Methods and compositions for reducing heparan sulfate proteoglycan-mediated clearance of factor viii

• US 20040248785A1
• Filed: 05/26/2004
• Published: 12/09/2004
• Est. Priority Date: 01/12/2001
• Status: Active Grant

• Current Assignee: University of Maryland Baltimore County (University of Maryland)
• Original Assignee: University of Maryland Baltimore County (University of Maryland)
• Inventors: Saenko, Evgueni L., Sarafanov, Andrey G.
• Granted Patent: US 7,615,622 B2
• Time in Patent Office: Days
• US Class Current: 514/12
• CPC Class Codes: A61K 2039/53 DNA (RNA) vaccination
• 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: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Abstract

The present invention provides methods of increasing the half-life factor VIII. More specifically, the invention methods of increasing the half-life of factor VIII by substituting amino acids in the A2 domain or in the C2 domain of factor VIII or in both domains. It further provides factor VIII mutants produced by these methods. The invention also provides a method of using receptor-associated protein (RAP) to increase the half-life of factor VIII. The invention also provides polynucleotides encoding the mutant factor VIII, polynucleotides encoding RAP, and methods of treating hemophilia using the polypeptides and polynucleotides of the invention.

Claims

1. A mutant factor VIII comprising a nonconservative amino acid substitution at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), Arg(571), and Lys(659);

wherein the mutant factor VIII has reduced heparan sulfate proteoglycan (HSPG)-dependent, receptor-independent clearance; and

wherein the mutant factor VIII has procoagulant activity.

2. The mutant factor VIII of claim 1, wherein said mutant factor VIII has reduced receptor-dependent clearance.

3. The mutant factor VIII of claim 2, which lacks the B domain.

4. The mutant factor VIII of claim 3, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Lys(659).

5. The mutant factor VIII of claim 4, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), and Lys(659).

6. The mutant factor VIII of claim 5, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), and Lys(659).

7. The mutant factor VIII of claim 6, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), and Lys(659).

8. The mutant factor VIII of claim 3, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Arg(571).

9. The mutant factor VIII of claim 3, comprising SEQ ID NO:

5.

10. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 3.

11. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 3.

12. The method of claim 11, which further comprises administering an effective amount of receptor-associated protein (RAP).

13. A polynucleotide encoding the mutant factor VIII of claim 3.

14. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 13.

15. The method of claim 14, which further comprises administering an effective amount of a polynucleotide encoding RAP.

16. The mutant factor VIII of claim 2, further comprising an amino acid substitution at one or more positions in the A2 domain;

wherein the mutant factor VIII has reduced receptor-dependent clearance; and

wherein the mutant factor VIII has procoagulant activity.

17. The mutant factor VIII of claim 16, which lacks the B domain.

18. The mutant factor VIII of claim 17, comprising an amino acid substitution at one or more of positions 484 to 509.

19. The mutant factor VIII of claim 17, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Lys(659).

20. The mutant factor VIII of claim 19, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), and Lys(659).

21. The mutant factor VIII of claim 20, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), and Lys(659).

22. The mutant factor VIII of claim 21, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), and Lys(659).

23. The mutant factor VIII of claim 17, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Arg(571).

24. The mutant factor VIII of claim 17, comprising SEQ ID NO:

5.

25. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 17.

26. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 17.

27. The method of claim 26, which further comprises administering an effective amount of RAP.

28. A polynucleotide encoding the mutant factor VIII of claim 17.

29. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 28.

30. The method of claim 29, which further comprises administering an effective amount of a polynucleotide encoding RAP.

31. The mutant factor VIII of claim 2, further comprising an amino acid substitution at one or more positions in the C2 domain;

wherein the mutant factor VIII has reduced receptor-independent clearance; and

wherein the mutant factor VIII has procoagulant activity.

32. The mutant factor VIII of claim 31, which lacks the B domain.

33. The mutant factor VIII of claim 32, comprising an amino acid substitution at one or more of positions 2303 to 2332.

34. The mutant factor VIII of claim 33, comprising an amino acid substitution at one or more of positions 2311 to 2319.

35. The mutant factor VIII of claim 32, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Lys(659).

36. The mutant factor VIII of claim 35, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), and Lys(659).

37. The mutant factor VIII of claim 36, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), and Lys(659).

38. The mutant factor VIII of claim 37, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), and Lys(659).

39. The mutant factor VIII of claim 32, wherein said nonconservative amino acid substitution is at one or more positions selected from the group consisting of Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Arg(571).

40. The mutant factor VIII of claim 32, comprising SEQ ID NO:

5.

41. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 32.

42. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 32.

