# US 10,306,874 B2 · Non-human animals having a humanized lymphocyte-activation gene 3

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- Number: US 10,306,874 B2
- Document ID: US10306874B2
- Status: Granted
- Kind: B2
- Application number: 15/355,392
- Filed: 2016-11-18
- Published: 2017-05-25
- Granted: 2019-06-04
- Earliest claimed: 2015-11-20
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Alexander O. Mujica; Elena Burova; Andrew J. Murphy
- Web page: https://patentgrep.com/patent/US10306874B2

## CPC

- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- A01K 67/0276: Knock-out vertebrates
- A61K 49/0008: {Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure}
- C07K 14/70503: {Immunoglobulin superfamily}
- A01K 2207/15: Humanized animals
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2217/075: inducing loss of function, i.e. knock out
- 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
- A01K 2267/03: Animal model, e.g. for test or diseases
- A01K 2267/0331: Animal model for proliferative diseases
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- C12N 2510/00: Genetically modified cells
- C12N 2517/02: Cells from transgenic animals
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- 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)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Non-human animals, and methods and compositions for making and using the same, are provided, wherein the non-human animals comprise a humanization of a Lymphocyte activation gene 3 (Lag3). The non-human animals may be described, in some embodiments, as having a genetic modification to an endogenous Lag3 locus so that the non-human animals express a Lag3 polypeptide that includes a human portion and an endogenous portion (e.g., a non-human portion).

## Claims

### Claim 1

A mouse whose genome comprises a humanized Lymphocyte activation gene 3 (Lag-3) gene at an endogenous Lag-3 locus,
wherein a humanized Lag-3 polypeptide is expressed from the humanized Lag-3 gene on the surface of activated T-lymphocytes in the mouse,
wherein the humanized Lag-3 polypeptide comprises (i) the first two N-terminal immunoglobulin-like domains of a human LAG-3 polypeptide and (ii) the transmembrane and intracellular domains of an endogenous mouse Lag-3 polypeptide, and
wherein the humanized Lag-3 gene is operably linked to the endogenous mouse Lag-3 promoter at the endogenous Lag-3 locus.

### Claim 2 (depends on 1)

The mouse of claim 1, wherein the humanized Lag-3 polypeptide comprises amino acids 29-260 of the human LAG-3 polypeptide, wherein the human LAG-3 polypeptide comprises the amino acid sequence as set forth in SEQ ID No. 6.

### Claim 3 (depends on 1)

The mouse of claim 1, wherein the humanized Lag-3 gene comprises endogenous mouse Lag-3 exons 1, 5, 6, 7 and 8.

### Claim 4 (depends on 1)

The mouse of claim 1, wherein the humanized Lag-3 gene comprises exons 2-4 of a human LAG-3 gene.

### Claim 5 (depends on 1)

A humanized Lag-3 polypeptide produced by the mouse of claim 1.

### Claim 6 (depends on 1)

An isolated cell or tissue of the mouse of claim 1, wherein the genome of the isolated cell or tissue comprises the humanized Lag-3 gene at the endogenous Lag-3 locus.

### Claim 7

A method of making a mouse comprising
modifying a mouse genome so that the modified genome comprises a humanized Lag-3 gene at an endogenous Lag-3 locus, thereby making the mouse,
wherein a humanized Lag-3 polypeptide is expressed from the humanized Lag-3 gene on the surface of activated T-lymphocytes in the mouse, and
wherein the humanized Lag-3 polypeptide comprises (i) the first two N-terminal immunoglobulin-like domains of a human LAG-3 polypeptide and (ii) the transmembrane and intracellular domains of an endogenous mouse Lag-3 polypeptide, and
wherein the humanized Lag-3 gene is operably linked to the endogenous mouse Lag-3 promoter at the endogenous Lag-3 locus.

### Claim 8 (depends on 7)

The method of claim 7, comprising
(a) integrating a genomic fragment into an endogenous Lag-3 gene in a mouse ES cell, the genomic fragment comprising a nucleotide sequence that encodes the first two N-terminal immunoglobulin-like domains of the human LAG-3 polypeptide, thereby obtaining a genetically modified mouse embryonic stem cell; and,
(c) creating a mouse using the genetically modified mouse ES cell of (a).

### Claim 9 (depends on 7)

The method of claim 7, wherein the humanized Lag-3 gene comprises exons 2-4 of a human LAG-3 gene.

### Claim 10 (depends on 7)

The method of claim 7, wherein the humanized Lag-3 gene encodes at least amino acids 29-260 of the human LAG-3 polypeptide, wherein the human LAG-3 polypeptide comprises the amino acid sequence as set forth in SEQ ID No. 6.