# US 12,006,353 B2 · Application of SCFV protein, car gene expression vector, CAR-T cell and application thereof

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- Number: US 12,006,353 B2
- Document ID: US12006353B2
- Status: Granted
- Kind: B2
- Application number: 17/872,131
- Filed: 2022-07-25
- Published: 2023-03-16
- Granted: 2024-06-11
- Earliest claimed: 2022-07-25
- Assignees: Long Chen
- Applicants: Long Chen
- Inventors: Long Chen; Fake Yang; Fukun Chen; Huatao Niu; Yongzhu Pu; Jindan Li; Conghui Yang; Dongxue Chen
- Web page: https://patentgrep.com/patent/US12006353B2

## CPC

- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- 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 40/4219: {Receptors for chemokines}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C12N 5/0636: {T lymphocytes}
- C12N 15/86: Viral vectors
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/03: containing a transmembrane segment
- C12N 2510/00: Genetically modified cells
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

An application of SCFV protein in preparation of a CAR gene expression vector is disclosed. The protein sequence of the SCFV protein is shown in SEQ ID No. 1. The 1st˜21st amino acids are the extracellular signal peptide amino acid sequences. The 22nd˜31st amino acids are Flag amino acid sequences. And the remaining sequences are CXCR4 scfv sequences. An application of the gene sequence encoding the SCFV protein in preparation of a CAR gene expression vector is disclosed, and the gene sequence is shown in SEQ ID No.2. A CAR gene expression vector and a CAR-T cell are disclosed. The expression vector is plent-EF1a-Flag-CXCR4 CAR, and CAR-T cells are used in the preparation of a drug for a targeted therapy of tumor cells with high expression of CXCR4.

## Claims

### Claim 1

A method of preparing a chimeric antigen receptor (CAR) gene expression vector, comprising ligating a nucleic acid that encodes a protein comprising the ScFv having the amino acid sequence set forth in SEQ ID NO: 1 into a vector, wherein amino acids 1-21 are extracellular signal peptide amino acid sequences; amino acids 22-31 are Flag amino acid sequences; and the remaining amino acid sequences are CXCR4 ScFv sequences.

### Claim 2

A chimeric antigen receptor (CAR) gene expression vector, which is a lentivirus vector comprising the nucleic acid sequence set forth in SEQ ID NO: 2.

### Claim 3 (depends on 2)

A CAR-T cell made by infecting T cells with the gene expression vector according to claim 2.

### Claim 4 (depends on 2)

A method of constructing the CAR gene expression vector according to claim 2, comprising:
separately performing enzymatic digestion on a vector comprising the nucleic acid sequence set forth in SEQ ID NO: 2 and the lentiviral vector to obtain digested products;
ligating the digested products at 22° C. for 2 hours to produce a ligated product; and
purifying and recovering the ligated product to obtain the CAR gene expression vector.

### Claim 5 (depends on 4)

The method of constructing the CAR gene expression vector according to claim 4, wherein the nucleic acid sequence set forth in SEQ ID NO: 2 is obtained by digesting with the enzymes AsisI and MIuI.

### Claim 6 (depends on 5)

The method of constructing the CAR gene expression vector according to claim 5, wherein the lentivirus vector is obtained by digesting with the enzymes AsisI and MIuI.