Factor VII: remodeling and glycoconjugation of Factor VII

• US 8,008,252 B2
• Filed: 04/09/2003
• Issued: 08/30/2011
• Est. Priority Date: 10/10/2001
• Status: Expired due to Fees

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: Chen, Xi, Hakes, David, Zopf, David, DeFrees, Shawn, Bayer, Robert, Bowe, Caryn
• Primary Examiner(s): Tsang; Cecilia
• Assistant Examiner(s): HEARD, THOMAS SWEENEY
• Application Number:
  US10/411,044
• Publication Number:
  US 20100015684A1
• Time in Patent Office: 3,065 Days
• Field of Search: None
• US Class Current: 514/14.3
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C12N 9/6437 Coagulation factor VIIa (3.4.21.21)
• CPC Class Codes: C12N 9/647 Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12Y 304/21021 Coagulation factor VIIa (3.4.21.21)

Abstract

The invention includes methods and compositions for remodeling a peptide molecule, including the addition or deletion of one or more glycosyl groups to a peptide, and/or the addition of a modifying group to a peptide.

Claims

1. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:

2. The method of claim 1, further comprising:

(c) prior to step (b), removing a group added to said saccharide during post-translational modification.

3. The method of claim 2, wherein said group is a member selected from phosphate, sulfate, carboxylate and esters thereof.

4. The method of claim 1, wherein said peptide has the formula:

5. The method of claim 1, wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol).

6. The method of claim 1, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).

7. The method of claim 1, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.

8. The method of claim 1, wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal1, ST3Gal3, ST6GalNAcI CST-II and combinations thereof.

9. The method of claim 1, further comprising:

(d) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.

10. The method of claim 1, wherein said peptide has the formula:

11. The method of claim 1, wherein said peptide has the formula:

12. The method of claim 11, wherein X¹¹ and X¹² are (mannose)_q, wherein

q is selected from the integers between 1 and 20, and when q is three or greater (mannose)_q is selected from linear and branched structures.

13. The method of claim 1, wherein said peptide has the formula:

14. The method of claim 13, wherein

X¹⁴ and X¹⁵ are members independently selected from GlcNAc and Sia;

and i and k are independently selected from the integers 0 and 1, with the proviso that at least one of i and k is 1, and when k is 1, g, h, and j are 0.

15. The method of claim 1, wherein said peptide has the formula:

16. The method of claim 1, wherein said removing of step (a) utilizes a glycosidase.

17. The method of claim 1, wherein said removing of step (a) involves removal of a sialic acid moiety using a sialidase.

87. A method of treating a patient having a condition, said method comprising administering to said patient a covalent conjugate of a Factor VIIa peptide,

wherein

said conjugate is formed by a method according to claim 1, 18, 24, 30, 36, 46, 70 or 76;

and

said condition is a member selected from hemophilia in combination with a bleeding episode, hemophilia A, hemophilia A in combination with antibodies to Factor III, hemophilia B, hemophilia B in combination with antibodies to Factor IX, liver cirrhosis, cirrhosis in combination with gastrointestinal bleeding, cirrhosis in combination with orthotopic liver transplant, bone marrow transplant and liver resection.

18. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:

19. The method of claim 18, wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol).

20. The method of claim 18, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).

21. The method of claim 18, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.

22. The method of claim 18, wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal1, ST3Gal3, ST6GalNAcI, CST-II and combinations thereof.

23. The method of claim 18, further comprising:

(b) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.

24. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:

25. The method of claim 24, further comprising:

(b) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.

26. The method according to claim 24, wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.

27. The method of claim 24, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).

28. The method of claim 24, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).

29. The method of claim 24, wherein said at least one glycosyltransferase is a member selected from the group consisting of GalT, ST3Gal3, CST-II and combinations thereof.

30. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:

31. The method of claim 30, further comprising:

(c) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.

32. The method according to claim 30, wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.

33. The method of claim 30, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).

34. The method of claim 30, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).

35. The method of claim 30, wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI, CST-II and combinations thereof.

36. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:

37. The method of claim 36, wherein said removing of step (a) produces a truncated glycan in which a, b, c, e and x are each 0.

38. The method of claim 36, wherein X³, X⁵, and X⁷, are selected from the group consisting of (mannose)_z and (mannose)_z-(X⁸)_y

wherein

X⁸ is a glycosyl moiety selected from mono- and oligo-saccharides;

y is an integer selected from 0 and 1; and

z is an integer between 1 and 20, wherein

when z is 3 or greater, (mannose)_z is selected from linear and branched structures.

39. The method of claim 36, wherein X⁴ is selected from the group consisting of GlcNAc and xylose.

40. The method of claim 36, wherein X³, X⁵, and X⁷ are (mannose)_u, wherein

u is selected from the integers between 1 and 20, and when u is 3 or greater, (mannose)_u is selected from linear and branched structures.

41. The method of claim 36, further comprising:

(c) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.

42. The method of claim 36, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.

43. The method of claim 36, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).

44. The method of claim 36, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).

45. The method of claim 36, wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI CST-II and combinations thereof.

46. A cell-free, in vitro method of forming a covalent conjugate between a poly(ethylene) glycol and a glycosylated or non-glycosylated Factor VIIa peptide, wherein said poly(ethylene) glycol is conjugated to said Factor VIIa peptide via a glycosyl linking group interposed between and covalently linked to both said Factor VIIa peptide and said poly(ethylene) glycol, said method comprising:

(a) contacting said Factor VIIa peptide with a mixture comprising a nucleotide sugar having a urethane linker attached to said poly(ethylene) glycol, and a glycosyltransferase for which said nucleotide sugar is a substrate under conditions suitable for said at least one glycosyltransferase to transfer a modified sugar moiety of said nucleotide sugar onto said Factor VIIa peptide, wherein said modified sugar moiety comprises a urethane linker attached to at least one poly(ethylene) glycol,

thereby forming said covalent conjugate of said Factor VIIa peptide.

