CETUXIMAB WITH MODIFIED GLYCOSYLATION AND USES THEREOF

• US 20160002330A1
• Filed: 02/13/2014
• Published: 01/07/2016
• Est. Priority Date: 02/13/2013
• Status: Abandoned Application

• Current Assignee: Laboratoire francais du Fractionnement et des Biotechnologies
• Original Assignee: Laboratoire francais du Fractionnement et des Biotechnologies
• Inventors: MEADE, Harry M.
• Application Number:
  US14/767,089
• Publication Number:
  US 20160002330A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A01K 2227/102 Caprine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 31/4745 condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines yohimbine derivatives, vinblastine A61K31/475; ergoline derivatives A61K31/48
• CPC Class Codes: A61K 31/513 having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
• CPC Class Codes: A61K 31/522 having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
• CPC Class Codes: A61K 39/39533 against materials from animals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 2317/12 isolated from milk
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation

Abstract

In one aspect, the disclosure relates to antibodies with altered glycosylation patterns, methods of production of said antibodies, and methods of use thereof. In some embodiments, the antibody is cetuximab.

Claims

1. A glycosylated antibody comprising:

(a) a heavy chain comprising SEQ ID NO;

1;

(b) a light chain comprising SEQ ID NO;

2;

wherein the glycosylated antibody has fewer galactose-alpha-1,3-galactose moieties than the glycosylated antibody produced in non-mammary cell culture.

2. The glycosylated antibody of claim 1, wherein the non-mammary cell culture is mouse myeloma cell culture.

3. The glycosylated antibody of claim 1 or 2, wherein the fewer galactose-alpha-1,3-galactose moieties are located in the variable region of the heavy chain.

4. The glycosylated antibody of any one of claims 1-3, wherein the fewer galactose-alpha-1,3-galactose moieties are located at position 107 of the heavy chain as shown in SEQ ID NO:

1.

5. The glycosylated antibody of claim 1, wherein the glycosylated antibody lacks galactose-alpha-1,3-galactose moieties.

6. The glycosylated antibody of any one of claims 1-5, wherein the glycosylated antibody is produced in mammary gland epithelial cells.

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

8. The glycosylated antibody of claim 7, wherein the non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama.

9. The glycosylated antibody of claim 7, wherein the non-human mammal is a goat.

10. The glycosylated antibody of any one of claims 1-9, wherein the antibody is chimeric, humanized or a fully human antibody.

11. The glycosylated antibody of any one of claims 1-10, wherein the antibody is cetuximab.

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

40. Mammary gland epithelial cells that produce the antibody of any one of claims 1-11.

41. The mammary gland epithelial cells of claim 40, wherein the mammary gland epithelial cells comprise a nucleic acid comprising SEQ ID NO:

3 and a nucleic acid comprising SEQ ID NO;

4.

42. The mammary gland epithelial cells of claim 41, wherein the nucleic acid comprising SEQ ID NO:

3 and the nucleic acid comprising SEQ ID NO;

4 are connected.

43. A transgenic non-human mammal comprising the mammary gland epithelial cells of any one of claims 40-42.

48. The transgenic non-human mammal of claim 43 or 47, wherein the transgenic non-human mammal is a goat.

49. A method comprising:

administering an effective amount of the antibody of any one of claims 1-11 or the composition of any one of claims 12-27 to a subject in need thereof.

50. The method of claim 49, wherein the subject has cancer.

51. The method of claim 50, wherein the cancer is head and neck cancer or colorectal cancer.

52. The method of any one of claims 49-51, wherein the method further comprises administering at least one additional therapeutic agent.

53. The method of claim 52, wherein the at least one additional therapeutic agent is irinotecan, leucovorin calcium, fluorouracil, or a combination thereof.

13. A composition comprising a population of glycosylated antibodies, wherein the glycosylated antibody comprises:

(a) a heavy chain comprising SEQ ID NO;

1;

(b) a light chain comprising SEQ ID NO;

2; and

wherein the population of antibodies has fewer galactose-alpha-1,3-galactose moieties than the population of glycosylated antibodies produced in non-mammary cell culture.

