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CAR-T cell for overcoming TGF-beta immunosuppression for solid tumor

A TGF-, immunosuppressive technology, applied in the field of genes, can solve the problems of off-target effect of tumor tissue, poor tumor killing effect, low anti-tumor activity, etc., achieve extensive immune response, improve killing effect, and increase the effect of proliferation ability

Active Publication Date: 2021-01-12
山东省成体细胞产业技术研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Existing immune cell therapy for solid tumors has the following problems: the combined therapy of checkpoint inhibitors and CAR-T cells, due to the high degree of heterogeneity of tumor tissue, during the treatment process , there will be off-target effects of tumor tissue and tumor immune escape problems during the treatment process, accompanied by toxic and side effects, making CAR-T ineffective in treating solid tumors
During the treatment of tumors, the existing tumor-specific CAR-T cells contain more TGF-β in the tumor microenvironment, which will lead to poor killing effect of CAR-T cells on tumors, resulting in low anti-tumor activity

Method used

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  • CAR-T cell for overcoming TGF-beta immunosuppression for solid tumor
  • CAR-T cell for overcoming TGF-beta immunosuppression for solid tumor
  • CAR-T cell for overcoming TGF-beta immunosuppression for solid tumor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Selection of solid tumor targets

[0053] The present invention uses gene editing technologies such as CAR-T cells, specifically as follows:

[0054] Construct a dual-target CAR, targeting TGF-β, using anti-TGF-β monoclonal antibodies to neutralize TGF-β factors in the tumor microenvironment, and inhibit tumor cell growth;

[0055] Construct GPC3 CAR-T co-expressing dominant negative transforming growth factor receptor II (Dn-TGFβRII), and use Dn-TGFBRII to block TGF-β signaling pathway;

[0056] Combine CAR-T cell therapy with transforming growth factor receptor II (TGFβRII), and use CRISPR / Cas9 technology to knock out TGFβRII in CAR-T cells to block the TGF-β signaling pathway.

Embodiment 2

[0057] Example 2 Construction of CAR expression vector

[0058] 1. Construction of Anti-TGF-β-GPC3-CAR double expression vector

[0059] Anti-TGF-β-GPC3-CAR module diagram figure 1 . The nucleic acid sequences of each module of Anti-TGF-β-GPC3-CAR are as follows:

[0060] (1) CD8 leader leader nucleic acid artificial sequence (SEQ ID NO.1)

[0061] (2) Anti-TGF-β (scFv) nucleic acid artificial sequence (SEQ ID NO.2)

[0062] (3) Linker nucleic acid artificial sequence (SEQ ID NO.3)

[0063] (4) Anti-GPC3 (scFv) nucleic acid artificial sequence (SEQ ID NO.4)

[0064] (5) CD8 Hinge region (CD8α) nucleic acid artificial sequence (SEQ ID NO.5)

[0065] (6) CD8 transmembrane region (CD8 TM) nucleic acid artificial sequence (SEQ ID NO.6)

[0066] (7) Nucleic acid artificial sequence of CD226 co-stimulatory region (SEQ ID NO.7)

[0067] (8) 41BB co-stimulatory region nucleic acid artificial sequence (SEQ ID NO.8)

[0068] (9) Nucleic acid artificial sequence of CD3ζ signalin...

Embodiment 3

[0096] Example 3 Lentivirus packaging and titer detection

[0097] 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-TGF-β-GPC3-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 CAR-T cell for overcoming TGF-beta immunosuppression for a solid tumor. The CAR-T cell is a CAR-T cell for blocking a TGF-beta signal and targeting the solid tumor. The double-target CAR is adopted to increase the number of effective antigens on the surface of the solid tumor, wider immune response is carried out, the activation of the T cell is enhanced through the synergistic effect, the binding efficiency of the CAR-T and the tumor cell is improved, and the problems of relapse and metastasis caused by tumor immune escape are solved to a greater extent. The CAR capable of keeping activity in TME and resisting immunosuppressive cytokines is designed, the expression of corresponding immunosuppressive cytokines (TGF beta RII) on the surface of the T cell is reducedthrough a gene editing technology (CRISPR / CAS9), the tumor microenvironment is overcome, the activity of the CAR-T cell is improved, and the CAR-T cell is more effective in solid tumor treatment.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to a novel CAR design method for overcoming TGF-β immunosuppression for solid tumors. Background technique [0002] In recent years, tumor immunotherapy has developed rapidly and has become the fourth new technology for tumor treatment after surgery, radiotherapy, and chemotherapy. It has attracted extensive attention from the medical community 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 is different from traditional immunotherapy. Tumor-specific CAR-T cells are generated after a large number of cultures, and then reinfused into the patient to attack cancer cells. The targeting is more precise, the lethal...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62A61K39/00A61P35/00
CPCC12N5/0636C07K16/22C07K16/303C07K14/7051C07K14/71A61K39/001134A61P35/00C12N2510/00C07K2317/622C07K2319/33C07K2319/03
Inventor 刘明录强邦明卢永灿王立新金海锋冯建海张传鹏许淼王亮
Owner 山东省成体细胞产业技术研究院有限公司
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