Method for making heteromultimeric polypeptides

• US 20020155537A1
• Filed: 12/07/2001
• Published: 10/24/2002
• Est. Priority Date: 03/01/1995
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Presta, Leonard G., Carter, Paul J., Ridgway, John B.
• Granted Patent: US 7,642,228 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• 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. A method of preparing a heteromultimer comprising a first polypeptide and a second polypeptide which meet at an interface, wherein the interface of the first polypeptide comprises a protuberance which is positionable in a cavity in the interface of the second polypeptide, the method comprising the steps of:

(a) culturing a host cell comprising nucleic acid encoding the first polypeptide and second polypeptide, wherein the nucleic acid encoding the first polypeptide has been altered from the original nucleic acid to encode the protuberance or the nucleic acid encoding the second polypeptide has been altered from the original nucleic acid to encode the cavity, or both, and wherein the culturing is such that the nucleic acid is expressed; and

(b) recovering the heteromultimer from the host cell culture.

2. The method of claim 1 wherein the nucleic acid encoding the first polypeptide has been altered from the original nucleic acid to encode the protuberance and the nucleic acid encoding the second polypeptide has been altered from the original nucleic acid to encode the cavity.

3. The method of claim 1 wherein step (a) is preceded by a step wherein nucleic acid encoding an original residue from the interface of the first polypeptide is replaced with nucleic acid encoding an import residue having a larger side chain volume than the original residue.

4. The method of claim 3 wherein the import residue is arginine (R).

5. The method of claim 3 wherein the import residue is phenylalanine (F).

6. The method of claim 3 wherein the import residue is tyrosine (Y).

7. The method of claim 3 wherein the import residue is tryptophan (W).

8. The method of claim 1 wherein step (a) is preceded by a step wherein nucleic acid encoding an original residue in the interface of the second polypeptide is replaced with nucleic acid encoding an import residue having a smaller side chain volume than the original residue.

9. The method of claim 8 wherein the import residue is not cysteine (C).

10. The method of claim 8 wherein the import residue is alanine (A).

11. The method of claim 8 wherein the import residue is serine (S).

12. The method of claim 8 wherein the import residue is threonine (T).

13. The method of claim 8 wherein the import residue is valine (V).

14. The method of claim 1 wherein the first and second polypeptide each comprise an antibody constant domain.

15. The method of claim 14 wherein the antibody constant domain is a C_H3 domain.

16. The method of claim 15 wherein the antibody constant domain is from an IgG.

17. The method of claim 16 wherein the IgG is hum an IgG₁.

18. The method of claim 1 wherein the heteromultimer is a bispecific antibody.

19. The method of claim 1 wherein the heteromultimer is a bispecific immunoadhesin.

20. The method of claim 1 wherein the heteromultimer is an antibody-immunoadhesin chimera.

21. The method of claim 3 wherein one original residue from the first polypeptide has been replaced with an import residue.

22. The method of claim 8 wherein one original residue from the second polypeptide has been replaced with an import residue.

23. The method of claim 1 wherein step (a) is preceded by a step wherein the nucleic acid encoding the first and second polypeptide is introduced into the host cell.

24. A heteromultimer prepared by the method of claim 1.

26. The heteromultimer of claim 25 wherein the protuberance and cavity have been introduced into the interface of the first and second polypeptides respectively.

27. The heteromultimer of claim 26 wherein the protuberance and cavity each comprise a naturally occurring amino acid residue.

28. A composition comprising the heteromultimer of claim 25 and a pharmaceutically acceptable carrier.

29. A host cell comprising nucleic acid encoding the heteromultimer of claim 25.

30. The host cell of claim 29 wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are present in a single vector.

31. The host cell of claim 29 wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are present in separate vectors.

32. A method of making a heteromultimer comprising culturing the host cell of claim 29 so that the nucleic acid is expressed and recovering the heteromultimer from the cell culture.

33. The method of claim 32 wherein the host cell is a mammalian cell.

34. The method of claim 32 wherein the heteromultimer is recovered from the cell culture media.

36. The method of claim 35 wherein the first and second polypeptide each comprise an antibody constant domain.

37. The method of claim 35 wherein the antibody constant domain is a C_H3 domain.

38. The method of claim 37 wherein the antibody constant domain is from a human IgG.

25. A heteromultimer comprising a first polypeptide and a second polypeptide which meet at an interface, wherein the interface of the first polypeptide comprises a protuberance which is positionable in a cavity in the interface of the second polypeptide, and wherein the protuberance or cavity, or both, have been introduced into the interface of the first and second polypeptides respectively.

35. A method of preparing a heteromultimer comprising:

(a) altering a first nucleic acid encoding a first polypeptide so that an amino acid residue in the interface of the first polypeptide is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance on the first polypeptide;

(b) altering a second nucleic acid encoding a second polypeptide so that an amino acid residue in the interface of the second polypeptide is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the second polypeptide, wherein the protuberance is positionable in the cavity;

(c) introducing into a host cell the first and second nucleic acids and culturing the cell so that expression of the first and second nucleic acid occurs;

(d) recovering the heteromultimer formed from the cell culture.