# US 9,516,868 B2 · Mice that make VL binding proteins

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- Number: US 9,516,868 B2
- Document ID: US9516868B2
- Status: Granted
- Kind: B2
- Application number: 13/195,951
- Filed: 2011-08-02
- Published: 2012-04-19
- Granted: 2016-12-13
- Earliest claimed: 2010-08-02
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Lynn Macdonald; Sean Stevens; Cagan Gurer; Karolina A. Hosiawa; Andrew J. Murphy
- Inventors: Lynn Macdonald; Sean Stevens; Cagan Gurer; Karolina A. Hosiawa; Andrew J. Murphy
- Web page: https://patentgrep.com/patent/US9516868B2

## CPC

- C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies}
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/082: Hepadnaviridae (F), e.g. hepatitis B virus
- C07K 16/461: {Igs containing Ig-regions, -domains or -residues form different species}
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2227/105: Murine
- A01K 2267/01: Animal expressing industrially exogenous proteins
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/21: from primates, e.g. man
- C07K 2317/31: multispecific
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C12N 2015/8518: {expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles}
- 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)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Genetically modified mice and methods for making an using them are provided, wherein the mice comprise a replacement of all or substantially all immunoglobulin heavy chain V gene segments, D gene segments, and J gene segments with at least one light chain V gene segment and at least one light chain J gene segment. Mice that make binding proteins that comprise a light chain variable domain operably linked to a heavy chain constant region are provided. Binding proteins that contain an immunoglobulin light chain variable domain, including a somatically hypermutated light chain variable domain, fused with a heavy chain constant region, are provided. Modified cells, embryos, and mice that encode sequences for making the binding proteins are provided.

## Claims

### Claim 1

A mouse whose germline genome comprises a modified endogenous mouse immunoglobulin heavy chain locus comprising a replacement of all functional endogenous mouse immunoglobulin heavy chain variable (VH) gene segments, all functional endogenous mouse immunoglobulin heavy chain diversity (DH) gene segments and all functional endogenous mouse immunoglobulin heavy chain joining (JH) gene segments at the endogenous mouse immunoglobulin heavy chain locus with a nucleotide sequence that comprises a plurality of contiguous unrearranged functional human immunoglobulin light chain variable Vκ (hVκ) gene segments and all five unrearranged functional human immunoglobulin light chain joining gene segments (hJκ1-hJκ5),
wherein the plurality of unrearranged functional hVκ gene segments and the five unrearranged functional human immunoglobulin light chain joining gene segments (hJκ1-hJκ5) are operably linked to an intact endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus,
wherein the plurality of unrearranged functional hVκ gene segments and the five unrearranged functional human immunoglobulin light chain joining gene segments (hJκ1-hJκ5) rearrange in a B cell during B cell development to form a rearranged human immunoglobulin light chain variable region Vκ/Jκ nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus, and
wherein the mouse comprises a CD19+ B cell comprising the rearranged human immunoglobulin light chain variable region Vκ/Jκ nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus.

### Claim 2 (depends on 1)

The mouse of claim 1, wherein all the functional endogenous mouse VH, DH, and JH gene segments are replaced with at least 6 human Vκ gene segments and the five unrearranged functional human immunoglobulin light chain joining gene segments (hJκ1-hJκ5).

### Claim 3 (depends on 1)

The mouse of claim 1, wherein all the functional endogenous mouse VH, DH, and JH gene segments are replaced with at least 16 human Vκ gene segments and the five contiguous unrearranged human immunoglobulin light chain joining gene segments (hJκ1-hJκ5).

### Claim 4 (depends on 1)

The mouse of claim 1, wherein all the functional endogenous mouse VH, DH, and JH gene segments are replaced with at least 30 human Vκ gene segments and the five contiguous unrearranged human immunoglobulin light chain joining gene segments (hJκ1-hJκ5).

### Claim 5 (depends on 1)

The mouse of claim 1, wherein all the functional endogenous mouse VH, DH, and JH gene segments are replaced with at least 40 human Vκ gene segments and the five contiguous unrearranged human immunoglobulin light chain joining gene segments (hJκ1-hJκ5).

### Claim 6 (depends on 1)

An isolated cell from the mouse of claim 1, wherein the cell comprises a modified endogenous mouse immunoglobulin heavy chain locus comprising a replacement of all functional endogenous mouse immunoglobulin heavy chain variable (VH) gene segments, all functional endogenous mouse immunoglobulin heavy chain diversity (DH) gene segments and all functional endogenous mouse immunoglobulin heavy chain joining (JH) gene segments at the endogenous mouse immunoglobulin heavy chain locus with a nucleotide sequence that comprises a plurality of unrearranged functional human immunoglobulin light chain variable Vκ (hVκ) gene segments and all five unrearranged functional human immunoglobulin light chain joining gene segments (hJκ1-hJκ5),
wherein the plurality of unrearranged functional hVκ gene segments and all five unrearranged functional human immunoglobulin light chain joining gene segments (hJκ1-hJκ5) are operably linked to an intact endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus,
wherein the plurality of unrearranged functional hVκ gene segments and the five unrearranged functional human immunoglobulin light chain joining gene segments (hJκ1-hJκ5) are capable of rearranging in a B cell during B cell development to form a rearranged human immunoglobulin light chain variable region Vκ/Jκ nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region at the endogenous mouse immunoglobulin heavy chain locus.

### Claim 7 (depends on 6)

The isolated cell of claim 6, wherein the cell is an embryonic stem cell.

### Claim 8 (depends on 1)

A cell isolated from the mouse of claim 1, wherein the cell is the CD19+ B cell.

### Claim 9 (depends on 8)

A hybridoma comprising a myeloma cell line fused with the CD19+ B cell of claim 8, wherein the hybridoma produces a polypeptide encoded by the rearranged human immunoglobulin light chain variable region Vκ/Jκ nucleotide sequence operably linked to the endogenous mouse immunoglobulin heavy chain constant region.

### Claim 10 (depends on 1)

The mouse of claim 1, wherein the rearranged human immunoglobulin light chain variable region Vκ/Jκ gene sequence comprises at least one N addition.

### Claim 11 (depends on 1)

The mouse of claim 1, wherein the mouse is homozygous or heterozygous for the modified endogenous immunoglobulin heavy chain locus.