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Construction and application of chimeric antigen receptor T cell having GM-CSF knockdown and secreting single chain antibody for neutralizing GM-CSF

A chimeric antigen receptor, GM-CSF technology, applied in the field of biotechnology engineering, can solve the problem of release syndrome, toxic and side effects, etc., and achieve the effect of improving overall safety, slowing down cytokine release syndrome and neurotoxicity

Active Publication Date: 2020-02-07
浙江启新生物技术有限公司
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Problems solved by technology

In recent years, CAR-T has shown amazing results in the treatment of malignant hematological diseases, but the application of this therapy is limited by its related toxic and side effects, mainly including cytokine release syndrome and neurotoxicity

Method used

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  • Construction and application of chimeric antigen receptor T cell having GM-CSF knockdown and secreting single chain antibody for neutralizing GM-CSF
  • Construction and application of chimeric antigen receptor T cell having GM-CSF knockdown and secreting single chain antibody for neutralizing GM-CSF
  • Construction and application of chimeric antigen receptor T cell having GM-CSF knockdown and secreting single chain antibody for neutralizing GM-CSF

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Experimental program
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Effect test

Embodiment Construction

[0019] Main experimental materials: EcoR V-HF, MluI-HF, NdeI restriction endonuclease (NEB company), seamless cloning enzyme (He Yuanbio), high-fidelity Prime GXL STAR enzyme (TAKARA company), TransStbl3 competent cell ( Quanshijin Biotechnology Co., 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 Sangon Bioengineering Co., Ltd.), primers (Jiangsu Jinweizhi Biotechnology Co., Ltd.).

[0020] 1. Construction of recombinant plasmids

[0021] ① Construction of plenti-CD19-anti-GM-CSF scFv recombinant plasmid:

[0022] anti-GM-CSF scFv (SEQ ID NO.5) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., using Ndel and MluI-HF double enzyme digestion plenti-EF1a-anti-CD19CAR vector and synthesized anti-GM-CSF scFv gene , so that both sides have Ndel and MluI cohesive ends, the react...

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Abstract

The invention discloses construction and application of a fourth-generation chimeric antigen receptor (CAR)-T cell secreting anti-GM-CSF scFv for neutralizing GM-CSF and having GM-CSF gene knockdown.The integral safety of CAR-T therapy can be improved by secreting anti-GM-CSF scFv neutralizing the GM-CSF, combining the GM-CSF gene knockdown in the CAR-T cell, preventing or retarding occurrence and development of the cytokine release syndrome and the neurotoxicity. Furthermore, a nucleotide sequence encoding microRNA is inserted into an EF1 alpha promoter, and the package titer of a chronic virus carrier is not influenced.

Description

[0001] 1. Technical field [0002] This patent belongs to the field of biotechnology engineering, and specifically relates to the construction of a fourth-generation chimeric antigen receptor (CAR)-T cell that secretes anti-GM-CSFscFv that neutralizes GM-CSF and incorporates GM-CSF gene knockdown and apply. [0003] 2. Technical background: [0004] CAR-T therapy is a cellular immunotherapy program based on chimeric antigen receptors. Through in vitro gene transfer technology, the gene sequence encoding chimeric antigen receptor (CAR) is transfected into T cells to generate tumor-specific T cells that can bind to target antigens. In recent years, CAR-T has shown amazing effects in the treatment of malignant hematological diseases, but the application of this therapy is limited by its related toxic and side effects, mainly including cytokine release syndrome and neurotoxicity. Cytokine release syndrome refers to the release of a large number of cytokines during the interaction...

Claims

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Application Information

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IPC IPC(8): C07K16/24C12N15/867C12N15/62C12N5/10A61K39/00A61P35/00
CPCC07K16/243C07K16/2803C07K14/7051C12N15/86C12N5/0636A61K39/001112A61P35/00C07K2317/622C07K2319/00C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156
Inventor 不公告发明人
Owner 浙江启新生物技术有限公司
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