43. The method of claim 42, which further comprises administering an effective amount of receptor associated protein (RAP).

44. A polynucleotide encoding the mutant factor VIII of claim 32.

45. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 44.

46. The method of claim 45, which further comprises administering an effective amount of a polynucleotide encoding RAP.

47. A mutant factor VIII comprising a nonconservative amino acid substitution at one or more positions in the A2 domain selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), Arg(571), and Lys(659);

and an amino acid substitution selected from the group consisting of;

(a) an amino acid substitution at one or more positions in the A2 domain;

(b) an amino acid substitution at one or more positions in the C2 domain as numbered in SEQ ID NO;

1;

(c) an amino acid substitution at one or more positions in the A2 domain, and at one or more positions in the C2 domain as numbered in SEQ ID NO;

1;

wherein the mutant factor VIII has reduced heparan sulfate proteoglycan (HSPG)-dependent, receptor-independent clearance; and

wherein the mutant factor VIII has procoagulant activity.

48. The mutant factor VIII of claim 47, wherein the mutant factor VIII has reduced receptor-dependent and reduced receptor-independent clearance.

49. The mutant factor VIII of claim 48, which lacks the B domain.

50. The mutant factor VIII of claim 49, comprising SEQ ID NO:

5.

51. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 49.

52. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 49.

53. The method of claim 52, which further comprises administering an effective amount of RAP.

54. A polynucleotide encoding the mutant factor VIII of claim 49.

55. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 54.

56. The method of claim 55, which further comprises administering an effective amount of a polynucleotide encoding RAP.

57. A method of increasing the half-life of factor VIII, comprising substituting a nonconservative amino acid for a residue at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), Arg(571), and Lys(659);

wherein the resulting factor VIII has reduced heparan sulfate proteoglycan (HSPG)-dependent, receptor-independent clearance; and

wherein the resulting factor VIII has procoagulant activity.

58. The method of claim 57, wherein the resulting factor VIII has reduced receptor-dependent clearance.

59. The method of claim 58, which further comprises a method selected from the group consisting of:

(a) method which comprises substituting an amino acid at one or more positions in the A2 domain;

wherein the resulting factor VIII has reduced receptor-dependent clearance; and

wherein the resulting factor VIII has procoagulant activity;

(b) a method which comprises substituting an amino acid at one or more positions in the C2 domain;

wherein the resulting factor VIII has reduced receptor-independent clearance; and

wherein the resulting factor VIII has procoagulant activity;

(c) a method which comprises administering to a patient in need thereof an effective amount of a fragment of RAP, wherein said fragment binds LRP;

(d) a method comprising methods (a) and (b);

(e) a method comprising methods (a) and (c); and

(f) a method comprising methods (b) and (c).

60. A mutant factor VIII comprising a nonconservative amino acid substitution at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), Arg(571), and Lys(659);

wherein the mutant factor VIII has reduced heparan sulfate proteoglycan (HSPG)-dependent, receptor-independent clearance; and

wherein the mutant factor VIII has procoagulant activity.

61. The mutant factor VIII of claim 60, wherein said mutant factor VIII has reduced receptor-dependent clearance.

62. The mutant factor VIII of claim 61, which lacks the B domain;

63. The mutant factor VIII of claim 62, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Lys(659).

64. The mutant factor VIII of claim 63, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), and Lys(659).

65. The mutant factor VIII of claim 64, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), and Lys(659).

66. The mutant factor VIII of claim 65, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), and Lys(659).

67. The mutant factor VIII of claim 62, wherein said nonconservative amino acid substitutions are at Arg(490) and at Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Arg(571).

68. The mutant factor VIII of claim 62, comprising SEQ ID NO:

5.

69. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 62.

70. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 62.

71. The method of claim 70, which further comprises administering an effective amount of receptor-associated protein (RAP).

72. A polynucleotide encoding the mutant factor VIII of claim 62.

73. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 72.

74. The method of claim 73, which further comprises administering an effective amount of a polynucleotide encoding RAP.

75. The mutant factor VIII of claim 61, further comprising an amino acid substitution at one or more positions in the A2 domain;

wherein the mutant factor VIII has reduced receptor-dependent clearance; and

wherein the mutant factor VIII has procoagulant activity.

76. The mutant factor VIII of claim 75, which lacks the B domain.

77. The mutant factor VIII of claim 76, comprising an amino acid substitution at one or more of positions 484 to 509.

78. The mutant factor VIII of claim 76, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Lys(659).

79. The mutant factor VIII of claim 78, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), and Lys(659).

80. The mutant factor VIII of claim 79, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), and Lys(659).

81. The mutant factor VIII of claim 80, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), and Lys(659).

82. The mutant factor VIII of claim 76, wherein said nonconservative amino acid substitutions are at Arg(490) and at Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Arg(571).

