Erythropoietin conjugates

• US 6,340,742 B1
• Filed: 06/28/2000
• Issued: 01/22/2002
• Est. Priority Date: 07/02/1999
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Original Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Inventors: Josel, Hans-Peter, Burg, Josef, Hilger, Bernd
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US09/604,871
• Time in Patent Office: 573 Days
• Field of Search: 424/85.1, 514/2, 514/8, 514/12, 514/21, 530/351, 530/404, 530/408
• US Class Current: 530/351
• 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 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]

Abstract

Erythropoietin glycoprotein products are disclosed which have the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells. The present conjugates have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo. In addition, compared with conventional PEG-EPO conjugates, the conjugates of this invention have superior clinical properties. The present invention also includes a method for the treatment of anemia in a human employing the novel erythropoietin glycoprotein products as well as a method for preparing the erythropoietin glycoprotein products.

Claims

1. A conjugate, said conjugate comprising an erythropoietin glycoprotein having at least one free amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells and selected from the group consisting of human erythropoietin and analogs thereof which have the primary structure of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or by the rearrangement of at least one glycosylation site;

said glycoprotein being covalently linked to from one to three lower-alkoxy poly(ethylene glycol) groups, each poly(ethylene glycol) group being covalently linked to the glycoprotein via a linker of the formula —

C(O)—

X—

S—

Y—

with the C(O) of the linker forming an amide bond with one of said amino groups, X is —

CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

, k is from 1 to 10, Y is

the average molecular weight of each poly(ethylene glycol) group is from about 20 kilodaltons to about 40 kilodaltons, and the molecular weight of the conjugate is from about 51 kilodaltons to about 175 kilodaltons.

2. The conjugate of claim 1, wherein the erythropoietin glycoprotein is a human erythropoietin protein.

3. The conjugate of claim 2, wherein the human erythropoietin protein is expressed by endogenous gene activation.

4. The conjugate of claim 2, wherein the erythropoietin glycoprotein has the amino acid sequence set out in (SEQ ID NO:

1) or (SEQ ID NO;

2).

5. The conjugate of claim 4, wherein X is —

(CH₂)_k—

.

6. The conjugate of claim 4, wherein k is from 1 to 4.

7. The conjugate of claim 4, wherein the average molecular weight of each poly(ethylene glycol) group is from about 24 kilodaltons to about 35 kilodaltons.

8. The conjugate of claim 7, wherein the average molecular weight of each poly(ethylene glycol) group is about 30 kilodaltons.

9. The conjugate of claim 4, wherein the glycoprotein is covalently linked to one or two lower-alkoxy capped poly(ethylene glycol) groups.

10. The conjugate of claim 4, wherein the poly(ethylene glycol) groups are capped by methoxy.

11. The conjugate of claim 2, of the formula:

wherein n is an integer from 1 to 3;

m is an integer from 450 to 900;

R is lower alkyl;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

, and P is the residue of the erythropoietin glycoprotein without the amino group or groups which form an amide linkage with X.

12. The conjugate of claim 11, wherein X is —

CH₂—

.

13. The conjugate of claim 11, wherein m is an integer from 550 to 800.

14. The conjugate of claim 13, wherein m is an integer from 650 to 700.

15. The conjugate of claim 11, wherein n is 1.

16. The conjugate of claim 11, wherein R is methyl.

17. The conjugate of claim 11, wherein P has the amino acid sequence set out in (SEQ ID NO:

1) or (SEQ ID NO;

2).

18. The conjugate of claim 2, of the formula:

wherein n is an integer from 1 to 3;

m is an integer from 450 to 900;

R is lower alkyl;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

, and P is the residue of the erythropoietin glycoprotein without the amino group or groups which form an amide linkage with X.

19. The conjugate of claim 18, wherein X is —

CH₂—

.

20. The conjugate of claim 18, wherein m is an integer from 550 to 800.

21. The conjugate of claim 20, wherein m is an integer from 650 to 700.

22. The conjugate of claim 18, wherein R is methyl.

23. The conjugate of claim 18, wherein P has the amino acid sequence set out in (SEQ ID NO:

1) or (SEQ ID NO;

2).

24. The conjugate of claim 1, wherein the erythropoietin glycoprotein has the primary structure of human erythropoietin modified by the addition of from 1 to 6 glyosylation sites.

