HIGHLY GALACTOSYLATED ANTI-TNF-ALPHA ANTIBODIES AND USES THEREOF

• US 20150368334A1
• Filed: 02/13/2014
• Published: 12/24/2015
• Est. Priority Date: 02/13/2013
• Status: Active Grant

• Current Assignee: Laboratoire francais du Fractionnement et des Biotechnologies
• Original Assignee: Laboratoire francais du Fractionnement et des Biotechnologies
• Inventors: Meade, Harry M., Chen, Li-How
• Granted Patent: US 10,174,110 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 39/39508 from milk, i.e. lactoglobulins
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• 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 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C07K 16/04 from milk
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/283 against Fc-receptors, e.g. CD16, CD32, CD64 CD23 C07K16/2851
• CPC Class Codes: C07K 2317/12 isolated from milk
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/734 Complement-dependent cytotoxicity [CDC]
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

In one aspect, the disclosure relates to highly galactosylated anti-TNF-alpha antibodies and compositions thereof. In one aspect, the disclosure relates to populations of anti-TNF-alpha antibodies with a high level of galactosylation, and compositions thereof. In one aspect, the disclosure relates to methods of production and use of highly galactosylated anti-TNF-alpha antibodies and populations of anti-TNF-alpha antibodies with a high level of galactosylation. In some embodiments, the anti-TNF-alpha antibody is adalimumab.

Claims

1. An anti-TNF-alpha antibody, wherein the antibody is highly galactosylated.

2. The antibody of claim 1, wherein the antibody is highly fucosylated.

3. The antibody of claim 1 or claim 2, wherein the antibody comprises mono-galactosylated N-glycans.

4. The antibody of any one of claims 1-3, wherein the antibody comprises bi-galactosylated N-glycans.

5. The antibody of any one of claims 1-4, wherein the heavy chain of the antibody comprises SEQ ID NO:

1, and wherein the light chain of the antibody comprises SEQ ID NO;

2.

6. The antibody of any one of claims 1-5, wherein the antibody is adalimumab.

7. The antibody of any one of claims 1-6, wherein the antibody is produced in mammary epithelial cells of a non-human mammal.

8. The antibody of any one of claims 1-7, wherein the antibody is produced in a transgenic non-human mammal.

9. The antibody of claim 8 or claim 9, wherein the non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama.

10. The antibody of claim 9, wherein the non-human mammal is a goat.

11. A composition comprising the antibody of any one of claims 1-10, further comprising milk.

12. A composition comprising the antibody of any one of claims 1-11, further comprising a pharmaceutically-acceptable carrier.

42. Mammary gland epithelial cells that produce the antibody of any one of claims 1-12 or the population of antibodies of the compositions of any one of claims 13-35.

43. A transgenic non-human mammal comprising the mammary gland epithelial cells of claim 42.

44. A method comprising administering the antibody of any one of claims 1-10 or the composition of any one claims 11-35, to a subject in need thereof.

45. The method of claim 44, wherein the subject has an inflammatory disorder or autoimmune disorder.

46. The method of claim 45, wherein the inflammatory disorder or autoimmune disorder is rheumatoid arthritis, psoriasis, Crohn'"'"'s disease, juvenile idiopathic arthritis, ankylozing spondylitis, ulcerative colitis, chronic inflammation, hepatitis, Behç

et'"'"'s disease, Wegener'"'"'s granulomatosis, or sarcoidosis.

13. A composition, comprising:

a population of antibodies,

wherein the antibody is an anti-TNF-alpha antibody, and

wherein the level of galactosylation of the antibodies in the population is at least 60%.

14. The composition of claim 13, wherein the level of galactosylation of the antibodies in the population is at least 70%.

15. The composition of claim 13, wherein the level of galactosylation of the antibodies in the population is at least 80%.

16. The composition of any one of claims 13-15, wherein the level of fucosylation of the antibodies in the population is at least 50%.

17. The composition of any one of claims 13-15, wherein the level of fucosylation of the antibodies in the population is at least 60%.

18. The composition of any one of claims 13-17, wherein the population comprises antibodies that comprise mono-galactosylated N-glycans.

19. The composition of any one of claims 13-18, wherein the population comprises antibodies that comprise bi-galactosylated N-glycans.

20. The composition of any one of claims 13-19, wherein the ratio of the level of galactosylation of the antibodies in the population to the level of fucosylation of the antibodies in the population is between 1.0 and 1.4.

21. The composition of any one of claims 13-20, wherein at least 35% of the antibodies in the population comprise bi-galactosylated N-glycans and at least 25% of the antibodies in the population comprise mono-galactosylated N-glycans.

22. The composition of any one of claims 13-21, wherein the heavy chain of the antibody comprises SEQ ID NO:

1, and wherein the light chain of the antibody comprises SEQ ID NO;

2.

23. The composition of any one of claims 13-22, wherein the antibody is adalimumab.

24. The composition of any one of claims 13-23, wherein the antibody is produced in mammary epithelial cells of a non-human mammal.

25. The composition of any one of claims 13-24, wherein the antibody is produced in a transgenic non-human mammal.

26. The composition of claim 24 or claim 25, wherein the non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama.

27. The composition of claim 26, wherein the non-human mammal is a goat.

28. The composition of any one of claims 13-27, wherein the composition further comprises milk.

29. The composition of any one of claims 13-28, wherein the composition further comprises a pharmaceutically-acceptable carrier.

30. The composition of any one of claims 24-29, wherein the population of antibodies has an increased level of complement dependent cytotoxicity (CDC) activity when compared to a population of antibodies not produced in mammary gland epithelial cells.

