# US 11,026,407 B2 · Mice expressing a limited immunoglobulin light chain repertoire

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- Number: US 11,026,407 B2
- Document ID: US11026407B2
- Status: Granted
- Kind: B2
- Application number: 16/530,030
- Filed: 2019-08-02
- Published: 2020-01-23
- Granted: 2021-06-08
- Earliest claimed: 2010-02-08
- Assignees: Regeneran Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: John McWhirter; Lynn Macdonald; Sean Stevens; Andrew J. Murphy
- Web page: https://patentgrep.com/patent/US11026407B2

## CPC

- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/462: {Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another}
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 2207/15: Humanized animals
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2217/15: Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
- A01K 2227/105: Murine
- A01K 2267/01: Animal expressing industrially exogenous proteins
- C07K 2317/21: from primates, e.g. man
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/567: Framework region [FR]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C12N 2800/204: of bacterial origin, e.g. BAC
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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 present a choice of two human light chain variable gene segments such that the immunoglobulin light chains expresses by the mouse comprise one of the two human light chain variable gene segments. 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.

## Claims

### Claim 1

A method for generating a human immunoglobulin heavy or light chain variable region sequence comprising the steps of:
(a) immunizing a genetically modified mouse with an antigen of interest, wherein the genetically modified mouse comprises in its germline genome:
(i) exactly two unrearranged human immunoglobulin Vκ gene segments and five unrearranged human immunoglobulin Jκ fix gene segments operably linked to a mouse immunoglobulin light chain constant region sequence at the endogenous kappa light chain loci of the mouse, wherein the two unrearranged human immunoglobulin Vκ gene segments are a human Vκ1-39 gene segment and a human Vκ3-20 gene segment; and
(ii) one or more unrearranged human immunoglobulin VH gene segments, one or more unrearranged human immunoglobulin DH gene segments, and one or more unrearranged human immunoglobulin JH gene segments operably linked to a mouse immunoglobulin heavy chain constant region sequence at the endogenous heavy chain loci of the mouse;
wherein the unrearranged human immunoglobulin heavy chain and kappa light chain gene segments of the genetically modified mouse are capable of rearranging and encoding human immunoglobulin variable domains of an antibody, wherein the genetically modified mouse does not comprise endogenous immunoglobulin Vκ or Jκ gene segments that are capable of rearranging to form an immunoglobulin light chain variable region sequence; and
(b) determining a human immunoglobulin heavy or light chain variable region sequence that encodes a human heavy or kappa light chain variable domain of an antibody, respectively, that specifically binds the antigen and that was generated by the genetically modified mouse.

### Claim 2 (depends on 1)

The method of claim 1, wherein the five unrearranged human immunoglobulin Jκ gene segments are a human Jκ1 gene segment, a human Jκ2 gene segment, a human Jκ3 gene segment, a human Jκ4 gene segment, and a human Jκ5 gene segment.

### Claim 3 (depends on 1)

The method of claim 1, wherein the mouse light chain constant region is a mouse Cκ constant region.

### Claim 4 (depends on 1)

The method of claim 1, wherein the human Vκ1-39 gene segment in the germline genome of the genetically modified mouse is a human germline Vκ1-39 gene segment and the human Vκ3-20 gene segment in the germline genome of the genetically modified mouse is a human germline Vκ3-20 gene segment.

### Claim 5 (depends on 1)

The method of claim 1, wherein the germline genome of the genetically modified mouse comprises in order: the human Vκ1-39 gene segment, the human Vκ3-20 gene segment, the human Jκ1 gene segment, the human Jκ2 gene segment, the human Jκ3 gene segment, the human Jκ4 gene segment, and the human Jκ5 gene segment.

### Claim 6 (depends on 5)

The method of claim 5, wherein the mouse light chain constant region is a mouse Cκ constant region.

### Claim 7 (depends on 1)

The method of claim 1, wherein the genetically modified mouse comprises a nonfunctional λ light chain locus.

### Claim 8 (depends on 1)

The method of claim 1, wherein the genetically modified mouse comprises a functional λ light chain locus.

### Claim 9

A mouse embryonic stem (ES) cell comprising in its genome:
a) exactly two unrearranged human immunoglobulin Vκ gene segments and five unrearranged human immunoglobulin Jκ gene segments operably linked to a mouse immunoglobulin light chain constant region sequence at the endogenous kappa light chain loci of the mouse, wherein the two unrearranged human immunoglobulin Vκ gene segments are a human Vκ1-39 gene segment and a human Vκ3-20 gene segment; and
b) one or more unrearranged human immunoglobulin VH gene segments, one or more unrearranged human immunoglobulin DH gene segments, and one or more unrearranged human immunoglobulin JH gene segments operably linked to a mouse immunoglobulin heavy chain constant region sequence at the endogenous heavy chain loci of the mouse ES cell.

### Claim 10 (depends on 9)

The ES cell of claim 9, wherein the five unrearranged human immunoglobulin Jκ gene segments are a human Jκ1 gene segment, a human Jκ2 gene segment, a human Jκ3 gene segment, a human Jκ4 gene segment, and a human Jκ5 gene segment.

### Claim 11 (depends on 9)

The ES cell of claim 9, wherein the mouse light chain constant region is a mouse Cκ constant region.

### Claim 12 (depends on 9)

The ES cell of claim 9, wherein the human Vκ1-39 gene segment is a human germline Vκ1-39 gene segment and the human Vκ3-20 gene segment is a human germline Vκ3-20 gene segment.

### Claim 13 (depends on 9)

The ES cell of claim 9, wherein the genome of the ES comprises in order: the human Vκ1-39 gene segment, the human Vκ3-20 gene segment, the human Jκ1 gene segment, the human Jκ2 gene segment, the human Jκ3 gene segment, the human Jκ4 gene segment, and the human Jκ5 gene segment.

### Claim 14 (depends on 9)

A mouse embryo derived from the ES cell of claim 9.