IGM production by transformed cells and methods of quantifying said IgM production

• US 20070154469A1
• Filed: 07/15/2004
• Published: 07/05/2007
• Est. Priority Date: 07/15/2003
• Status: Active Grant

• Current Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Original Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Inventors: Irie, Reiko
• Granted Patent: US 7,709,615 B2
• Time in Patent Office: Days
• US Class Current: 424/133.100
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/3084 against tumour-associated gangliosides
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/734 Complement-dependent cytotoxicity [CDC]
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: G01N 33/686 Anti-idiotype

Abstract

IgM can be obtained in the form of a pentamer by placing the genes encoding the H, L, and J chains on the same vector to transform appropriate host cells. The gene encoding the J chain may be introduced by co-transfection. When no J chain is expressed, the IgM is produced as a hexamer. The transformants obtained according to the present invention achieve a high yield of IgM. The present invention also provides methods which enable separation and quantification of polymeric IgM.

Claims

1. A transformed cell producing IgM of a) 100 mg/L or more;

or b) 35 pg/cell/day or more.

3. The transformed cell of claim 1, which is a eukaryotic cell.

4. The transformed cell of claim 1, which is a prokaryotic cell.

5. The transformed cell of claim 3, which is a mammalian cell.

6. The transformed cell of claim 1, which is an established cell line.

7. The transformed cell of claim 6, which is a non-lymphoid cell line.

8. The transformed cell of claim 7, which is a CHO cell line.

24. A method for producing an IgM, comprising a step of culturing the cell of claim 1 and then collecting the IgM.

25. A method for producing a substantially pure IgM, comprising a step of purifying an IgM from a culture supernatant obtained from culture of the cell of claim 1.

26. An IgM obtained by the method of claim 24.

27. A substantially pure IgM obtained by the method of claim 25.

28. The IgM of claim 26, which is a human, mouse, human chimeric, or humanized antibody.

29. The IgM of claim 26, which is a substantially pure pentamer or hexamer.

31. The IgM of claim 26, which is an anti-sugar chain antibody.

32. The IgM of claim 31, which is an anti-ganglioside antobody.

33. The IgM of claim 32, which is an anti-GM2 or GM3 antobody.

48. A pharmaceutical composition comprising the IgM of claim 26.

2. (canceled)

9. An expression vector comprising both (1) a nucleotide sequence encoding an IgM H chain and (2) a nucleotide sequence encoding an IgM L chain in the same vector, or a gene fragment comprising the genes (1) and (2).

10. The expression vector of claim 9, wherein the vector comprises (3) a nucleotide sequence encoding an IgM J chain in the same vector, or a gene fragment comprising the genes (1), (2), and (3).

11. The expression vector or gene fragment of claim 9, wherein IgM secretion is controlled by a transcriptional regulatory sequence.

12. The expression vector or gene fragment of claim 11, wherein the transcriptional regulatory sequence is selected from the group consisting of:

major late promoter of adenovirus 2;

early promoter of simian birus 40;

mouse mammary tumor virus (MMTV)-LTR promoter;

thymidine kinase promoter of herpes simplex virus;

cytomegalovirus promoter;

polypeptide chain elongation factor 1 α

promoter;

bovine growth hormoone promoter;

β

actin gene promoter; and

CAG promoter.

13. The expression vector or gene fragment of claim 12, wherein the transcriptional regulatory sequence is selected from the group consisting of:

early promoter of simian birus 40;

cytomegalovirus promoter;

polypeptide chain elongation factor 1 α

promoter;

CAG promoter.

14. A transformed cell transformed by the vector or gene fragment of claim 9.

16. The transformed cell of claim 14, wherein the expression vector or gene fragment comprises a nucleotide sequence encoding a J chain.

17. The transformed cell of claim 14, wherein the vector or gene fragment comprises a nucleotide sequence encoding an IgM J chain and the cell produces pentamer IgM with a content of 60% or more.

19. The transformed cell of claim 14, wherein the vector or gene fragment comprises no nucleotide sequence encoding an IgM J chain and the cell produces hexamer IgM with a content of 50% or more.

20. The tramsformed cell of claim 19, which produces hexamer IgM with a content of 80% or more.

21. The tramsformed cell of claim 14, wherein the vector or gene fragment comprises a nucleotide sequence encoding an IgM J chain and the cell produces IgM for which the ratio of the produced pentamer and hexamer (pentamer/hexamer ratio) is 1.5 or more.

