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T cell receptor for recognizing EBV antigen and application of T cell receptor

A cell receptor, cell technology, applied in the field of medicine, can solve the problem of limited function and achieve the effect of strong killing effect

Active Publication Date: 2021-06-11
HENAN CANCER HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the existence of the local immunosuppressive microenvironment of the tumor, the function of the patient's own T cells to kill the tumor is limited

Method used

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  • T cell receptor for recognizing EBV antigen and application of T cell receptor
  • T cell receptor for recognizing EBV antigen and application of T cell receptor
  • T cell receptor for recognizing EBV antigen and application of T cell receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1: Cloning of EBV LMP2A antigen short peptide-specific T cells and TCR gene acquisition

[0136] Peripheral blood lymphocytes from healthy volunteers with genotype HLA-A1101 were stimulated with a synthetic short peptide SSCSSCPLSK (SEQ ID NO: 15; Jiangsu GenScript Biotechnology Co., Ltd.). The SSCSSCPLSK short peptide was refolded with biotin-labeled HLA-A1101 to prepare pHLA monomer. These monomers are combined with PE-labeled streptavidin (BD Company) to form PE-labeled tetramers, the tetramers and anti-CD8-FITC double-positive cells are enriched, and the obtained double-positive cells are subjected to flow cytometry Single cells were obtained by sorting, and the single cells obtained by sorting were amplified with a one-step RT-PCR kit (QIAGEN, catalog number 210212) to amplify TCR α chain and β chain respectively, and the PCR products were sequenced. Comparing the sequencing results with the sequences in the public database of IMGT (International Immunogen...

Embodiment 2

[0150] Example 2: EBV LMP2A antigen short peptide-specific TCR lentiviral vector construction and lentiviral packaging

[0151] (1) TCR lentiviral vector construction (named pLKO-TCR048)

[0152] The EBV LMP2A TCR α and β chain variable region sequences were cloned into a pLKO-based expression plasmid (Addgene), and the α or β variable domains were cloned by a multi-fragment recombination cloning kit (Novazyme Biotechnology Company, catalog number C113) Into PLKO-based expression plasmids containing α or β constant regions, the ligated plasmids were transformed into competent E. coli strain Stbl3 cells (Shanghai Weidi Biotechnology Co., Ltd.) and inoculated in LB / ml containing 100 μg / ml ampicillin on agar plates. After overnight incubation at 37°C, single colonies were picked and grown overnight at 37°C in 10 ml LB containing 100 μg / ml ampicillin with shaking. The cloned plasmid was purified using a mini-prep kit (TIANGEN Cat. #DP118-02) and sequenced to obtain pLKO-TCR048. ...

Embodiment 3

[0157] Example 3: Construction and functional identification of Jurkat cell line expressing EBV LMP2A antigen short peptide-specific TCR

[0158] Use T2-A11 cells (T2 cells are preserved in ATCC, the preservation catalog number is CRL-1992, T2-A11 cells are constructed on the basis of T2 cells with reference to Cancer Biology & Therapy, 8:21, 2025-2032) to verify the transduction of the TCR of the present invention function of effector cells

[0159] ELISA protocol

[0160] The following experiments were performed to demonstrate the specific activation response of TCR-transduced T cells to target cells. The amount of IL-2 secretion detected by ELISA assay was used as the readout value of T cell activation.

[0161] (1) Reagent

[0162] Assay medium: 10% FBS (ThermoFisher, Cat. No. 10099-044), RPMI1640 (ThermoFisher, Cat. No. C11875500BT)

[0163] Wash buffer (PBST): 1xPBS (Sigma, Cat. P3813) containing 0.05% Tween-20, prepared in deionized water.

[0164] Human IL-2 Uncoa...

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Abstract

The invention discloses a T cell receptor for recognizing an EBV antigen and application of the T cell receptor. The T cell receptor (TCR) comprises an alpha chain containing a variable region and / or a beta chain containing a variable region, wherein the variable region of the alpha chain comprises a complementarity determining region 3 (CDR3) containing an amino acid sequence of AVVNNNDMR (SEQ ID NO: 3); and / or the variable region of the beta chain comprises a complementarity determining region 3 (CDR3) comprising ASSPGRWYEQY (SEQ ID NO: 6). The TCR provided by the invention can be specifically combined with an EBV antigen short peptide, and T cells transducing the TCR can be specifically activated and have a very strong killing effect on target cells.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a T cell receptor for recognizing EBV antigen and the application of the T cell receptor. Background technique [0002] EBV (Epstein–Barr virus), also known as human herpesvirus 4, is a double-stranded DNA virus. Humans are the sole reservoir of EBV, with approximately >90% of the global adult population harboring the virus. [0003] EBV is transmitted through contact with saliva. Most primary infections occur in infancy and are usually asymptomatic or nonspecific, whereas infections in late adolescence or adulthood result in infectious mononucleosis. Once infection occurs, EBV usually exists latently in peripheral lymphocytes, making the infected person a lifelong EBV carrier. Carrying EBV has no serious consequences in most cases, as long as the virus remains in a latent, inactive state. However, in rare cases, the latent virus is activated, which can lead to chronic ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/725C12N15/12C12N15/867C12N5/10A61K38/17A61P35/00A61P31/22
CPCC07K14/7051C12N15/86C12N5/0636A61P35/00A61P31/22C12N2740/15043C12N2510/00A61K38/00
Inventor 王子兵陈琳张建功李红乐寇贺丹
Owner HENAN CANCER HOSPITAL
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