises an orthogonal tRNA synthetase and an orthogonal tRNA.

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

and purifying the fEPO polypeptide from the cells.

67. A method of increasing serum half-life or circulation time of fEPO, the method comprising substituting a non-naturally encoded amino acid for any one or more amino acids in naturally occurring fEPO.

68. A fEPO polypeptide encoded by a polynucleotide having a sequence shown in SEQ ID NO:

24;

SEQ ID NO;

25;

SEQ ID NO;

26 or SEQ ID NO;

27, wherein at least one amino acid is substituted by a non-naturally encoded amino acid.

69. The fEPO polypeptide of claim 68, wherein the non-naturally encoded amino acid is linked to a water soluble polymer.

70. The fEPO polypeptide of claim 68, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety.

71. The fEPO polypeptide of claim 68, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 1, 2, 3, 4, 5, 6, 8, 9, 17, 21, 24, 25, 26, 27, 28, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 43, 45, 47, 50, 51, 52, 53, 54, 55, 56, 57, 58, 68, 72, 76, 77, 78, 79, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 107, 110, 111, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 154, 157, 158, 159, 160, 162, 163, 164, 165 and 166 from SEQ ID NO:

3.

72. The fEPO polypeptide of claim 68, 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.

73. The fEPO polypeptide of claim 70, wherein the poly(ethylene glycol) moiety has a molecular weight of between about 1 and about 100 kDa.

74. The fEPO polypeptide of claim 70, wherein the poly(ethylene glycol) moiety has a molecular weight of between 5 kDa and 40 kDa.

75. The fEPO polypeptide of claim 70, wherein the polyethylene glycol moiety is a branched or multiarmed polymer.

76. A pharmaceutical composition comprising the fEPO polypeptide of claim 68 and a pharmaceutically acceptable carrier.

77. A non-human erythropoietin polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:

30;

SEQ ID NO;

31;

SEQ ID NO;

32 and SEQ ID NO;

33, wherein said non-human erythropoietin polypeptide comprises a non-naturally encoded amino acid substitution or addition.