31. The composition of any one of claims 24-30, wherein the population of antibodies has an increased level of antibody-dependent cellular cytotoxicity (ADCC) activity when compared to a population of antibodies not produced in mammary gland epithelial cells.

32. The composition of any one of claims 30-31, wherein the population of antibodies not produced in mammary gland epithelial cells is produced in cell culture.

33. The composition of any one of claims 30-32, wherein the level of galactosylation of the antibodies not produced in mammary gland epithelial cells is 50% or lower when compared to the level of galactosylation of the antibodies produced in mammary gland epithelial cells.

34. The composition of any one of claims 30-32, wherein the level of galactosylation of the antibodies not produced in mammary gland epithelial cells is 30% or lower when compared to the level of galactosylation of the antibodies produced in mammary gland epithelial cells.

35. The composition of any one of claims 30-32, wherein the level of galactosylation of the antibodies not produced in mammary gland epithelial cells is 10% or lower when compared to the level of galactosylation of the antibodies produced in mammary gland epithelial cells.

36. A method for producing a population of antibodies, comprising:

expressing the population of antibodies in mammary gland epithelial cells of a non-human mammal such that a population of antibodies is produced,

wherein the antibody is an anti-TNF-alpha antibody,

wherein the level of galactosylation of the antibodies in the population is at least 60%.

37. The method of claim 36, wherein the mammary gland epithelial cells are in culture and are transfected with a nucleic acid that comprises a sequence that encodes the antibody.

38. The method of claim 36, wherein the mammary gland epithelial cells are in a non-human mammal engineered to express a nucleic acid that comprises a sequence that encodes the antibody in its mammary gland.

39. The method of claim 37 or claim 38, wherein the nucleic acid comprises SEQ ID NO:

3 and SEQ ID NO;

4.

40. The method of any of claims 36-39, wherein the mammary gland epithelial cells are goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama mammary gland epithelial cells.

41. The method of claim 40, wherein the mammary gland epithelial cells are goat mammary gland epithelial cells.

47. A monoclonal anti-TNF antibody composition comprising monoclonal anti-TNF antibodies having glycan structures on the Fc glycosylation sites (Asn297, EU numbering),

wherein said glycan structures have a galactose content of more than 60%.

48. An anti-TNF-alpha antibody, wherein the antibody contains an oligomannose.

49. The antibody of claim 48, wherein the heavy chain of the antibody comprises SEQ ID NO:

1, and wherein the light chain of the antibody comprises SEQ ID NO;

2.

50. The antibody of claim 48 or 49, wherein the antibody is adalimumab.

51. The antibody of any one of claims 48-50, wherein the antibody is produced in mammary epithelial cells of a non-human mammal.

52. The antibody of any one of claims 48-51, wherein the antibody is produced in a transgenic non-human mammal.

53. The antibody of claim 51 or claim 52, wherein the non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama.

54. The antibody of claim 53, wherein the non-human mammal is a goat.

55. A composition comprising the antibody of any one of claims 48-54, further comprising milk.

56. A composition comprising the antibody of any one of claims 48-55, further comprising a pharmaceutically-acceptable carrier.

72. The antibody of any one of claims 48-54, wherein the oligomannose is Man5.

73. The antibody of any one of claims 48-54, wherein the oligomannose is Man6.

74. The antibody of any one of claims 48-54, wherein the oligomannose is Man7.

57. A composition, comprising:

a population of antibodies,

wherein the antibody is an anti-TNF-alpha antibody, and

wherein the at least 30% of the antibodies contain at least one oligomannose.

58. The composition of claim 57, wherein the antibodies exhibit a high mannose glycosylation pattern.

59. The composition of claim 58, wherein at least one chain of the milk-derived antibodies contains an oligomannose and is non-fucosylated.

60. The composition of claim 57, wherein the major carbohydrate of the milk-derived antibodies is non-fucosylated.

61. The composition of any one of claims 57-60, wherein the heavy chain of the antibody comprises SEQ ID NO:

1, and wherein the light chain of the antibody comprises SEQ ID NO;

2.

62. The composition of any one of claims 57-61, wherein the antibody is adalimumab.

63. The composition of any one of claims 57-62, wherein the antibody is produced in mammary epithelial cells of a non-human mammal.

64. The composition of any one of claims 57-63, wherein the antibody is produced in a transgenic non-human mammal.

65. The composition of claim 63 or claim 64, wherein the non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama.

66. The composition of claim 65, wherein the non-human mammal is a goat.

67. The composition of any one of claims 57-66, wherein the composition further comprises milk.

68. The composition of any one of claims 57-67, wherein the composition further comprises a pharmaceutically-acceptable carrier.

69. The composition of any one of claims 63-68, wherein the population of antibodies has an increased level of complement dependent cytotoxicity (CDC) activity when compared to a population of antibodies not produced in mammary gland epithelial cells.

70. The composition of any one of claims 63-69, wherein the population of antibodies has an increased level of antibody-dependent cellular cytotoxicity (ADCC) activity when compared to a population of antibodies not produced in mammary gland epithelial cells.

71. The composition of claim 69 or 70, wherein the population of antibodies not produced in mammary gland epithelial cells is produced in cell culture.

75. The composition of any one of claims 57-71, wherein the oligomannose is Man5.

76. The composition of any one of claims 57-71, wherein the oligomannose is Man6.

77. The composition of any one of claims 57-71, wherein the oligomannose is Man7.