14. The composition of claim 13, wherein the non-mammary cell culture is mouse myeloma cell culture.

15. The composition of claim 13 or 14, wherein the fewer galactose-alpha-1,3-galactose moieties are located in the variable region of the heavy chain.

16. The composition of any one of claims 13-15, wherein the fewer galactose-alpha-1,3-galactose moieties are located at position 107 of the heavy chain as shown in SEQ ID NO:

1.

17. The composition of claim 13, wherein the glycosylated antibodies lack galactose-alpha-1,3-galactose moieties.

18. The composition of any one of claims 13-17, wherein the population of glycosylated antibodies is produced in mammary gland epithelial cells.

19. The composition of any one of claims 13-18, wherein the population of glycosylated antibodies is produced in the mammary gland epithelial cells of a non-human transgenic mammal.

20. The composition of claim 19, wherein the non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama.

21. The composition of claim 19, wherein the non-human mammal is a goat.

22. The composition of any one of claims 13-21, wherein the glycosylated antibodies in the population are chimeric, humanized or fully human antibodies.

23. The composition of any one of claims 13-22, wherein the glycosylated antibodies in the population are cetuximab antibodies.

24. The composition of any one of claims 13-23, wherein the composition further comprises milk.

25. The composition of any one of claims 13-24, wherein the composition further comprises a pharmaceutically acceptable carrier.

44. Mammary gland epithelial cells that produce the population of antibodies of any one of claims 13-27.

45. The mammary gland epithelial cells of claim 44, wherein the mammary gland epithelial cells comprise a nucleic acid comprising SEQ ID NO:

3 and a nucleic acid comprising SEQ ID NO;

4.

46. The mammary gland epithelial cells of claim 45, wherein the nucleic acid comprising SEQ ID NO:

3 and the nucleic acid comprising SEQ ID NO;

4 are connected.

47. A transgenic non-human mammal comprising the mammary gland epithelial cells of any one of claims 44-46.

26. A composition comprising monoclonal antibodies, wherein the monoclonal antibodies comprise:

(a) a heavy chain comprising SEQ ID NO;

1;

(b) a light chain comprising SEQ ID NO;

2; and

wherein the composition is produced in a mammary gland of a transgenic goat.

27. A composition comprising monoclonal antibodies, wherein the monoclonal antibodies comprise:

(a) a heavy chain comprising SEQ ID NO;

1;

(b) a light chain comprising SEQ ID NO;

2; and

wherein the monoclonal antibodies lack galactose-alpha-1,3-galactose.

28. A method comprising:

producing a population of glycosylated antibodies in mammary gland epithelial cells such that the population of glycosylated antibodies produced comprises fewer galactose-alpha-1,3-galactose moieties than the population of glycosylated antibodies produced in non-mammary cell culture, and

wherein the glycosylated antibodies comprise a heavy chain comprising SEQ ID NO;

1 and a light chain comprising SEQ ID NO;

2.

29. The method of claim 28, wherein the non-mammary cell culture is mouse myeloma cell culture.

30. The method of claim 28 or 29, wherein the population of glycosylated antibodies lacks galactose-alpha-1,3-galactose.

31. The method of any one of claims 28-30, wherein the method further comprises collecting the population of glycosylated antibodies.

32. The method of any one of claims 28-31, wherein the producing occurs in vitro.

33. The method of any one of claims 28-31, wherein the producing occurs in vivo.

34. The method of claim 33, wherein the producing occurs in a non-human transgenic mammal.

35. The method of claim 34, wherein the non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama.

36. The method of claim 34, wherein the non-human mammal is a goat.

37. The method of any one of claims 28-36, the method further comprising purifying the population of glycosylated antibodies.

38. The method of any one of claim 28-37, wherein the method further comprises

comparing the number of galactose-alpha-1,3-galactose moieties present in the population of glycosylated antibodies to the number of galactose-alpha-1,3-galactose moieties present in a population of glycosylated antibodies produced in non-mammary cell culture.

39. A population of glycosylated antibodies produced by the method of any one of claims 28-38.