22. The tramsformed cell of claim 14, wherein the vector or gene fragment comprises no nucleotide sequence encoding an IgM J chain and the cell produces IgM for which the ratio of the produced pentamer and hexamer (pentamer/hexamer ratio) is 1.5 or more.

23. The transformed cell of claim 14, wherein the expression vector or gene fragment comprising a gene encoding IgM H and L chains comprises no nucleotide sequence encoding a J chain and the nucleotide sequence encoding the J chain has been expressively introduced by co-transfection.

66. A method for producing an IgM, comprising a step of culturing the cell of claim 14 and then collecting the IgM.

67. A method for producing a substantially pure IgM, comprising a step of purifying an IgM from a culture supernatant obtained from culture of the cell of claim 14.

15. (canceled)

18. The transformed cell of chain 17, which produces pentamer IgM with a content of 80% or more.

30. A substantially pure pentamer or hexamer IgM comprising a sugar chain added bt a CHO cell.

68. A pharmaceutical composition comprising the IgM of claim 30.

34. An isolated polynocleotide comprising:

a) the nucleotide sequence SEQ ID NO;

1 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO;

2;

b) the nucleotide sequence SEQ ID NO;

3 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO;

4;

c) the nucleotide sequence SEQ ID NO;

19 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO;

20;

d) the nucleotide sequence SEQ ID NO;

21 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO;

22.

36. An insolated protein comprising an amino acid sequence encoding by the polynucleotide of claim 34.

38. An IgM comprising one or more proteins of claim 36.

39. The IgM of claim 38, further comprising an IgM J chain.

40. The IgM of claim 39, which is a pentamer.

69. A pharmaceutical composition comprising the IgM of claim 38.

35. (canceled)

37. (canceled)

41-47. -47. (canceled)

49. A pharmaceutical composition comprising 80% or more pentamer IgM.

50. A pharmaceutical composition comprising 50% or more hexamer IgM.

51. The pharmaceutical composition of claim 50, comprising 80% or more hexamer IgM.

52. A pharmaceutical composition comprising an IgM for which pentamer/hexamer ratio is 1.5 or more.

53. A pharmaceutical composition comprising an IgM for which hexamer/pentamer ratio is 1.5 or more.

54. A method for analyzing an IgM polymer, comprsing a step of separating an IgM by SDS-polyacrylamide gel electrophoresis using as a carrier polyacrylamide gel satisfying at least one condition selected from the group consisting of:

a) a polyacrylamide gel polymerized at a high temperature;

b) a polyacrylamide gel containing a high concentration of ammonium persulfate and glycerol; and

c) a polyacrylamide gel homogenized by stirring and degassed prior to polymerization.

55. The method of claim 54, wherein the temperature in condition a) is 37°

C. or higher.

56. The method of claim 54, wherein the concentration of ammonium persulfate in condition b) is 0.25% or more.

57. The method of claim 54, wherein the polyacrylamide gel satisfies at lease two conditions selected from the group consisting of conditions a) to c).

58. The method of claim 54, wherein the polyacrylamide gel satisfies all the conditions a) to c).

59. The method of claim 54, wherein a buffer for electrophoresis is a Tris-acetate SDS electrophoresis buffer.

60. The method of claim 54, wherein the IgM polymer is an IgM pentamer and/or hexamer.

61. The method of claim 54, wherein the method comprises analyzing an IgM aggregate.

62. The method of claim 54, wherein the method is free from use of RI.

63. The method of claim 54, comprising a step of quantifying the IgM polymer separated after electrophoresis.

64. An electrophoresis gel for separating an IgM polymer by SDS-polyacrylamide gel electrophoresis, comprising a polyacrylamide gel satisfying at least one condition selected from the group consisting of:

a) a polyacrylamide gel polymerized at a high temperature;

b) a polyacrylamide gel containing a high concentration of ammonium persulfate and glycerol; and

c) a polyacrylamide gel homogenized by stirring and degassed prior to polymerization.

65. A method for producing an electrophoresis gel for sepersting an IgM polymer by SDS-polyacrylamide gel electrophoresis, comprising at least one step selected from the group consisting of:

a) polymerizing an acrylamide at a high temperture;

b) adding a high concentration of ammonium persulfate to an acrylamide, and

c) homogenizing an acrylamide by stirring and degassed prior to polymerization.