IgM Production by Transformed Cell and Method of Quantifying the Same

• US 20100172899A1
• Filed: 03/18/2010
• Published: 07/08/2010
• 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: Tsunoda, Hiroyuki, Tsuchiya, Masayuki, Igawa, Tomoyuki, Sekimori, Yasuo, Irie, Reiko
• Granted Patent: US 8,257,703 B2
• Time in Patent Office: Days
• US Class Current: 424/130.100
• 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 substantially pure IgM obtained by a method comprising

culturing transformed cells so that they produce an IgM at a level of at least 100 mg/L or at a level of at least 35 pg/cell/day;

and

collecting the IgM from the culture or a supernatant of the culture.

3. The substantially pure IgM of claim 1, wherein the IgM is a human, mouse, human chimeric, or humanized antibody.

4. The substantially pure IgM of claim 1, which is in the form of a substantially pure pentamer or hexamer.

6. The substantially pure IgM of claim 1, wherein the IgM is an anti-sugar chain antibody.

7. The substantially pure IgM of claim 6, wherein the IgM is an anti-ganglioside antibody.

8. The substantially pure IgM of claim 7, wherein the IgM is an anti-GM2 or GM3 antibody.

20. A pharmaceutical composition comprising the substantially purified IgM of claim 1.

2. A substantially pure IgM obtained by purifying an IgM from a culture supernatant obtained from culturing transformed cells producing the IgM at a level of at least 100 mg/L or at a level of at least 35 pg/cell/day.

5. A substantially pure pentamer or hexamer IgM comprising a sugar chain added by a CHO cell.

9. The substantially pure IgM of claim 5, wherein the IgM is an anti-sugar chain antibody.

10. The substantially pure IgM of claim 9, wherein the IgM is an anti-ganglioside antibody.

11. The substantially pure IgM of claim 10, wherein the IgM is an anti-GM2 or GM3 antibody.

26. A pharmaceutical composition comprising the substantially purified IgM of claim 5.

12. An isolated protein comprising:

a) the amino acid sequence of SEQ ID NO;

2;

b) the amino acid sequence of SEQ ID NO;

4;

c) the amino acid sequence of SEQ ID NO;

20;

or

d) the amino acid sequence of SEQ ID NO;

22.

13. The isolated protein of claim 12, wherein the protein comprises the amino acid sequence of SEQ ID NO:

2.

14. The isolated protein of claim 12, wherein the protein comprises the amino acid sequence of SEQ ID NO:

4.

15. The isolated protein of claim 12, wherein the protein comprises the amino acid sequence of SEQ ID NO:

20.

16. The isolated protein of claim 12, wherein the protein comprises the amino acid sequence of SEQ ID NO:

22.

17. An IgM comprising the amino acid sequence of one or more of SEQ ID NOs:

2, 4, 20 and 22.

18. The IgM of claim 17, further comprising an IgM J chain.

19. The IgM of claim 17, which is a pentamer.

27. A pharmaceutical composition comprising the IgM of claim 17.

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

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

23. The pharmaceutical composition of claim 22, comprising 80% or more hexamer IgM.

24. A pharmaceutical composition comprising an IgM having a pentamer/hexamer ratio of at least 1.5.

25. A pharmaceutical composition comprising an IgM having a hexamer/pentamer ratio of at least 1.5.