Modified Bovine G-CSF Polypeptides And Their Uses

• US 20100035812A1
• Filed: 07/22/2009
• Published: 02/11/2010
• Est. Priority Date: 07/23/2008
• 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: CANNING, Peter C., KODER, Alan, HO, Lillian, HAYS PUTNAM, Anna-Maria A., NORMAN, Thea, KNUDSEN, Nick, KRAYNOV, Vadim
• Granted Patent: US 10,138,283 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61P 11/16 Central respiratory analeptics
• CPC Class Codes: A61P 15/14 for lactation disorders, e.g. galactorrhoea
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF

Abstract

Modified bovine G-CSF polypeptides and uses thereof are provided.

Claims

1. A bG-CSF polypeptide comprising one or more non-naturally encoded amino acids.

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

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

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

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

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

7. The bG-CSF polypeptide of claim 6, wherein the second polypeptide is a bG-CSF polypeptide.

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

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

10. The bG-CSF 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 (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 NO;

     2).

11. The bG-CSF polypeptide of claim 10, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues:

3, 7, 11, 33, 43, 58, 62, 67, 69, 98, 99, 123, 124, 125, 133, 134, 136, 141, 159, 166, 169, 170, 173, and any combination thereof of SEQ ID NO;

1 or the corresponding amino acids in SEQ ID NO;

2.

12. The bG-CSF polypeptide of claim 10, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues:

3, 7, 33, 43, 58, 62, 67, 69, 99, 123, 124, 133, 134, 141, 166, and any combination thereof (SEQ ID NO;

1 or the corresponding amino acids in SEQ ID NO;

     2).

13. The bG-CSF polypeptide of claim 10, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 3, 7, 62, 133, 166, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NO;

     2).

14. The bG-CSF polypeptide of claim 10, wherein the non-naturally encoded amino acid is substituted at position 62 (SEQ ID NO:

1 or the corresponding amino acid of SEQ ID NO;

     2).

15. The bG-CSF polypeptide of claim 10, wherein the non-naturally encoded amino acid is substituted at position 133 (SEQ ID NO:

1 or the corresponding amino acid of SEQ ID NO;

     2).

16. The bG-CSF 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 I (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 (i.e., at the carboxyl terminals of the protein), and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NO;

     2).

17. The bG-CSF polypeptide of claim 16, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 3, 7, 11, 33, 43, 58, 62, 67, 69, 98, 99, 123, 124, 125, 133, 134, 136, 141, 159, 166, 169, 170, 173, and any combination thereof of SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NO;

2.

18. The bG-CSF polypeptide of claim 16, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 3, 7, 33, 43, 58, 62, 67, 69, 99, 123, 124, 133, 134, 141, 166, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NO;

     2).

19. The bG-CSF polypeptide of claim 16, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 3, 7, 62, 133, 166, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NO;

     2).

20. The bG-CSF polypeptide of claim 16, wherein the non-naturally encoded amino acid is substituted at position 62 (SEQ ID NO:

1 or the corresponding amino acid of SEQ ID NO;

     2).

21. The bG-CSF polypeptide of claim 4, wherein the non-naturally encoded amino acid is substituted at position 133 (SEQ ID NO:

1 or the corresponding amino acid of SEQ ID NO;

     2).

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

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

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

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

26. The bG-CSF 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 bG-CSF 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 bG-CSF polypeptide of claim 27, wherein the non-naturally encoded amino acid comprises a carbonyl group.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

47. The bG-CSF 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 bG-CSF polypeptide of claim 1, wherein the polypeptide is a bG-CSF antagonist.

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

50. The bG-CSF 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 bG-CSF polypeptide according to claim 48, wherein the polypeptide prevents activation of the bG-CSF receptor.

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

53. The bG-CSF 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 bG-CSF polypeptide of claim 3, the method comprising contacting an isolated bG-CSF 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(ethylone glycol) moiety is a branched or multiarmed polymer.

66. A composition comprising the bG-CSF 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 an animal having a disorder modulated by bG-CSF comprising administering to the animal a therapeutically-effective amount of the composition of claim 66.

