Methods For Making Fully Human Bispecific Antibodies Using A Common Light Chain

• US 20130045492A1
• Filed: 06/05/2012
• Published: 02/21/2013
• Est. Priority Date: 02/08/2010
• Status: Abandoned Application

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Babb, Robert, McWhirter, John, MacDonald, Lynn, Stevens, Sean, Davis, Samuel, Buckler, David R., Hosiawa, Karolina A., Murphy, Andrew J.
• Application Number:
  US13/488,628
• Publication Number:
  US 20130045492A1
• Time in Patent Office: Days
• US Class Current: 435/7.92
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/15 Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• 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 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• 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
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/204 : of bacterial origin, e.g. BAC

Abstract

A genetically modified mouse is provided, wherein the mouse expresses an immunoglobulin light chain repertoire characterized by a limited number of light chain variable domains. Mice are provided that express just one or a few immunoglobulin light chain variable domains from a limited repertoire in their germline. Methods for making bispecific antibodies having universal light chains using mice as described herein, including human light chain variable regions, are provided. Methods for making human variable regions suitable for use in multispecific binding proteins, e.g., bispecific antibodies, and host cells are provided. Bispecific antibodies capable of binding first and second antigens are provided, wherein the first and second antigens are separate epitopes of a single protein or separate epitopes on two different proteins are provided.

Claims

1. A method for making a bispecific antigen-binding protein, comprising

exposing a first mouse that expresses a single human immunoglobulin light chain to a first antigen of interest that comprises a first epitope,

exposing a second mouse that expresses a single human immunoglobulin light chain to a second antigen of interest that comprises a second epitope,

allowing the first and the second mouse to each mount immune responses to the antigens of interest,

identifying in the first mouse a first human heavy chain variable region that binds the first epitope of the first antigen of interest,

identifying in the second mouse a second human heavy chain variable region that binds the second epitope of the second antigen of interest,

making a first fully human heavy chain gene that encodes a first heavy chain that binds the first epitope of the first antigen of interest,

making a second fully human heavy chain gene that encodes a second heavy chain that binds the second epitope of the second antigen of interest,

expressing the first heavy chain and the second heavy chain in a cell that expresses a single fully human light chain to form a bispecific antigen-binding protein, and

isolating the bispecific antigen-binding protein.

2. The method of claim 1, wherein the single fully human light chain is derived from a rearranged human Vκ

1-39 or a human Vκ

3-20 gene segment.

3. The method of claim 1, wherein

(a) the first antigen and the second antigen are not identical;

or

(b) the first antigen and the second antigen are identical, and the first epitope and the second epitope are not identical.

4. The method of claim 1, wherein the human light chain when paired with the first heavy chain specifically binds the first epitope of the first antigen and when paired with the second heavy chain specifically binds the second epitope of the second antigen.

5. The method of claim 1, wherein the first fully human heavy chain gene bears an amino acid modification that reduces its affinity to protein A, and the second fully human heavy chain does not comprise a modification that reduces its affinity to protein A.

6. The method of claim 5, wherein the modification is selected from a 95R (EUR 435R), 96F (EUR 436F), and a combination thereof.

7. The method of claim 1, wherein the human light chain of the cell comprises a germline sequence.

8. A method for making a human bispecific antibody comprising a step of employing in the bispecific antibody two human heavy chain variable region sequences of two different B cells a mouse that expresses a single human light chain variable domain.

9. The method of claim 8, wherein the single human light chain variable domain comprises a rearranged human Vκ

1-39 or human Vκ

3-20 gene segment.

10. The method of claim 8, wherein in the single human light chain variable domain further comprises a rearranged human Jκ

5 or human Jκ

1 gene segment.

11. The method of claim 8, wherein the mouse further comprises a humanized heavy chain locus containing one or more unrearranged human V_H gene segments, one or more unrearranged human D_H gene segments, and one or more unrearranged human J_H gene segments, operably linked to one or more non-human heavy chain constant region genes.

12. The method of claim 11, wherein the humanized heavy chain locus comprises 80 unrearranged human V_H gene segments, 27 unrearranged human D_H gene segments and six unrearranged human J_H gene segments operably linked to one or more mouse heavy chain constant region genes.

13. The method of claim 12, wherein the one or more human V_H gene segments comprises human V_H1-2, V_H1-8, V_H1-24, V_H1-69, V_H2-5, V_H3-7, V_H3-9, V_H3-11, V_H3-13, V_H3-15, V_H3-20, V_H3-23, V_H3-30, V_H3-33, V_H3-48, V_H3-53, V_H4-31, V_H4-39, V_H4-59, V_H5-51, V_H6-1, or a combination thereof.

14. The method of claim 8, wherein the mouse does not contain an endogenous light chain variable gene segment that is capable of rearranging to form an immunoglobulin light chain.

15. A method of selecting two human immunoglobulin heavy chain variable domains for use in a bispecific antibody comprising immunizing a mouse with an antigen of interest, wherein the mouse expresses a single human light chain variable domain, and wherein the two human immunoglobulin heavy chain variable domains independently associate with the single human light chain variable domain to bind the antigen of interest.

16. The method of claim 15, wherein the single human light chain variable domain is derived from a rearranged human Vκ

1-39 gene segment.

17. The method of claim 16, wherein the single human light chain variable domain further comprises a rearranged human Jκ

5 gene segment.

18. The method of claim 15, wherein the single human light chain variable domain is derived from a rearranged human Vκ

3-20 gene segment.

19. The method of claim 18, wherein the single human light chain variable domain further comprises a rearranged human Jκ

1 gene segment.

20. The method of claim 15, wherein the mouse does not contain an endogenous light chain variable gene segment that is capable of rearranging to form an immunoglobulin light chain.