Fourth-generation CAR-T cell as well as construction method and application thereof

An XCAR1 & CAR2-Y-Z, cell technology, applied in the field of biotechnology engineering, can solve the problems of lack of target antigen, immune escape, short duration of CAR-T cells, etc., and achieve the effect of reducing the recurrence of malignant tumors

Active Publication Date: 2019-03-22
温州启星生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the achievements of CAR-T therapy in the treatment of malignant hematological tumors are obvious to all (such as Kymriah for refractory / relapsed acute B-lymphoblastic leukemia and refractory / relapsed non-Hodgkin's Lymphoma Yescarta was launched in the United States last year), but at least 50% of the recurrence rate, and CAR-T cells are not effective in treating solid tumors, the main reason is that there is a lack of suitable target antigens, CAR-T cells persist Challenging problems such as short time, immune escape, and immunosuppressive tumor microenvironment

Method used

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  • Fourth-generation CAR-T cell as well as construction method and application thereof
  • Fourth-generation CAR-T cell as well as construction method and application thereof
  • Fourth-generation CAR-T cell as well as construction method and application thereof

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Embodiment Construction

[0043] The following describes the embodiments of the present invention in detail with reference to the accompanying drawings, but the present invention can be implemented in a variety of different ways defined and covered by the claims.

[0044] Main experimental materials:

[0045] EcoRI-HF, MluI-HF, NdeI restriction endonucleases (NEB), seamless cloning enzyme (Heyuan Bio), high-fidelity Prime GXL STAR enzyme (TAKARA), TransStbl3 Competent Cells (Quanshijin Biotech) Ltd.), Plasmid Mini Kit I (OMEGA), Plasmid Maxi Kit (QIAGEN), DMEM, RPMI-1640, Opti-MEM medium, Gibco FBS (Thermo Fisher Scientific), Sanger sequencing (Shanghai Sunny Biological Co., Ltd.), Nacl, yeast powder, peptone, EDTA, NaOH (Shanghai Shenggong Biological Engineering Co., Ltd.), primer (Jiangsu Jinweizhi Biological Technology Co., Ltd.).

[0046] 1. Construction of recombinant plasmid

[0047] (1) Construction of Plenti-Nectin4CAR1 and Plenti-Nectin4CAR2 recombinant plasmids:

[0048] Nectin4CAR1 (hCD8leader-VL1...

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Abstract

The invention provides construction and application of a fourth-generation chimeric antigen receptor T (CAR-T) cell (expressing IL-7 and CCL19) specific to Nectin-4 dual targets on the surface of a malignant tumor. Two spatial epitopes of a Nectin-4 antigen are taken as target points, and the constructed fourth-generation CAR-T is used for treating and expressing a malignant solid tumor of the Nectin-4 antigen to solve the immune escape problem of the CAR-T during treatment of the solid tumor; and the constructed fourth-generation CAR-T can enhance proliferative capacity and sustained survivability, so that the solid tumor is effectively treated, and a new strategy is provided for effective prevention and treatment of postoperative recurrence / metastasis of the solid tumor.

Description

Technical field [0001] The present invention belongs to the field of biotechnology engineering, and specifically relates to the construction and application of fourth-generation chimeric antigen receptor T (CAR-T) cells (expressing IL-7 and CCL19) targeting Nectin-4 dual targets on the surface of malignant tumors. Background technique [0002] With the continuous deterioration of global environmental factors, the incidence and mortality of malignant tumors are on the rise in both developed and developing countries, and malignant tumors have become the greatest hazard to human life and health. About 4.3 million people were diagnosed with cancer in China in 2015, and 2.8 million people died of cancer. Most of them were solid tumors (such as lung cancer, breast cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, etc.). There will be 5.5 million new cancer cases, of which the death toll will reach 4 million. Due to the inherent shortcomings of traditional treatm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/52C07K14/5418C07K14/7051C07K14/70517C07K16/2803C07K2317/622C07K2319/00C07K2319/02C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 高基民李帆帆
Owner 温州启星生物技术有限公司
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