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Chimeric antigen receptor taking TIGIT and PD-1 as targets, CAR-T cell and preparation method thereof

A chimeric antigen receptor, PD-1 technology, applied in the field of genes, can solve the problems of insignificant anti-tumor effect, large side effects, and easy emergence of drug resistance, so as to improve the killing ability, improve the effectiveness, and avoid immune escape. Effect

Active Publication Date: 2021-03-12
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Existing tumor immune technologies mostly use anti-PD-1 antibody, anti-PD-l1 antibody and CTLA-4 antibody to treat tumors, but these drugs also have other inhibitory proteins including TIGIT when they are treated with anti-checkpoint drugs. Long-term use of antibody drugs has relatively large side effects, drug resistance is prone to occur, and the anti-tumor effect is not significant

Method used

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  • Chimeric antigen receptor taking TIGIT and PD-1 as targets, CAR-T cell and preparation method thereof
  • Chimeric antigen receptor taking TIGIT and PD-1 as targets, CAR-T cell and preparation method thereof
  • Chimeric antigen receptor taking TIGIT and PD-1 as targets, CAR-T cell and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of TIGIT and PD-1 dual-target CAR expression vector

[0037] Anti-TIGIT-PD-1-CAR module diagram figure 1 . The nucleic acid sequences of each module of the Anti-TIGIT-PD-1-CAR vector are as follows:

[0038] (1) CD8 leader leader nucleic acid artificial sequence (SEQ ID NO.1)

[0039] (2) Nucleic acid artificial sequence of TIGIT extracellular region (SEQ ID NO.2)

[0040] (3) Linker nucleic acid artificial sequence (SEQ ID NO.3)

[0041] (4) Nucleic acid artificial sequence of PD-1 extracellular region (SEQ ID NO.4)

[0042] (5) CD8 Hinge region (CD8α) nucleic acid artificial sequence (SEQ ID NO.5)

[0043] (6) CD8 transmembrane region (CD8 TM) nucleic acid artificial sequence (SEQ ID NO.6)

[0044] (7) 41BB co-stimulatory region nucleic acid artificial sequence (SEQ ID NO.7)

[0045] (8) Nucleic acid artificial sequence of CD3ζ signaling region (SEQ ID NO.8)

[0046] (9) pLent-C-GFP vector (SEQ ID NO.9).

[0047] Order the sequences of SE...

Embodiment 2

[0049] Example 2 Preparation of TIGIT and PD-1 double gene knockout T cells

[0050] 1. Construction of CRISPR / CAS9 Knockout TIGIT and PD-1 Expression Vectors

[0051] Design the sgRNA according to the website http: / / chopchop.cbu.uib.no / (see Table 1 below), and screen the sgRNA fragment with few off-target sites and strong specificity from the TIGIT gene (SEQ ID NO.10) (TIGIT sgRNA ), the sgRNA fragment (PD-1 sgRNA) was screened out from the PD-1 gene (SEQ ID NO.11).

[0052] Table 1

[0053]

[0054] The TIGIT sgRNA nucleic acid sequence and PD-1 sgRNA nucleic acid sequence obtained by screening were entrusted to Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize and insert them into standard vectors and connect them to the CRISPR / CAS9 expression vector pX330A (purchased from Shanghai Yayong Pharmaceutical Technology Co., Ltd., SEQ ID NO.12) BbsI restriction site, respectively to obtain pX330A-TIGIT vector and pX330A-PD-1 vector (see figure 2 with 3 ).

[0055...

Embodiment 3

[0066] Example 3 Lentivirus packaging and titer detection

[0067] Inoculate the lentiviral packaging cell line 293T in a 10cm culture dish containing DMEM+10% FBS, culture at 37°C and 5% CO2, and prepare for transfection when the adherence rate is 70%-80%. Take a sterile 1.5ml EP tube and prepare the reaction system according to the following components: serum-free DMEM: 3ml; anti-TIGIT-PD-1-CAR plasmid: 10μg; GM easyTM Lentiviral Mix: 10μL (10μg); HG Transgene TM Reagent : 60 μL. After mixing, place it at room temperature for 20 minutes, evenly drop it into a culture dish containing 293T cells, and place it in a CO2 incubator for cultivation. After 24 hours of transfection, carefully suck off the cell culture medium and discard it in a waste liquid cup filled with disinfectant solution, and then add 15ml of fresh medium containing 10% serum to continue culturing. After changing the medium for 48 hours, draw the cell supernatant into a 50ml centrifuge tube, centrifuge at 50...

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Abstract

The invention provides a chimeric antigen receptor taking TIGIT and PD-1 as targets, and the chimeric antigen receptor comprises a TIGIT extracellular region and a PD-1 extracellular region; the invention also provides a CAR-T cell containing the chimeric antigen receptor and a preparation method thereof. The CAR-T cell prepared from the chimeric antigen receptor taking the immune checkpoints TIGIT and PD-1 as the targets can promote the proliferation of the CAR-T cell, improve the killing ability of the CAR-T cell, avoid immune escape of tumor cells and improve the effectiveness of CAR-T celltreatment. The Anti-TIGIT-PD-1-CAR-T cells prepared by the method disclosed by the invention is best in proliferation capacity and the most remarkable in effect of killing target cells, and the killing capacity of the Anti-TIGIT-PD-1-CAR-T cells is about three times that of single-target Anti-TIGIT-CAR or Anti-PD1-CAR.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to a novel CAR construction method and application for solid tumors targeting immune checkpoints TIGIT and PD-1. Background technique [0002] Tumors have always been a serious threat to human health. In recent years, tumor immunotherapy has developed rapidly and has become a hot spot and breakthrough in the field of tumor treatment. Tumor immunotherapy includes immune cell therapy, immune checkpoint inhibitors, cytokines, cellular vaccines, etc. Among them, chimeric antigen receptor T cell (Chimeric Antigen Receptor T-Cell, CAR-T cell) therapy is a new type of cell therapy, which mainly artificially transforms T cells of tumor patients through genetic engineering technology, and generates them after mass culture Tumor-specific CAR-T cells, which are then infused back into the patient to attack cancer cells, have more precise targeting, stronger lethality, and longer-lasting effects....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N5/10C12N5/0783C12N15/85
CPCC07K14/7051C07K16/28C07K16/2818C12N5/0636C12N15/85C07K2319/00
Inventor 刘明录金海锋卢永灿王立新强邦明张传鹏韩庆梅许淼冯建海王亮
Owner SHANDONG XINRUI BIOTECH CO LTD
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