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

96. The bG-CSF polypeptide of claim 1, wherein the bG-CSF polypeptide comprises one or more amino acid substitution, addition, or deletion that modulates haematopoiesis in an animal after administration of the polypeptide.

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

98. The bG-CSF polypeptide of claim 1, wherein the bG-CSF polypeptide comprises one or more amino acid substitution, addition, or deletion that modulates neutrophil proliferation in an animal after administration of the polypeptide.

99. The bG-CSF polypeptide of claim 1, wherein the polypeptide also comprises a naturally encoded amino acid substitution.

100. The bG-CSF polypeptide of claim 1, wherein the bc-CSF polypeptide comprises one or more amino acid substitution, addition, or deletion that modulates neutrophil maturation in an animal after administration of the polypeptide.

101. The bG-CSF polypeptide of claim 1, wherein the bG-CSF polypeptide comprises one or more amino acid substitution, addition, or deletion that modulates neutrophil function in an animal after administration of the polypeptide.

102. A method of treating an animal with an infection modulated by bG-CSF comprising administering to the animal a therapeutically-effective amount of the composition of claim 66.

103. The bG-CSF polypeptide of claim 4, wherein the water soluble polymer is linked to said polypeptide by an oxime bond.

104. A method of preventing infection in an animal comprising administering to the animal a therapeutically-effective amount of the composition of claim 66.

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

3 or 4 or a polynucleotide sequence encoding SEQ ID NO;

1 or 2, 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 bG-CSF polypeptide comprising a non-naturally encoded amino acid, the method comprising, culturing cells comprising a polynucleotide or polynucleotides encoding a bG-CSF polypeptide comprising a selector codon, an orthogonal RNA synthetase and an orthogonal tRNA under conditions to permit expression of the bG-CSF polypeptide comprising a non-naturally encoded amino acid;

and purifying the bG-CSF polypeptide.

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

73. A bG-CSF polypeptide encoded by a polynucleotide having a sequence shown in SEQ ID NO:

3 or 4 or encoding a polypeptide shown as SEQ ID NO;

1 or 2, wherein said polynucleotide comprises a selector codon, and wherein said polypeptide comprises at least one non-naturally encoded amino acid.

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

76. The bG-CSF 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 (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 NO;

     2).

77. The bG-CSF polypeptide of claim 76, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 3, 7, 11, 33, 43, 58, 62, 67, 69, 98, 99, 123, 124, 125, 133, 134, 136, 141, 159, 166, 169, 170, 173, and any combination thereof of SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NO;

2.

78. The bG-CSF polypeptide of claim 76, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 3, 7, 33, 43, 58, 62, 67, 69, 99, 123, 124, 133, 134, 141, 166, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NO;

     2).

79. The bG-CSF polypeptide of claim 76, wherein the non-naturally encoded amino acid is substituted at a position selected from the group consisting of residues 3, 7, 62, 133, 166, and any combination thereof (SEQ ID NO:

1 or the corresponding amino acids in SEQ ID NO;

     2).

80. The bG-CSF polypeptide of claim 76, wherein the non-naturally encoded amino acid is substituted at position 62 (SEQ ID NO:

1 or the corresponding amino acid of SEQ ID NO;

     2).

81. The bG-CSF polypeptide of claim 73, wherein the non-naturally encoded amino acid is substituted at position 133 (SEQ ID NO:

1 or the corresponding amino acid of SEQ ID NO;

     2).

82. The bG-CSF 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 bG-CSF 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 bG-CSF polypeptide of claim 75, wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer.

85. The bG-CSF 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 bG-CSF polypeptide of claim 73 and a pharmaceutically acceptable carrier.

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

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

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

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

92. A bG-CSF 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 bG-CSF polypeptide of claim 92, wherein said bG-CSF polypeptide is monoPEGylated.

94. A bG-CSF 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 bG-CSF polypeptide of claim 94, wherein the bG-CSF polypeptide comprises one said linker, polymer, or biologically active molecule.