47. The method of claim 46, wherein said glycosyl linking group is covalently attached to a glycosyl residue covalently attached to said peptide.

48. The method of claim 46, wherein said glycosyl linking group is covalently attached to an amino acid residue of said peptide.

49. The method of claim 46, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 40,000.

50. The method of claim 46, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 80,000, and further wherein said poly(ethylene glycol) is a branched structure.

51. The method of claim 46, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 30,000, and further wherein said poly(ethylene glycol) is a linear structure.

52. The method of claim 49, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 5,000.

53. The method of claim 52, wherein said poly(ethylene glycol) has a degree of polymerization from about 1 to about 1,000.

54. The method of claim 46, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).

55. The method of claim 46, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.

56. The method of claim 46, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).

57. The method of claim 46, wherein said glycosyltransferase is selected from the group consisting of ST3Gal3, ST3Gal1, ST6GalNAcI, CST-II and combinations thereof.

58. The method of claim 49, further comprising:

(b) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.

59. The method of claim 46, wherein said glycosyltransferase is selected from the group consisting of sialyltransferase, galactosyltransferase, glucosyltransferase, GalNAc transferase, GlcNAc transferase, fucosyltransferase, mannosyltransferase and xylosyltransferase.

60. The method of claim 46, wherein said glycosyltransferase is recombinantly produced.

61. The method of claim 60, wherein said glycosyltransferase is a recombinant prokaryotic enzyme.

62. The method of claim 60, wherein said glycosyltransferase is a recombinant eukaryotic enzyme.

63. The method of claim 60, wherein said glycosylated peptide is partially deglycosylated prior to said contacting.

64. The method of claim 46, wherein said intact glycosyl linking group is a sialic acid residue.

65. The method of claim 46, wherein said method is performed in a cell-free environment.

66. The method of claim 46, wherein said covalent conjugate is isolated.

67. The method of claim 66, wherein said covalent conjugate is isolated by membrane filtration.

68. The covalent conjugate of claim 46, wherein said modified sugar donor is a nucleotide sugar selected from the group consisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside.

69. The covalent conjugate of claim 68, wherein said nucleotide sugar is selected from the group consisting of UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid and CMP-NeuAc.

70. A cell-free, in vitro method of forming a covalent conjugate of a Factor VIIa peptide, said peptide having the formula:

71. The method of claim 70, further comprising:

(b) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.

72. The method according to claim 70, wherein said poly(ethylene glycol) has a molecular weight that is essentially homodisperse.

73. The method of claim 70, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).

74. The method of claim 70, wherein said poly(ethylene glycol) is monomethoxy-poly(ethylene glycol).

75. The method of claim 70, wherein said at least one glycosyltransferase is selected from the group consisting of GalNAcT2, ST3Gal1, ST3Gal3, ST6GalNAcI, CST-I, CST-II and combinations thereof.

76. A method of forming a covalent conjugate between a Factor VIIa peptide and a poly(ethylene) glycol, wherein said poly(ethylene) glycol is covalently attached to said Factor VIIa peptide through an intact glycosyl linking group, said Factor VIIa peptide comprising a glycosyl residue having a formula which is a member selected from:

77. The method of claim 76, further comprising:

(b) prior to step (a), contacting said Factor VIIa peptide with a sialidase under conditions appropriate to remove a sialic acid from said Factor VIIa peptide, thereby removing said sialic acid from said Factor VIIa peptide.

78. The method of claim 76, further comprising:

(c) prior to step (a), contacting said Factor VIIa peptide with a galactosidase under conditions appropriate to remove a galactose from said Factor VIIa peptide, thereby removing said galactose from said Factor VIIa peptide.

79. The method of claim 76, further comprising:

(d) prior to step (a), contacting said Factor VIIa peptide with a galactosyl transferase and a galactose donor under conditions appropriate to transfer a galactose to said Factor VIIa peptide, thereby transferring said galactose to said Factor VIIa peptide.

80. The method of claim 76, wherein said poly(ethylene glycol) is a monomethoxy-poly(ethylene glycol).

81. The method of claim 76, wherein said poly(ethylene glycol) is a member selected from linear poly(ethylene glycol) and branched poly(ethylene glycol).

82. The method of claim 76, wherein said poly(ethylene glycol) has a molecular weight distribution that is essentially homodisperse.

83. The method of claim 76, wherein said at least one glycosyltransferase is selected from the group consisting of ST3Gal3, CST-II and combinations thereof.

84. The method of claim 76, further comprising:

(e) contacting said covalent conjugate with a sialic acid donor and a sialyltransferase under conditions suitable for said sialyltransferase to transfer a sialic acid residue onto said covalent conjugate, thereby transferring a sialic acid moiety onto said covalent conjugate.

85. The method of claim 76, wherein

a, b, c, d, e, g, i, j, l, o, p and q members independently selected from 0 and 1;

r and t are l;

f, h, k, m, s, u, v, w, x and y are 0; and

n is selected from the integers from 0 to 4.

86. The method of claim 76, wherein

a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p, q, r, s, t and u are members independently selected from 0 and 1;

v, w, x and y are 0; and

n is a member selected from the integers from 0 to 4.