83. The mutant factor VIII of claim 76, comprising SEQ ID NO:

5.

84. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 76.

85. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 76.

86. The method of claim 85, which further comprises administering an effective amount of RAP.

87. A polynucleotide encoding the mutant factor VIII of claim 76.

88. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 87.

89. The method of claim 88, which further comprises administering an effective amount of a polynucleotide encoding RAP.

90. The mutant factor VIII of claim 61, further comprising an amino acid substitution at one or more positions in the C2 domain;

wherein the mutant factor VIII has reduced receptor-independent clearance; and

wherein the mutant factor VIII has procoagulant activity.

91. The mutant factor VIII of claim 90, which lacks the B domain.

92. The mutant factor VIII of claim 91, comprising an amino acid substitution at one or more of positions 2303 to 2332.

93. The mutant factor VIII of claim 92, comprising an amino acid substitution at one or more of positions 2311 to 2319.

94. The mutant factor VIII of claim 91, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Lys(659).

95. The mutant factor VIII of claim 94, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), and Lys(659).

96. The mutant factor VIII of claim 95, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), and Lys(659).

97. The mutant factor VIII of claim 96, wherein said nonconservative amino acid substitutions are at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), and Lys(659).

98. The mutant factor VIII of claim 91, wherein said nonconservative amino acid substitutions are at Arg(490) and at Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), and Arg(571).

99. The mutant factor VIII of claim 91, comprising SEQ ID NO:

5.

100. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 91.

101. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 91.

102. The method of claim 101, which further comprises administering an effective amount of receptor associated protein (RAP).

103. A polynucleotide encoding the mutant factor VIII of claim 91.

104. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 103.

105. The method of claim 104, which further comprises administering an effective amount of a polynucleotide encoding RAP.

106. A mutant factor VIII, comprising a nonconservative amino acid substitution at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), Arg(571), and Lys(659) and an amino acid substitution selected from the group consisting of:

(a) an amino acid substitution at one or more positions in the A2 domain;

(b) an amino acid substitution at one or more positions in the C2 domain as numbered in SEQ ID NO;

1;

(c) an amino acid substitution at one or more positions in the A2 domain, and at one or more positions in the C2 domain as numbered in SEQ ID NO;

1;

wherein the mutant factor VIII has reduced heparan sulfate proteoglycan (HSPG)-dependent, receptor-independent clearance; and

wherein the mutant factor VIII has procoagulant activity.

107. The mutant factor VIII of claim 106, wherein the mutant factor VIII has reduced receptor-dependent and reduced receptor-independent clearance.

108. The mutant factor VIII of claim 107, which lacks the B domain.

109. The mutant factor VIII of claim 108, comprising SEQ ID NO:

5.

110. A pharmaceutically acceptable composition comprising the mutant factor VIII of claim 108.

111. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the mutant factor VIII of claim 108.

112. The method of claim 111, which further comprises administering an effective amount of RAP.

113. A polynucleotide encoding the mutant factor VIII of claim 108.

114. A method of treating hemophilia which comprises administering to a patient in need thereof an effective amount of the polynucleotide of claim 113.

115. The method of claim 114, which further comprises administering an effective amount of a polynucleotide encoding RAP.

116. A method of increasing the half-life of factor VIII, comprising substituting a nonconservative amino acid for a residue at Arg(490) and at one or more positions selected from the group consisting of Lys(380), Lys(512), Lys(523), Arg(527), Lys(556), Arg(562), Lys(570), Arg(571), and Lys(659);

wherein the resulting factor VIII has reduced heparan sulfate proteoglycan (HSPG)-dependent, receptor-independent clearance; and

wherein the resulting factor VIII has procoagulant activity.

117. The method of claim 116, wherein the resulting factor VIII has reduced receptor-dependent clearance.

118. The method of claim 117, which further comprises a method selected from the group consisting of

(a) method which comprises substituting an amino acid at one or more positions in the A2 domain;

wherein the resulting factor VIII has reduced receptor-dependent clearance; and

wherein the resulting factor VIII has procoagulant activity;

(b) a method which comprises substituting an amino acid at one or more positions in the C2 domain;

wherein the resulting factor VIII has reduced receptor-independent clearance; and

wherein the resulting factor VIII has procoagulant activity;

(c) a method which comprises administering to a patient in need thereof an effective amount of a fragment of RAP, wherein said fragment binds LRP;

(d) a method comprising methods (a) and (b);

(e) a method comprising methods (a) and (c); and

(f) a method comprising methods (b) and (c).