25. The conjugate of claim 1, wherein the glycoprotein has a sequence selected from the group consisting of:

Asn³⁰Thr³² (SEQ ID NO;

1);

Asn⁵¹Thr⁵³ (SEQ ID NO;

1),

Asn⁵⁷Thr⁵⁹ (SEQ ID NO;

1);

Asn⁶⁹(SEQ ID NO;

1);

Asn⁶⁹Thr⁷¹ (SEQ ID NO;

1);

Ser⁶⁸Asn⁶⁹Thr⁷¹ (SEQ ID NO;

1);

Val⁸⁷Asn⁸⁸Thr⁹⁰ (SEQ ID NO;

1);

Ser⁸⁷Asn⁸⁸Thr⁹⁰ (SEQ ID NO;

1);

Ser⁸⁷Asn⁸⁸Gly⁸⁹Thr⁹⁰ (SEQ ID NO;

1);

Ser⁸⁷Asn⁸⁸Thr⁹⁰Thr⁹² (SEQ ID NO;

1);

Ser⁸⁷Asn⁸⁸Thr⁹⁰Ala¹⁶² (SEQ ID NO;

1);

Asn⁶⁹Thr⁷¹Ser⁸⁷Asn⁸⁸Thr⁹⁰ (SEQ ID NO;

1);

Asn³⁰Thr³²Val⁸⁷Asn⁸⁸Thr⁹⁰ (SEQ ID NO;

1);

Asn⁸⁹Ile⁹⁰Thr⁹¹(SEQ ID NO;

1);

Ser⁸⁷Asn⁸⁹Ile⁹⁰Thr⁹¹ (SEQ ID NO;

1);

Asn¹³⁶Thr¹³⁸ (SEQ ID NO;

1);

Asn¹³⁸Thr¹⁴⁰ (SEQ ID NO;

1);

Thr¹²⁵ (SEQ ID NO;

1); and

Pro¹²⁴Thr¹²⁵ (SEQ ID NO;

1).

26. The conjugate of claim 25, wherein X is —

(CH₂)_k—

.

27. The conjugate of claim 25, wherein k is from 1 to 4.

28. The conjugate of claim 25, wherein the average molecular weight of each poly(ethylene glycol) group is from about 24 kilodaltons to about 35 kilodaltons.

29. The conjugate of claim 28, wherein the average molecular weight of each poly(ethylene glycol) group is about 30 kilodaltons.

30. The conjugate of claim 25, wherein the glycoprotein is covalently linked to one or two lower-alkoxy capped poly(ethylene glycol) groups.

31. The conjugate of claim 25, wherein the poly(ethylene glycol) groups are capped by methoxy.

32. The conjugate of claim 25, of the formula:

wherein n is an integer from 1 to 3;

m is an integer from 450 to 900;

R is lower alkyl;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

, and P is the residue of the erythropoietin glycoprotein without the amino group or groups which form an amide linkage with X.

33. The conjugate of claim 32, wherein X is —

CH₂—

.

34. The conjugate of claim 32, wherein m is an integer from 550 to 800.

35. The conjugate of claim 34, wherein m is an integer from 650 to 700.

36. The conjugate of claim 32, wherein n is 1.

37. The conjugate of claim 32, wherein R is methyl.

38. The conjugate of claim 25, of the formula:

wherein n is an integer from 1 to 3;

m is an integer from 450 to 900;

R is lower alkyl;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

, and P is the residue of the erythropoietin glycoprotein without the amino group or groups which form an amide linkage with X.

39. The conjugate of claim 38, wherein X is —

CH₂—

.

40. The conjugate of claim 38, wherein m is an integer from 550 to 800.

41. The conjugate of claim 40, wherein m is an integer from 650 to 700.

42. The conjugate of claim 38, wherein R is methyl.

43. The conjugate of claim 1, wherein the glycoprotein has a sequence comprising the sequence of human erythropoietin and a second sequence at the carboxy terminus of the human erythropoietin sequence, wherein the second sequence contains at least one glycosylation site.

44. The conjugate of claim 43, wherein the second sequence comprises a sequence derived from the carboxy terminal sequence of human chorionic gonadotropin.

45. The conjugate of claim 44, wherein the glycoprotein has a sequence selected from the group consisting of:

(a) the sequence of human erythropoietin and the sequence (SEQ ID NO;

3) at the carboxy terminus of the human erythropoietin sequence;

(b) the sequence in (a) modified by Ser⁸⁷ Asn⁸⁸ Thr⁹⁰; and

(c) the sequence in (a) modified by Asn³⁰ Thr³² Val⁸⁷ Asn⁸⁸ Thr⁹⁰.

46. The conjugate of claim 45, wherein X is —

(CH₂)_k—

.

47. The conjugate of claim 45, wherein k is from 1 to 4.

48. The conjugate of claim 45, wherein the average molecular weight of each poly(ethylene glycol) group is from about 24 kilodaltons to about 35 kilodaltons.

49. The conjugate of claim 48, wherein the average molecular weight of each poly(ethylene glycol) group is about 30 kilodaltons.

50. The conjugate of claim 45, wherein the glycoprotein is covalently linked to one or two lower-alkoxy capped poly(ethylene glycol) groups.

51. The conjugate of claim 45, wherein the poly(ethylene glycol) groups are capped by methoxy.

52. The conjugate of claim 45, of the formula:

wherein n is an integer from 1 to 3;

m is an integer from 450 to 900;

R is lower alkyl;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

, and P is the residue of the erythropoietin glycoprotein without the amino group or groups which form an amide linkage with X.

53. The conjugate of claim 52, wherein X is —

CH₂—

.

