# US 12,004,495 B2 · Genetically modified non-human animals with humanized immunoglobulin locus

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- Number: US 12,004,495 B2
- Document ID: US12004495B2
- Status: Granted
- Kind: B2
- Application number: 17/010,587
- Filed: 2020-09-02
- Published: 2020-12-17
- Granted: 2024-06-11
- Earliest claimed: 2019-02-18
- Assignees: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
- Applicants: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
- Inventors: Yuelei Shen; Jiawei Yao; Huizhen Zhao; Yabo Zhang; Lili Liu; Hui Lu; Shuwen Huang
- Web page: https://patentgrep.com/patent/US12004495B2

## CPC

- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2818: {against CD28 or CD152}
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- 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 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/565: Complementarity determining region [CDR]
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- 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

The present disclosure relates to genetically modified animals and cells with humanized heavy chain immunoglobulin locus and/or humanized light chain immunoglobulin locus.

## Claims

### Claim 1

A genetically-modified rodent comprising at an endogenous heavy chain immunoglobulin gene locus human Immunoglobulin Heavy Variable (IGHV) genes, human Immunoglobulin Heavy Diversity (IGHD) genes, and human Immunoglobulin Heavy Joining (IGHJ) genes at the heavy chain immunoglobulin gene locus of human chromosome 14 from a human cell, wherein the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked and can undergo VDJ rearrangement, wherein a contiguous human sequence is integrated into the endogenous chromosome by recombination, and the human sequence is at least 500 kb, wherein the modification can be detected by PCR using one or more primer pairs selected from the group consisting of:
(a) SEQ ID NO: 13 and SEQ ID NO: 14;
(b) SEQ ID NO: 15 and SEQ ID NO: 16;
(c) SEQ ID NO: 17 and SEQ ID NO: 18;
(d) SEQ ID NO: 19 and SEQ ID NO: 20;
(e) SEQ ID NO: 21 and SEQ ID NO: 22;
(f) SEQ ID NO: 23 and SEQ ID NO: 24; and
(g) SEQ ID NO: 25 and SEQ ID NO: 26.

### Claim 2 (depends on 1)

The rodent of claim 1, wherein the rodent comprises a disruption in the rodent's endogenous heavy chain immunoglobulin gene locus.

### Claim 3 (depends on 2)

The rodent of claim 2, wherein the rodent is a mouse and the disruption in the endogenous heavy chain immunoglobulin gene locus comprises a deletion of a contiguous sequence starting from mouse IGHV1-85 gene to mouse IGHJ4 gene.

### Claim 4 (depends on 1)

The rodent of claim 1, wherein the rodent is homozygous with respect to the integrated human sequence at the endogenous heavy chain immunoglobulin gene locus.

### Claim 5 (depends on 1)

The rodent of claim 1, wherein the rodent comprises a disruption in the rodent's endogenous light chain immunoglobulin gene locus.

### Claim 6 (depends on 1)

The rodent of claim 1, wherein the rodent further comprises at an endogenous light chain immunoglobulin gene locus, one or more human Immunoglobulin Kappa Variable (IGKV) genes, and one or more human Immunoglobulin Kappa Joining (IGKJ) genes.

### Claim 7 (depends on 6)

The rodent of claim 6, wherein the rodent further comprises all human IGKV genes and all human IGKJ genes at the endogenous light chain immunoglobulin gene locus.

### Claim 8 (depends on 7)

The rodent of claim 7, wherein the rodent can detectably express IGKVID-12, IGKV2D-30, IGKV1-27, IGKV6-21, IGKV1-6, IGKV1-39, IGKV1-16, IGKV1-17, IGKV2D-29, IGKV3-11, IGKV1-5, IGKV1-12, IGKV1-9, IGKV3-20, IGKV3-15, IGKV2-28, IGKV2-30, IGKV1-33, IGKV4-1, IGKV6D-41, IGKV3D-11, IGKV2D-24, IGKVID-43, IGKVID-17, IGKV7-3, IGKV2D-26, IGKV1-37, IGKV2-29, IGKV1-13, IGKV5-2, IGKV2-40, IGKV3-7, IGKV3D-20, IGKV1D-8, IGKV6D-21, IGKV1-8, IGKV2-24, and IGKV3D-15.

### Claim 9 (depends on 1)

The rodent of claim 1, wherein the rodent can produce a humanized antibody.

### Claim 10 (depends on 1)

A cell obtained from the rodent of claim 1.

### Claim 11 (depends on 1)

The rodent of claim 1, wherein the rodent is a mouse.

### Claim 12 (depends on 1)

The rodent of claim 1, wherein the rodent comprises human IGHV3-64D and IGHV5-10-1 at the endogenous heavy chain immunoglobulin gene locus.

### Claim 13 (depends on 1)

The rodent of claim 1, wherein the rodent comprises human IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, IGHV3-75, and IGHV(II)-74-1 at the endogenous heavy chain immunoglobulin gene locus.

### Claim 14 (depends on 1)

The rodent of claim 1, wherein the rodent can detectably express IGHV3-15, IGHV3-53, IGHV3-66, IGHV5-51, IGHV1-24, IGHV1-18, IGHV1-69, IGHV3-7, IGHV3-74, IGHV3-23, IGHV3-43, IGHV3-21, IGHV3-30-3, IGHV4-59, IGHV6-1, IGHV3-48, IGHV4-34, IGHV4-39, IGHV3-33, IGHV3-30, IGHV4-61, IGHV3-47, IGHV3-NL1, IGHV3-69-1, IGHV7-4-1, IGHV1-58, IGHV4-28, IGHV2-26, IGHV2-5, IGHV3-64, IGHV3-20, IGHV2-70, IGHV3-11, IGHV4-30-2, IGHV3-13, IGHV3-49, IGHV5-10-1, IGHV3-72, IGHV1-2, IGHV4-30-4, IGHV1-46, IGHV3-64D, IGHV1-3, IGHV3-73, IGHV4-4, and IGHV4-31.

### Claim 15 (depends on 1)

The rodent of claim 1, wherein the rodent comprises at the endogenous heavy chain immunoglobulin gene locus one short flippase recognition target (FRT) site and one loxP recognition sequence, wherein the integrated human sequence is between the FRT site and the loxP recognition sequence.

### Claim 16 (depends on 1)

The rodent of claim 1, wherein the modification can be detected by PCR using the primer pair of SEQ ID NO: 13 and SEQ ID NO: 14.

### Claim 17 (depends on 1)

The rodent of claim 1, wherein the modification can be detected by PCR using the primer pair of SEQ ID NO: 15 and SEQ ID NO: 16.

### Claim 18 (depends on 1)

The rodent of claim 1, wherein the modification can be detected by PCR using the primer pair of SEQ ID NO: 17 and SEQ ID NO: 18.

### Claim 19 (depends on 1)

The rodent of claim 1, wherein the modification can be detected by PCR using the primer pair of SEQ ID NO: 19 and SEQ ID NO: 20.

### Claim 20 (depends on 1)

The rodent of claim 1, wherein the modification can be detected by PCR using the primer pair of SEQ ID NO: 21 and SEQ ID NO: 22.

### Claim 21 (depends on 1)

The rodent of claim 1, wherein the modification can be detected by PCR using the primer pair of SEQ ID NO: 23 and SEQ ID NO: 24.

### Claim 22 (depends on 1)

The rodent of claim 1, wherein the modification can be detected by PCR using the primer pair of SEQ ID NO: 25 and SEQ ID NO: 26.