Method for making heteromultimeric polypeptides

• US 7,642,228 B2
• Filed: 12/07/2001
• Issued: 01/05/2010
• Est. Priority Date: 03/01/1995
• Status: Expired due to Fees

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Carter, Paul J., Ridgway, John B., Presta, Leonard G.
• Primary Examiner(s): Stucker; Jeffrey
• Assistant Examiner(s): Gucker; Stephen
• Application Number:
  US10/010,245
• Publication Number:
  US 20020155537A1
• Time in Patent Office: 2,951 Days
• Field of Search: 435/69.1, 530/350, 514/2, 514/12
• US Class Current: 514/1.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: C07K 14/70514 CD4
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins and antibody-immunoadhesin chimeras. The invention also relates to the heteromultimers prepared using the method. Generally, the method involves introducing a protuberance at the interface of a first polypeptide and a corresponding cavity in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heteromultimer formation and hinder homomultimer formation. “Protuberances” are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). The protuberance and cavity can be made by synthetic means such as altering the nucleic acid encoding the polypeptides or by peptide synthesis.

Claims

1. An isolated heteromultimer comprising at least a first polypeptide and a second polypeptide, whereinthe first polypeptide is associated with the second polypeptide via an interface, said interface having introduced therein at least one protuberance or cavity such that:

(a) the interface of the first polypeptide comprises a protuberance that is positionable in a cavity in the iterface of the second polypeptide and/or

(b) the interface of the first polypeptide comprises a cavity that accommodates a protuberance of the second polypepetide, such that a greater ratio of heteromultimer;

homomultimer forms than for a multitimer having a wild-type interface, and

2) polypeptide interface via a CH3 constant damain of an antibody.

2. The heteromultimer of claim 1 wherein the interface comprises both (a) and (b).

3. The heteromultimer of claim 1 wherein the protuberance has been introduced into the interface.

4. The heteromultimer of claim 1 wherein the cavity has been introduced into the interface.

5. The heteromultimer of claim 3, wherein the protuberance comprises a non-naturally occurring amino acid residue.

6. The heteromultimer of claim 3, wherein the protuberance comprises a naturally occurring amino acid residue.

7. The heteromultimer of claim 6, wherein the protuberance comprises an armine (R) residue.

8. The heteromultimer of claim 6, wherein the protuberance comprises a phenylalanine (F) residue.

9. The heteromultimer of claim 6, wherein the protuberance comprises a tyrosine (Y) residue.

10. The heteromultimer of claim 6, wherein the protuberance comprises a tryptophan (W) residue.

11. The heteromultimer of claim 4, wherein the cavity comprises a non-naturally occurring amino acid residue.

12. The heteromultimer of claim 4, wherein the cavity comprises a naturally occurring amino acid residue.

13. The heteromultimer of claim 12, wherein the cavity comprises an alanine (A) residue.

14. The heteromultimer of claim 12, wherein the cavity comprises a serine (S) residue.

15. The heteromultimer of claim 12, wherein the cavity comprises a threonine (T) residue.

16. The heteromultimer of claim 12, wherein the cavity comprises a valine (V) residue.

17. The heteromultimer of claim 1, wherein the CH3 domain is from an IgG.

18. The heteromultimer of claim 17, wherein the IgG is of the IgG1 subtype.

19. The heteromultimer of claim 17, wherein the IgG is of the IgG2 subtype.

20. The heteromultimer of claim 17, wherein the IgG is of the IgG2A subtype.

21. The heteromultimer of claim 17, wherein the IgG is Of the IgG2B subtype.

22. The heteromultimer of claim 17, wherein the IgG is of the IgG3 subtype.

23. The heteromultimer of claim 17, wherein the IgG is Of the IgG4 subtype.

24. The heteromultimer of claim 1, wherein the first or second polypeptide further comprises a binding domain.

25. The heteromultimer of claim 24, wherein the binding domain is an antigen binding domain.

26. The heteromultimer of claim 24, wherein the binding domain is a ligand binding domain.

27. The heteromultimer of claim 24, wherein the binding domain is a receptor binding domain.

28. The heteromultimer of claim 24, wherein the binding domain is an enzymatic domain.

29. The heteromultimer of claim 24, wherein the binding domain is an antibody variable domain.

30. The heteromultimer of claim 1 which is a multi-specific antibody.

31. The heteromultimer of claim 30 which is a bi-specific antibody.

32. The heteromultimer of claim 30 which is a tri-specific antibody.

33. The heteromultimer of claim 1 which is an immunoadhesin.

34. The heteromultimer of claim 33 which is a multi-specific immunoadhesin.

35. The heteromultimer of claim 34 which is a bi-specific immunoadhesin.

36. The heteromultimer of claim 34 which is a heterodimer.

37. The heteromultimer of claim 34 which is a heterotrimer.

38. The heteromultimer of claim 34 which is a heterotetramer.

39. The heteromultimer of claim 1 which is an antibody-immunoadhesin chimera.

40. A composition comprising the heteromultimer of any of claims 1, 2, 17, 24, 33 and 39 and a pharmaceutically acceptable carrier.