# US 10,945,418 B2 · Genetically modified non-human animal with human or chimeric PD-L1

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- Number: US 10,945,418 B2
- Document ID: US10945418B2
- Status: Granted
- Kind: B2
- Application number: 16/428,941
- Filed: 2019-05-31
- Published: 2020-01-23
- Granted: 2021-03-16
- Earliest claimed: 2016-08-31
- Assignees: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
- Applicants: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
- Inventors: Yuelei Shen; Yang Bai; Yanan Guo; Rui Huang; Xiaofei Zhou; Chaoshe Guo
- Web page: https://patentgrep.com/patent/US10945418B2

## CPC

- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 14/70532: {B7 molecules, e.g. CD80, CD86}
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C12N 15/907: {in mammalian cells}
- 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 2267/0331: Animal model for proliferative diseases
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- 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)
- 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)

## Abstract

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) programmed death-ligand 1 (PD-L1, PDL1, or B7-H1), and methods of use thereof. In one aspect, the disclosure relates to genetically-modified, non-human animals whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric programmed death-ligand 1 (PD-L1).

## Claims

### Claim 1

A genetically-modified mouse whose genome comprises at least one chromosome comprising a nucleotide sequence encoding a chimeric programmed death-ligand 1 (PD-L1) at an endogenous PD-L1 gene locus, wherein the chimeric PD-L1 comprises SEQ ID NO: 33, wherein the mouse expresses the chimeric PD-L1 on the surface of one or more spleen cells after the genetically modified mouse is challenged by anti-CD3 antibody.

### Claim 2 (depends on 1)

The mouse of claim 1, wherein the sequence encoding the chimeric PD-L1 is operably linked to an endogenous regulatory element at the endogenous PD-L1 gene locus in the at least one chromosome.

### Claim 3 (depends on 1)

The mouse of claim 1, wherein the mouse does not express endogenous PD-L1.

### Claim 4 (depends on 1)

The mouse of claim 1, wherein the mouse is homozygous with respect to the sequence encoding the chimeric PD-L1 at the endogenous PD-L1 gene locus.

### Claim 5 (depends on 1)

The mouse of claim 1, wherein the mouse whose genome further comprises a nucleotide sequence encoding a humanized PD-1 protein at the endogenous PD-1 gene locus, wherein the mouse expresses the humanized PD-1 protein on the surface of one or more spleen cell after the genetically modified mouse is challenged by anti-CD3 antibody.

### Claim 6 (depends on 1)

The mouse of claim 1, wherein exon 3 at the endogenous PD-L1 gene locus is modified by CRISPR with sgRNAs that target SEQ ID NO: 1 and SEQ ID NO: 11.

### Claim 7

A genetically modified mouse or a progeny thereof, wherein the genetically modified mouse is made by a method comprising the steps of: modifying genome of an embryo of a mouse by CRISPR with sgRNAs that target SEQ ID NO: 1 and SEQ ID NO: 11 to introduce a fragment of human PD-L1 gene, wherein an endogenous PD-L1 gene locus in the genome of the embryo is modified; and transplanting the embryo to a recipient mouse to produce the genetically-modified mouse, wherein the genetically-modified mouse expresses a chimeric PD-L1 comprising a fragment of human PD-L1 protein on the surface of one or more spleen cells after the genetically-modified mouse is challenged by anti-CD3 antibody.

### Claim 8 (depends on 7)

The genetically-modified mouse or the progeny thereof of claim 7, wherein the mouse has a C57BL/6 background.

### Claim 9

A genetically-modified mouse whose genome comprises at least one chromosome comprising a sequence encoding a human PD-L1 at an endogenous PD-L1 gene locus, wherein the sequence encoding the human PD-L1 is operably linked to an endogenous regulatory element at the endogenous PD-L1 gene locus in the at least one chromosome, wherein the mouse expresses a human PD-L1 on the surface of one or more spleen cells after the genetically modified mouse is challenged by anti-CD3 antibody.

### Claim 10 (depends on 9)

The mouse of claim 9, wherein the mouse does not express endogenous PD-L1.

### Claim 11 (depends on 9)

The mouse of claim 9, wherein the mouse is homozygous with respect to the sequence encoding the human PD-L1 at the endogenous PD-L1 gene locus.