Common Light Chain Mouse

• US 20110195454A1
• Filed: 02/08/2011
• Published: 08/11/2011
• Est. Priority Date: 02/08/2010
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: MURPHY, ANDREW J., DAVIS, SAMUEL, STEVENS, SEAN, BUCKLER, DAVID R., MCWHIRTER, JOHN, MACDONALD, LYNN
• Granted Patent: US 10,143,186 B2
• Time in Patent Office: Days
• US Class Current: 435/69.6
• 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 67/0276 Knock-out vertebrates
• 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 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• 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 2317/515 Complete light chain, i.e. VL + CL
• 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
• CPC Class Codes: C12N 5/0606 : Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/10 : Cells modified by introduction of foreign genetic material

Abstract

A genetically modified mouse is provided, wherein the mouse is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa (κ) constant gene at the endogenous mouse κ locus, wherein the mouse expresses a reverse chimeric antibody having a light chain variable domain derived from one of only two human light chain variable region gene segments and a mouse κ constant domain, and a human heavy chain variable domain and a mouse heavy chain constant domain, from an endogenous mouse heavy chain locus. Bispecific epitope-binding proteins that are fully human are provided, comprising two different heavy chains that associate with an identical light chain that comprises a variable domain derived from one of two different human light chain variable region gene segments.

Claims

1. A mouse, comprising:

(a) a replacement at the endogenous mouse κ

immunoglobulin light chain variable region locus of all or substantially all endogenous mouse κ

immunoglobulin light chain variable region gene segments with a human Vκ

1-39/J gene segment, a human Vκ

3-20/J gene segment, or a combination thereof, wherein the human gene segment is operably linked to an endogenous mouse κ

constant gene; and

,

(b) a replacement of all or substantially all of the endogenous mouse heavy chain variable region gene locus with a plurality of human heavy chain variable region gene segments, wherein the human heavy chain variable region gene segments are operably linked to an endogenous mouse heavy chain constant gene, and the human heavy chain variable region gene segments are capable of rearranging and forming a rearranged human/mouse chimeric heavy chain gene.

2. The mouse of claim 1, wherein the mouse lacks an endogenous mouse κ

immunoglobulin light chain variable region locus that is capable of rearranging and forming a gene that encodes a mouse κ

variable region.

3. The mouse of claim 1, further comprising a mouse κ

intronic enhancer 5′

with respect to the mouse light chain constant region.

4. The mouse of claim 1, further comprising a mouse κ

3′

enhancer.

5. The mouse of claim 1, wherein the plurality of human heavy chain variable region gene segments comprises a segment selected from a 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51, a 6-1 human immunoglobulin variable region gene segment.

6. The mouse of claim 1, wherein the plurality of human heavy chain variable region gene segments comprises a segment selected from a D1-7, D1-26, D3-3, D3-10, D3-16, D3-22, D5-5, D5-12, D6-6, D6-13, D7-27 gene segment, and a combination thereof.

7. The mouse of claim 1, wherein the mouse comprises a B cell that comprises a rearranged immunoglobulin heavy chain variable region gene sequence comprising a human heavy chain variable region gene derived from a VH gene segment selected from a VH2-5, VH3-23, VH3-30, VH4-39, VH4-59, VH5-51, and derived from a D gene segment selected from a D1-7, D1-26, D3-3, D3-16, D3-10, D3-22, D5-5, D5-12, D6-6, D6-13, and D7-27.

8. The mouse of claim 1, wherein the human Vκ

1-39 gene segment is present in a rearrangement with a human Jκ

5 gene segment.

9. The mouse of claim 1, wherein the human Vκ

3-20 gene segment is present in a rearrangement with a human Jκ

1 gene segment.

10. A method for selecting a human variable region for making a bispecific antibody, comprising:

(a) immunizing a genetically modified mouse with an antigen of interest, wherein the mouse comprises (i) a replacement at the endogenous mouse κ

immunoglobulin light chain variable region locus of all or substantially all endogenous mouse κ

immunoglobulin light chain variable region gene segments with a human Vκ

1-39/J gene segment, a human Vκ

3-20/J gene segment, or a combination thereof, wherein the human gene segment is operably linked to an endogenous mouse κ

constant gene; and

, (ii) a replacement of all or substantially all of the endogenous mouse heavy chain variable region gene locus with a plurality of human heavy chain variable region gene segments, wherein the human heavy chain variable region gene segments are operably linked to an endogenous mouse heavy chain constant gene, and the human heavy chain variable region gene segments are capable of rearranging and forming a rearranged human/mouse chimeric heavy chain gene;

(b) allowing the mouse to develop an immune response to the antigen of interest; and

,

(c) identifying a clonally selected lymphocyte of the mouse that expresses an antibody that specifically binds the antigen of interest, and obtaining from the lymphocyte or the antibody a nucleotide sequence that encodes a human heavy chain variable region that specifically binds the antigen of interest; and

,

(d) employing the nucleotide sequence of (c) in making a bispecific antibody.

11. The method of claim 10, wherein the mouse lacks an endogenous mouse κ

immunoglobulin light chain variable region locus that is capable of rearranging and forming a gene that encodes a mouse κ

variable region.

12. The method of claim 10, further comprising a mouse κ

intronic enhancer 5′

with respect to the mouse light chain constant region.

13. The method of claim 10, further comprising a mouse κ

3′

enhancer.

14. The method of claim 10, wherein the plurality of human heavy chain variable region gene segments comprises a segment selected from a 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51, a 6-1 human immunoglobulin variable region gene segment.

15. The method of claim 10, wherein the plurality of human heavy chain variable region gene segments comprises a segment selected from a D1-7, D1-26, D3-3, D3-10, D3-16, D3-22, D5-5, D5-12, D6-6, D6-13, D7-27 gene segment, and a combination thereof.

16. The method of claim 10, wherein the mouse comprises a B cell that comprises a rearranged immunoglobulin heavy chain variable region gene sequence comprising a human heavy chain variable region gene derived from a VH gene segment selected from a VH2-5, VH3-23, VH3-30, VH4-39, VH4-59, VH5-51, and derived from a D gene segment selected from a D1-7, D1-26, D3-3, D3-16, D3-10, D3-22, D5-5, D5-12, D6-6, D6-13, and D7-27.

17. The method of claim 10, wherein the human Vκ

1-39 gene segment is present in a rearrangement with a human Jκ

5 gene segment.

18. The method of claim 10, wherein the human Vκ

3-20 gene segment is present in a rearrangement with a human Jκ

1 gene segment.

19. The method of claim 10, wherein steps (a) through (d) are performed a first time for a first antigen of interest to generate a first human heavy chain variable region sequence, and steps (a) through (d) are performed a second time for a second antigen of interest to generate a second human heavy chain variable region sequence, and wherein the first human heavy chain variable region sequence is expressed fused with a first human heavy chain constant region to form a first human heavy chain, the second human heavy chain variable region sequence is expressed fused with a second human heavy chain constant region to form a second human heavy chain, wherein the first and the second human heavy chains are expressed in the presence of a single human light chain derived from a Vκ

1-39 or a Vκ

3-20 gene segment.

20. The method of claim 19, wherein the first human heavy chain comprises a modification that eliminates or substantially reduces the affinity of the first human heavy chain to protein A, and the second human heavy chain retains the ability to bind protein A.

21. The method of claim 20, wherein the modification that eliminates or substantially reduces affinity of the first human heavy chain to protein A is selected from a 95R (EU 435R), a 96F (EU 436F), and a combination thereof.