Knobs and holes heteromeric polypeptides

• US 8,216,805 B2
• Filed: 02/04/2010
• Issued: 07/10/2012
• 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., Presta, Leonard G., Ridgway, John B.
• Primary Examiner(s): Stucker, Jeffrey
• Assistant Examiner(s): Gucker, Stephen
• Application Number:
  US12/700,618
• Publication Number:
  US 20100254986A1
• Time in Patent Office: 887 Days
• Field of Search: None
• 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 each comprising a C_H3 antibody constant domain, wherein said polypeptides meet at an engineered interface within the C_H3 domain, the method comprising the steps of:

(a) creating an engineered protuberance in the interface of the first polypeptide by replacing at least one contact residue of said polypeptide within its C_H3 domain;

(b) creating, an engineered cavity in the interface of the second polypeptide by replacing at least one contact residue of said polypeptide within its C_H3 domain, wherein said protuberance in the first polypeptide is positional in said cavity of the second polypeptide so as to form a protuberance-into-cavity mutant pair, and wherein the contact residue to be replaced on the first polypeptide corresponds to an IgG residue selected from the group consisting of amino acid residues 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407 and 409, according to the amino acid numbering as shown in FIG. 5;

(c) expressing said first and second polypeptides in a host cell to obtain the heteromultimer;

(d) recovering the heteromultimer from the host cell culture;

wherein said creating an engineered protuberance comprises replacing the nucleic acid encoding the original residue with nucleic acid encoding an import residue having a larger side chain volume than the original residue; and

wherein said creating an engineered cavity comprises replacing the nucleic acid encoding an original residue with nucleic acid encoding an import residue having a smaller side chain volume than the original residue.

2. The method of claim 1, wherein the contact residue to be replaced on the first polypeptide is selected from the group consisting of amino acid residues 366, 368, 370, 405, 407 and 409, according to the amino acid numbering as shown in FIG. 5.

3. The method of claim 1, wherein said engineered interface within the C_H3 domain comprises at least two protuberance-into-cavity mutant pairs created by repeating steps (a) and (b).

4. The method of claim 3, wherein said at least two protuberance-into-cavity mutant pairs are created by creating at least one protuberance and at least one cavity on the first polypeptide and creating at least one cavity and at least one protuberance on the second polypeptide.

5. The method of claim 3, wherein said at least two protuberance-into-cavity mutant pairs are created by creating more than one protuberance on the first polypeptide and creating more than one cavity on the second polypeptide.

6. The method of claim 1, wherein the C_H3 antibody constant domain is from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

7. The method of claim 6, wherein the immunoglobulin is IgG.

8. The method of claim 7, wherein the IgG is of a subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3 and IgG4.

9. The method of claim 7, wherein the IgG is a human IgG.

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

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

12. The method of claim 1, wherein the heteromultimer is an antibody-immunoadhesin chimera (Ab/Ia).

13. The method of claim 12, wherein the Ab/Ia is anti-CD3/CD4-IgG.

14. An isolated heteromultimer prepared by the method of claim 1.

15. An isolated heteromultimer comprising a first polypeptide and a second polypeptide each comprising a C_H3 antibody constant domain, said heteromultimer having, the following characteristics:

(a) said first and second polypeptides meet at an engineered interface within the C_H3 domain;

(b) the first polypeptide comprises at least one engineered protuberance in said interface, said protuberance comprising at least one altered contact residue;

(c) the second polypeptide comprises at least one engineered cavity in its said interface, said cavity comprising at least one altered contact residue;

(d) said protuberance is positional in said cavity so as to form a protuberance-into-cavity mutant pair; and

(e) said at least one altered contact residue of the first polypeptide corresponds to an IgG residue selected from the group consisting of amino acid residues 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407 and 409, according to the amino acid numbering as, shown in FIG. 5.

16. The heteromultimer of claim 15, wherein said protuberance comprises the replacement of the original contact residue with an import residue having a larger side chain volume.

17. The heteromultimer of claim 15, wherein said cavity comprises the replacement of the original contact residue with an import residue having a smaller side chain volume.

18. The heteromultimer of claim 15, wherein the altered contact residue is selected from the group consisting of amino acid residues 366, 368, 370, 405, 407 and 409, according to the amino acid numbering as shown in FIG. 5.

19. The heteromultimer of claim 15, wherein said engineered interface within the C_H3 domain comprises at least two protuberance-into-cavity mutant pairs between the first and second polypeptides.

20. The heteromultimer of claim 19, wherein said at least two protuberance-into-cavity mutant pairs comprise at least one protuberance and at least one cavity on the first polypeptide and at least one cavity and at least one protuberance on the second polypeptide.

21. The heteromultimer of claim 19, wherein said at least two protuberance-into-cavity mutant pairs comprise more than one protuberance on the first polypeptide and more than one cavity on the second polypeptide.

22. The heteromultimer of claim 15, wherein the C_H3 antibody constant domain is from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

23. The heteromultimer of claim 22, wherein the immunoglobulin is IgG.

24. The heteromultimer of claim 23, wherein the IgG is of a subtype selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3 and IgG4.

25. The heteromultimer of claim 23, wherein the IgG is a human IgG.

26. The heteromultimer of claim 15, wherein the heteromultimer is a bispecific antibody.

27. The heteromultimer of claim 15, wherein the heteromultimer is a bispecific immunoadhesin.

28. The heteromultimer of claim 15, wherein the heteromultimer is an antibody-immunoadhesin chimera (Ab/Ia).

29. The heteromultimer of claim 28, wherein the Ab/Ia is anti-CD3/CD4-IgG.

30. A composition comprising the heteromultimer of claim 15 and a pharmaceutically acceptable carrier.

31. A host cell comprising nucleic acid encoding the heteromultimer of claim 15.

32. The host cell of claim 31, wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are present in a single vector.

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

34. A method of making a heteromultimer comprising culturing the host cell of claim 31 so that the nucleic acid is expressed and recovering the heteromultimer from the cell culture.

35. The method of claim 34, wherein the host cell is a mammalian cell.

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