# US 12,760,087 B2 · Modified T cell, preparation method therefor and use thereof

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- Number: US 12,760,087 B2
- Document ID: US12760087B2
- Status: Granted
- Kind: B2
- Application number: 17/277,274
- Filed: 2019-09-17
- Published: 2022-01-13
- Granted: 2026-10-13
- Earliest claimed: 2018-09-17
- Assignees: Institute of Zoology, Chinese Academy of Sciences
- Applicants: Institute of Zoology, Chinese Academy of Sciences
- Inventors: Haoyi Wang; Xingying Zhang; Chen Cheng; Na Tang; Na Li
- Web page: https://patentgrep.com/patent/US12760087B2

## CPC

- C07K 14/70575: {NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525)}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4255: {Mesothelin [MSLN]}
- A61P 35/00: Antineoplastic agents
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C07K 14/7155: {for interleukins [IL]}
- C07K 16/30: from tumour cells
- C12N 5/0636: {T lymphocytes}
- A61K 2239/59: Reproductive system, e.g. uterus, ovaries, cervix or testes
- 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)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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 invention relates to the field of gene editing and tumor immunotherapy. In particular, the invention relates to methods for preparing modified T cells, such as CAR-T cells, by gene editing, and modified T cells prepared by the methods and uses thereof.

## Claims

### Claim 1

A method for preparing a modified T cell, comprising a step of eliminating the expression of at least one inhibitory protein in the T cell and a step of eliminating the expression of PD1 in the T cell, wherein the inhibitory protein is a T cell surface inhibitory receptor and/or a T cell exhaustion-related protein, wherein the inhibitory protein is a TGFBRII receptor, wherein said elimination of at least one inhibitory protein and elimination of the expression of PD1 is achieved by antisense RNA, antagomir, siRNA, shRNA, meganuclease, zinc finger nuclease, transcription activator-like effector nuclease, or a CRISPR system, wherein said T cell is a T cell comprising a chimeric antigen receptor (CAR).

### Claim 2 (depends on 1)

The method of claim 1, wherein said CRISPR system is a CRISPR/Cas9 system.

### Claim 3 (depends on 2)

The method of claim 2, wherein said CRISPR/Cas9 system targets one or more of the nucleotide sequences in the cells selected from the group consisting of SEQ ID NOs: 28-31.

### Claim 4 (depends on 1)

The method of claim 1, wherein the CAR comprises an antigen binding domain against a tumor associated antigen.

### Claim 5 (depends on 4)

The method of claim 4, wherein the tumor associated antigen is selected from the group consisting of CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialylganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipid, glioma-related antigens, p-human chorionic gonadotropin, a-fetoglobulin (AFP), lectin-responsive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, major histocompatibility complex (MHC) molecules that present tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, fibronectin extra domain A (EDA) and extra domain B (EDB), tenascin-C Al domain (TnC A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1, B7-2, GM-CSF, cytokine receptor, endothelial factor, BCMA, TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8 and FCRL5.

### Claim 6 (depends on 4)

The method of claim 4, wherein the antigen binding domain is selected from the group consisting of a monoclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a single domain antibody, an antibody single-chain variable region, and an antigen binding fragment thereof.

### Claim 7 (depends on 1)

The method of claim 1, wherein the CAR comprises a scFv against mesothelin, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD37 signal transduction domain.

### Claim 8 (depends on 7)

The method of claim 7, wherein the CAR comprises an amino acid sequence set forth in SEQ ID NO:27.

### Claim 9 (depends on 1)

A modified T cell prepared by the method of claim 1.