Readily Isolated Bispecific Antibodies with Native Immunoglobulin Format

• US 20100331527A1
• Filed: 06/25/2010
• Published: 12/30/2010
• Est. Priority Date: 06/26/2009
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: OLSON, KARA LOUISE, SMITH, ERIC, DAVIS, SAMUEL, MACDONALD, DOUGLAS
• Granted Patent: US 8,586,713 B2
• Time in Patent Office: Days
• US Class Current: 530/387.3
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C07K 16/247 IL-4
• CPC Class Codes: C07K 16/248 IL-6
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• 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 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Abstract

A bispecific antibody format providing ease of isolation is provided, comprising immunoglobulin heavy chain variable domains that are differentially modified in the CH3 domain, wherein the differential modifications are non-immunogenic or substantially non-immunogenic with respect to the CH3 modifications, and at least one of the modifications results in a differential affinity for the bispecific antibody for an affinity reagent such as Protein A, and the bispecific antibody is isolable from a disrupted cell, from medium, or from a mixture of antibodies based on its affinity for Protein A.

Claims

1. An heterodimeric bispecific antigen-binding protein, comprising:

a. a first polypeptide comprising, from N-terminal to C-terminal, a first epitope-binding region that selectively binds a first epitope, an immunoglobulin constant region that comprises a first CH3 region of a human IgG selected from IgG1, IgG2, and IgG4; and

,

b. a second polypeptide comprising, from N-terminal to C-terminal, a second epitope-binding region that selectively binds a second epitope, an immunoglobulin constant region that comprises a second CH3 region of a human IgG selected from IgG1, IgG2, and IgG4, wherein the second CH3 region comprises a modification that reduces or eliminates binding of the second CH3 domain to Protein A.

2. The bispecific protein of claim 1, wherein the first polypeptide and the second polypeptide are human IgG heavy chains.

3. The bispecific protein of claim 1, further comprising an immunoglobulin light chain.

4. The bispecific protein of claim 3, wherein the immunoglobulin light chain is a human immunoglobulin light chain.

5. The bispecific protein of claim 1, wherein the first and the second polypeptides are each human IgG1 heavy chains.

6. The bispecific protein of claim 1, wherein the modification is selected from the group consisting of (a) 95R, and (b) 95R and 96F in the IMGT exon numbering system, or (a′

) 435R, and (b′

) 435R and 436F in the EU numbering system.

7. The bispecific protein of claim 6, further comprising one to five modifications selected from the group consisting of 16E, 18M, 44S, 52N, 57M, and 82I in the IMGT exon numbering system, or 356E, 358M, 384S, 392N, 397M, and 422I in the EU numbering system.

8. The bispecific protein of claim 6, wherein the CH3 domain of the bispecific antibody is non-immunogenic or substantially non-immunogenic in a human.

9. The bispecific protein of claim 7, wherein the CH3 domain of the bispecific antibody is non-immunogenic or substantially non-immunogenic in a human.

10. A method for making a bispecific antibody, comprising:

a. obtaining a nucleic acid sequence encoding a first immunoglobulin heavy chain comprising a first variable domain that recognizes a first epitope, wherein the first immunoglobulin heavy chain comprises an IgG1, IgG2, or IgG4 isotype constant domain;

b. obtaining a second nucleic acid sequence encoding a second immunoglobulin heavy chain comprising a second variable domain that recognizes a second epitope, wherein the second immunoglobulin heavy chain comprises an IgG1, IgG2, or IgG4 isotype constant domain that comprises a modification in its CH3 domain that eradicates or reduces binding to Protein A;

c. obtaining a third nucleic acid sequence encoding an immunoglobulin a light chain that pairs with the first and the second immunoglobulin heavy chain;

d. introducing the first, second, and third nucleic acid sequences into a mammalian cell;

e. allowing the cell to express a bispecific antibody; and

,

f. isolating the bispecific antibody based on the ability of the bispecific antibody to bind Protein A.

11. The method of claim 10, wherein the modification is selected from the group consisting of (a) 95R, and (b) 95R and 96F in the IMGT exon numbering system, or (a′

) 435R, and (b′

) 435R and 436F in the EU numbering system.

12. The method of claim 11, further comprising one to five modifications selected from the group consisting of 16E, 18M, 44S, 52N, 57M, and 82I in the IMGT exon numbering system, or 356E, 358M, 384S, 392N, 397M, and 422I in the EU numbering system.

13. The method of claim 11, wherein the CH3 domain of the bispecific antibody is non-immunogenic or substantially non-immunogenic in a human.

14. The method of claim 12, wherein the CH3 domain of the bispecific antibody is non-immunogenic or substantially non-immunogenic in a human.

15. The method of claim 10, where the bispecific antibody is isolated on a solid support comprising Protein A.

16. The method of claim 15, wherein the solid support comprises a Protein A affinity column, and the bispecific antibody is isolated employing a pH gradient.

17. The method of claim 16, wherein the pH gradient is a step gradient comprising one or more pH steps between pH 3 and pH 5.

18. A method for isolating a bispecific antibody, comprising:

isolating from a disrupted cell or a mixture of antibodies a bispecific antibody having differentially modified IgG1, IgG2, or IgG4 CH3 domains, wherein the differentially modified CH3 domains are non-immunogenic or substantially non-immunogenic in a human, and wherein the modification results in a bispecific antibody with a heterodimeric heavy chain constant region whose monomers have a differential affinity for Protein A, and the bispecific antibody is isolated from the disrupted cell or the mixture based on its affinity for Protein A.

19. The method of claim 18, wherein one monomer of the heterodimeric heavy chain constant region is a human IgG1, and the other monomer of the heterodimeric heavy chain constant region is a modified human IgG1 comprising a modification selected from the group consisting of (a) H95R, and (b) H95R and Y96F in the IMGT exon numbering system, or (a′

) H435R, and (b′

) H435R and Y436F in the EU numbering system.

20. The method of claim 19, wherein the modified human IgG1 further comprises a modification selected from the group consisting of D16E, L18M, N44S, K52N, V57M, and V82I in the IMGT exon numbering system, or D356E, L358M, N384S, K392N, V397M, and V422I in the EU numbering system.