Production of tetravalent antibodies

• US 6,897,044 B1
• Filed: 01/28/1999
• Issued: 05/24/2005
• Est. Priority Date: 01/28/1999
• Status: Expired due to Fees

• Current Assignee: Biogen, Inc.
• Original Assignee: Biogen, Inc.
• Inventors: LaBarre, Michael J, Huynh, Tri B, Hanna, Nabil, Hariharan, Kandasamy, Braslawsky, Gary R
• Primary Examiner(s): HELMS, LARRY RONALD
• Application Number:
  US09/238,741
• Time in Patent Office: 2,308 Days
• Field of Search: 530/387.1, 530/387.3, 530/383.8, 530/388.85, 435/69.2, 435/69.6, 536/23.1, 536/23.53
• US Class Current: 435/69.6
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 47/6835 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/02 Nasal agents, e.g. decongestants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: C07K 16/2851 against the lectin superfamily, e.g. CD23, CD72
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/734 Complement-dependent cytotoxicity [CDC]

Abstract

The present invention relates to a novel process for the preparation of biologically active antibody dimers in a pharmaceutically acceptable composition. The dimers can be composed of two antibody molecules having the same antigen binding specificity and linked through reducible, disulfide, or a non-reducible thioether, bond (homodimer). Alternatively, the dimers can be composed of two different antibody molecules having binding specificity for two distinct antigens (heterodimer). These dimers are useful for inducing hyper-cross-linking of membrane antigens. The present invention further relates to the use of biologically active antibody dimers for the preferential killing or inhibition of selected cell populations in the treatment of diseases such as cancer and autoimmune disorders.

Claims

1. A method for producing an antibody heterodimer comprising an anti-CD20 antibody and an anti-CD23 antibody, comprising:

(i) obtaining or constructing a DNA molecule that encodes an antibody molecule heavy chain that has binding specificity to a CD20 or CD23 antigen when said heavy chain is paired with a corresponding light chain, and introducing at least one cysteine codon into said antibody molecule heavy chain via recombinant DNA mutagenesis;

(ii) expressing said DNA molecule in a suitable host cell, or expression system, together with a DNA molecule that encodes an antibody molecule light chain having the same specificity as the heavy chain, to produce a first anti-CD20 or anti-CD23 antibody molecule containing said introduced cysteine residue;

(iii) purifying said first antibody molecule from said host cell or expression system;

(iv) contacting said purified antibody molecule with an amount of a suitable reducing agent sufficient to partially reduce the intra or inter molecular disulfide bonds of said antibody molecule and thereby enhance dimerization of said first antibody molecule with a second antibody molecule; and

(v) contacting said purified first antibody molecule with a second antibody molecule that has a binding specificity to CD20 when said first antibody is an anti-CD23 antibody, or to CD23 when said first antibody is an anti-CD20 antibody, and allowing sufficient time for dimerization to proceed to thereby to produce a tetravalent antibody heterodimer comprising both an anti-CD20 antibody and an anti-CD23 antibody, wherein following dimerization, the anti-CD20 antibody of the heterodimer has binding specificity to CD20 and the anti-CD23 antibody of the heterodimer has binding specificity to CD23.

2. A method according to claim 1, wherein said first and second antibodies are both IgG antibodies.

3. A method according to claim 2, wherein said first and second antibodies are both IgG-1 antibodies.

4. A method according to claim 1, wherein the anti-CD20 antibody has binding specificity to a human CD20 antigen and the anti-CD23 antibody has binding specificity to a human CD23 antigen.

5. A method according to claim 4, wherein the anti-human CD20 antibody is a chimeric antibody consisting of light and heavy chains of a human IgG antibody in which the variable regions are replaced with the light and heavy chain variable regions of murine C2B8 antibody, and

the anti-human CD23 antibody is a chimeric antibody consisting of light and heavy chains of a human IgG antibody in which the variable regions are replaced with the light and heavy chain variable regions of monkey 5E8 antibody.

6. A method according to claim 5, wherein said first and second antibodies are both IgG-1 antibodies.

7. A method according to claim 1, wherein the location of said cysteine molecule is such that it prevents or inhibits formation of an intramolecular disulfide bridge between sister heavy chains on the same antibody molecule.

8. A method according to claim 1, wherein step (iv) further comprises terminating the reducing reaction by the addition of cysteine blocking reagent.

9. A method according to claim 1, wherein step (v) comprises cross-linking the reduced antibody molecules using a BIS-maleimido cross-linker.

10. A method according to claim 1, wherein the second antibody contains a thiol reactive group other than a cysteine group introduced therein.

11. The method of claim 10, wherein the thiol reactive group is selected from the group consisting of a maleimido group, a dithiopyridal group, and a reactive thiol.

12. A method of claim 10, wherein step (iv) further comprises terminating the reducing reaction by the addition of thiol blocking reagent.

13. A tetravalent antibody heterodimer produced by the method of claim 1.

14. An antibody heterodimer according to claim 13, wherein said first and second antibodies are both IgG antibodies.

15. An antibody heterodimer according to claim 14, wherein said first and second antibodies are both IgG-1 antibodies.

16. An antibody heterodimer produced by the method of claim 5.

17. An antibody heterodimer according to claim 16, wherein said first and second antibodies are both IgG-1 antibodies.

18. A tetravalent antibody heterodimer comprising a first antibody that has binding specificity to a CD20 antigen and a second antibody that has binding specificity to a CD23 antigen.

19. The antibody heterodimer of claim 18, wherein said first or said second antibody comprises an antibody heavy chain polypeptide that is mutated by introduction of a cysteine residue.

20. The antibody heterodimer of claim 18, wherein said first and second antibodies are both IgG antibodies.

21. The antibody heterodimer of claim 20, wherein said first and second antibodies are both IgG-1 antibodies.

22. The antibody heterodimer of claim 18, wherein said first and second antibodies are monoclonal antibodies.

23. The antibody heterodimer of claim 18, wherein the anti-CD20 antibody has binding specificity to a human CD20 antigen and the anti-CD23 antibody has binding specificity to a human CD23 antigen.

24. The antibody heterodimer of claim 23,

wherein the anti-human CD20 antibody is a chimeric antibody consisting of light and heavy chains of a human IgG antibody in which the variable regions are replaced with the light and heavy chain variable regions of murine C2B8 antibody, and

the anti-human CD23 antibody is a chimeric antibody consisting of light and heavy chains of a human IgG antibody in which the variable regions are replaced with the light and heavy chain variable regions of monkey 5E8 antibody.

25. The antibody heterodimer of claim 24, wherein said first and second antibodies are both IgG-1 antibodies.

26. The antibody heterodimer of claim 24, wherein said first and second antibodies are monoclonal antibodies.

27. The antibody heterodimer of claim 24, which activates components of the complement system.

28. The antibody heterodimer of claim 24, which promotes killing of cells by the complement cascade.

29. The antibody heterodimer of claim 24, which binds to Fcγ

receptors on cytotoxic effector cells.

30. The antibody heterodimer of claim 24, which binds to Fcγ

receptors on host immune cells.

31. The antibody heterodimer of claim 24, which induces B lymphoma cells to initiate apoptosis.

32. The antibody heterodimer of claim 24, which induces initiation of apoptosis of leukemic cells of a patient with chronic lymphocytic leukemia.