# US 2023/0265154 A1 · Universal chimeric antigen receptor t-cell preparation technique

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- Number: US 2023/0265154 A1
- Document ID: US20230265154A1
- Status: Published application
- Kind: A1
- Application number: 16/645,290
- Filed: 2018-09-06
- Published: 2023-08-24
- Earliest claimed: 2017-09-06
- Assignees: Gracell Biotechnologies (Shanghai) Co., Ltd.
- Applicants: Gracell Biotechnologies (Shanghai) Co., Ltd.
- Inventors: Xinxin WANG; Zhongdong SHI; Liping LIU; Chunhui YANG; Jing SUN; Songbai CAI; Wei CAO
- Web page: https://patentgrep.com/patent/US20230265154A1

## CPC

- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/22: Immunosuppressive or immunotolerising
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/418: {Antigens related to induction of tolerance to non-self}
- A61K 40/4211: {CD19 or B4}
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 14/70539: {MHC-molecules, e.g. HLA-molecules}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C12N 5/0636: {T lymphocytes}
- C12N 15/1138: {against receptors or cell surface proteins}
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/53: Hinge
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/03: containing a transmembrane segment
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2510/00: Genetically modified cells
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- 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)
- 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

Provided are a universal chimeric antigen receptor (CAR) T cell and preparation method and application thereof. The binding of the cell HLA-A/HLA-B to the TCR is inhibited; the TCR gene expression is silenced; the invention man be used for allogeneic tumor treatment and does not cause GVHD and HVG reaction during allogeneic infusion, and thus improves the survival and antineoplastic effect of allogeneic CAR-T cells in the receptor.

## Claims

### Claim 1

A universal chimeric antigen receptor T cell (CAR-T cell) with the following characteristics:
(a) the CAR-T cell expresses a chimeric antigen receptor CAR, and the CAR targets a marker of tumor cells; and
(b) the binding of HLA-A and/or HLA-B of the CAR-T cell to TCR (T cell receptor) is inhibited;
and the HLA-A and/or HLA-B gene expression of the CAR-T cell is silenced, so that the binding of HLA-A and/or HLA-B to TCR (T cell receptor) is inhibited.

### Claim 2 (depends on 1)

The CAR-T cell of claim 1, wherein endogenous β2m gene of the CAR-T cell is expressed and/or functioned normally.

### Claim 3 (depends on 1)

The CAR-T cell of claim 1, wherein β2m gene expression of the CAR-T cell is silenced, so that the binding of HLA-A and/or HLA-B to TCR (T cell receptor) is inhibited.

### Claim 4 (depends on 1)

The CAR-T cell of claim 1, wherein the CAR-T cell expresses an exogenous ligand fragment or antibody fragment of NK cell inhibitory receptor (killer-cell immunoglobulin-like receptor, KIR).

### Claim 5 (depends on 1)

The CAR-T cell of claim 1, wherein the TCR gene expression of the CAR-T cell is silenced.

### Claim 6 (depends on 1)

The CAR-T cell of claim 1, wherein the structure of the CAR is shown in formula I as below:
wherein,
each “-” is independently a linker or peptide bond;
L1 is an optional signal peptide sequence;
scFv is an antigen binding domain;
H1 is an optional hinge region;
TM1 is a transmembrane domain;
C is a co-stimulatory signaling molecule;
CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;
K is an optional KIR ligand element.

### Claim 7 (depends on 1)

A method for preparing the CAR-T cell of claim 1, comprising the following steps:
(A) providing a T cell to be modified; and
(B) modifying the T cell to express the CAR, and inhibiting the binding of HLA-A and/or HLA-B of the CAR-T cell to TCR, thereby obtaining the CAR-T cell of claim 1.

### Claim 8 (depends on 1)

A preparation comprising the CAR-T cell of claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.

### Claim 9

(canceled)

### Claim 10 (depends on 1)

A kit for preparing the CAR-T cell of claim 1 comprising a container and following components located in the container:
(1) a first nucleic acid sequence comprising a first expression cassette for expressing the CAR;
(2) a second nucleic acid sequence comprising a second expression cassette or a first gRNA for silencing β2m, or a fifth expression cassette or a second gRNA and/or a third gRNA for silencing HLA-A and/or HLA-B.

### Claim 11 (depends on 1)

The CAR-T cell of claim 1, wherein the HLA-C gene expression of the CAR-T cell is not affected.

### Claim 12 (depends on 4)

The CAR-T cell of claim 4, wherein the ligand fragment of the inhibitory receptor comprises a full-length HLA-C or an HLA-C fragment.

### Claim 13 (depends on 4)

The CAR-T cell of claim 4, wherein the antibody fragment of the inhibitory receptor is scFv of a KIR antibody, preferably 1-7F9VL and/or 1-7F9VH.

### Claim 14 (depends on 1)

The CAR-T cell of claim 1, wherein the “binding of HLA-A and/or HLA-B to TCR is inhibited” means that HLA-A does not bind or substantially does not bind to TCR, and/or HLA-B does not bind or substantially does not bind to TCR.