54. The conjugate of claim 52, wherein m is an integer from 550 to 800.

55. The conjugate of claim 54, wherein m is an integer from 650 to 700.

56. The conjugate of claim 52, wherein n is 1.

57. The conjugate of claim 52, wherein R is methyl.

58. The conjugate of claim 52, wherein the sequence of human erythropoietin is the sequence set out in (SEQ ID NO:

1) or (SEQ ID NO;

2).

59. The conjugate of claim 45, of the formula:

wherein n is an integer from 1 to 3;

m is an integer from 450 to 900;

R is lower alkyl;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

, and P is the residue of the erythropoietin glycoprotein without the amino group or groups which form an amide linkage with X.

60. The conjugate of claim 59, wherein X is —

CH₂—

.

61. The conjugate of claim 59, wherein m is an integer from 550 to 800.

62. The conjugate of claim 61, wherein m is an integer from 650 to 700.

63. The conjugate of claim 59, wherein R is methyl.

64. The conjugate of claim 59, wherein the sequence of human erythropoietin is the sequence set out in (SEQ ID NO:

1) or (SEQ ID NO;

2).

65. The conjugate of claim 1, wherein the glycoprotein has the sequence of human erythropoietin modified by a rearrangement of at least one glycosylation site.

66. The conjugate of claim 65, wherein the rearrangement comprises deletion of any of the N-linked glycosylation sites in human erythropoietin and addition of an N-linked glycosylation site at position 88 of the sequence of human erythropoietin.

67. The conjugate of claim 66, wherein the glycoprotein has the sequence of human erythropoietin modified by a modification selected from the group consisting of:

Gln²⁴ Ser⁸⁷ Asn⁸⁸ Thr⁹⁰;

Gln³⁸ Ser⁸⁷ Asn⁸⁸ Thr⁹⁰; and

Gln⁸³ Ser⁸⁷ Asn⁸⁸ Thr⁹⁰.

68. The conjugate of claim 67, wherein X is —

(CH₂)_k—

.

69. The conjugate of claim 67, wherein k is from 1 to 4.

70. The conjugate of claim 67, wherein the average molecular weight of each poly(ethylene glycol) group is from about 24 kilodaltons to about 35 kilodaltons.

71. The conjugate of claim 70, wherein the average molecular weight of each poly(ethylene glycol) group is about 30 kilodaltons.

72. The conjugate of claim 67, wherein the glycoprotein is covalently linked to one or two lower-alkoxy capped poly(ethylene glycol) groups.

73. The conjugate of claim 67, wherein the poly(ethylene glycol) groups are capped by methoxy.

74. The conjugate of claim 67, of the formula:

wherein n is an integer from 1 to 3;

m is an integer from 450 to 900;

R is lower alkyl;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

, and P is the residue of the erythropoietin glycoprotein without the amino group or groups which form an amide linkage with X.

75. The conjugate of claim 74, wherein X is —

CH₂—

.

76. The conjugate of claim 74, wherein m is an integer from 550 to 800.

77. The conjugate of claim 76, wherein m is an integer from 650 to 700.

78. The conjugate of claim 74, wherein n is 1.

79. The conjugate of claim 74, wherein R is methyl.

80. The conjugate of claim 67, of the formula:

wherein n is an integer from 1 to 3;

m is an integer from 450 to 900;

R is lower alkyl;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

, and P is the residue of the erythropoietin glycoprotein without the amino group or groups which form an amide linkage with X.

81. The conjugate of claim 80, wherein X is —

CH₂—

.

82. The conjugate of claim 80, wherein m is an integer from 550 to 800.

83. The conjugate of claim 82, wherein m is an integer from 650 to 700.

84. The conjugate of claim 80, wherein R is methyl.

85. A composition comprising conjugates, each of said conjugates comprising an erythropoietin glycoprotein having at least one free amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells and selected from the group consisting of human erythropoietin and analogs thereof which have the primary structure of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or by the rearrangement of at least one glycosylation site;

said glycoprotein being covalently linked to from one to three lower-alkoxy poly(ethylene glycol) groups, each poly(ethylene glycol) group being covalently linked to the glycoprotein via a linker of the formula —

C(O)—

X—

S—

Y—

with the C(O) of the linker forming an amide bond with one of said amino groups,

X is —

(CH₂ )_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

,

k is from 1 to 10,

Y is

 

the average molecular weight of each poly(ethylene glycol) group is from about 20 kilodaltons to about 40 kilodaltons, and the molecular weight of the conjugate is from about 51 kilodaltons to about 175 kilodaltons; and

the percentage of conjugates where n is 1 is at least ninety percent.

86. The composition of claim 85 comprising conjugates of the formula

wherein n is an integer from 1 to 3; m is an integer from 450 to 900;

R is lower alkyl;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

, and P is the residue of the erythropoietin glycoprotein without the amino group or groups which form an amide linkage with X.

87. The composition of according to claim 86 wherein the percentage of conjugates where n is 1 is at least ninety-two percent.

88. The composition of claim 86 wherein the percentage of conjugates where n is 1 is at least ninety-six percent.

89. The composition of claim 85 wherein the percentage of conjugates where n is 1 is from ninety percent to ninety-six percent.