Expression technology for proteins containing a hybrid isotype antibody moiety

• US 20030044423A1
• Filed: 03/07/2002
• Published: 03/06/2003
• Est. Priority Date: 03/07/2001
• Status: Active Grant

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Lexigen Pharmaceuticals Corporation
• Inventors: Lo, King-Ming, Gillies, Stephen D., Way, Jeffrey
• Granted Patent: US 7,148,321 B2
• Time in Patent Office: Days
• US Class Current: 424/192.1
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/53 Hinge
• 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

Disclosed are methods and compositions for efficiently expressing antibody fusion proteins. Antibody fusion proteins of the invention include a hybrid antibody moiety containing sequences from more than one type of antibody and/or mutant antibody sequences. Hybrid antibody fusion proteins of the invention may be produced at high levels and may combine functional properties characteristic of different antibody types in addition to functional properties of a non-antibody moiety.

Claims

1. A fusion protein comprising an immunoglobulin moiety fused to a non-immunoglobulin moiety, wherein said immunoglobulin moiety comprises a first domain from a first antibody isotype and a second domain from a second antibody isotype.

2. A fusion protein of claim 1, wherein said immunoglobulin region comprises a hinge region from a first antibody isotype and a constant region from a second antibody isotype.

3. The fusion protein of claim 2, wherein said constant region is a CH2 domain.

4. The fusion protein of claim 3, wherein said CH2 domain is from a human IgG2 heavy chain, and said hinge region contains three or fewer cysteine residues.

5. The fusion protein of claim 3, wherein said CH2 domain is from a human IgG4 heavy chain.

6. The fusion protein of claim 2, wherein the hinge region is a non-IgG2 hinge region.

7. The fusion protein of claim 2, wherein the hinge region is derived from human IGG1.

8. The fusion protein of claim 3-6, wherein the hinge region has two or fewer cysteines.

9. The fusion protein of claim 1, wherein said immunoglobulin domain lacks an antigen-binding site.

10. The fusion protein of claim 1, wherein said immunoglobulin domain comprises an antigen-binding site.

11. The fusion protein of claim 1, wherein the non-Ig moiety is fused by genetic engineering to the C-terminus of a heavy chain of the Ig domain.

12. The fusion protein of claim 1, wherein the non-Ig moiety is fused by genetic engineering to the N-terminus of a heavy chain of the Ig domain.

13. The fusion protein of claim 1, wherein the non-Ig moiety is fused to a light chain polypeptide.

14. The fusion protein of claim 2, wherein the hinge region is from a human IgA.

15. The fusion protein of claim 1, wherein said first immunoglobulin domain is a CH2 domain and said second immunoglobulin domain is a CH3 domain.

16. The fusion protein of claim 15, wherein the first antibody isotype is an IgG and the second antibody isotype is an IgM or an IgA.

20. A nucleic acid encoding a fusion protein of claim 1, 17, or 18.

21. A stably transfected cell expressing a protein of claim 1, 17, or 18.

24. The fusion protein of claim 1, wherein the immunoglobulin moeity comprises an Fc heavy chain region and the non-immunoglobulin moeity comprises an erythropoietin molecule.

25. A nucleic acid encoding the fusion protein of claim 24.

26. The fusion protein of claim 1, wherein the non-immunoglobulin moeity comprises an erythropoietin molecule.

17. An antibody or Ig fusion protein comprising an IgG2 antibody moiety in which the IgG2 hinge has been mutated two contain two cysteines.

18. An immunoglobulin moiety comprising a V domain directed against a tumor-associated antigen, said immunoglobulin moiety comprising a first domain from a first antibody isotype and a second domain from a second antibody isotype.

19. The protein of claim 18, comprising an inactive Fc receptor I binding site and a hinge region with three or fewer cysteines.

22. A method for improving the assembly of an antibody or Ig fusion protein, the method comprising the steps of:

a. replacing the hinge region of said protein with a different hinge region; and

,

b. testing the assembly of the altered protein.

23. The method of claim 22, wherein said protein and said altered protein are produced by genetic engineering techniques.

27. A method for improving the production of an antibody or Ig fusion protein from a cell, the method comprising the steps of:

a. replacing the hinge region of said protein with a different hinge region; and

,

b. testing the production of the altered protein.

28. The method of claim 27, wherein said protein and said altered protein are produced by genetic engineering techniques.