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Construction and application of third-generation NKG2D chimeric antigen receptor T or NK cell

A chimeric antigen receptor and cell technology, which is used in the construction and application of third-generation NKG2DCAR-T or CAR-NK cells, and can solve problems such as increased ligand expression.

Active Publication Date: 2020-08-04
浙江启新生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] NKG2DL is the ligand of NKG2D, which is not expressed or expressed very little on the surface of normal cells, but when cells are infected by pathogens or become cancerous or senescent, the expression of these ligands will increase significantly

Method used

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  • Construction and application of third-generation NKG2D chimeric antigen receptor T or NK cell
  • Construction and application of third-generation NKG2D chimeric antigen receptor T or NK cell
  • Construction and application of third-generation NKG2D chimeric antigen receptor T or NK cell

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Experimental program
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Embodiment

[0018] Main experimental materials:

[0019] EcoRI-HF, MluI-HF (NEB Company), Seamless Cloning Enzyme (He Yuanbio), High Fidelity Prime GXL STAR Enzyme (TAKARA Company), TransStbl3 Competent Cells (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.), yeast powder, peptone, EDTA, NaOH (Shanghai Sangong Biological Engineering Co., Ltd.), primers (Jiangsu Jinweizhi Biotechnology Co., Ltd.).

[0020] 1. Construction of recombinant plasmids

[0021] ①Construction of Plenti-anti-NKG2DL-CAR (SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 nucleotide sequences connected end to end) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. using Ndel and MluI-HF The plenti-EF1a--MCS vector and the synthetic NKG2D CAR gene were double-digested so that both had Ndel and MluI cohesive ends. The reaction conditions were 37°C for...

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Abstract

The invention discloses construction and application of a third-generation NKG2D chimeric antigen receptor T or NK cell. The third-generation NKG2D chimeric antigen receptor T or NK cell mainly comprises a total length NKG2D,41BB costimulatory molecules, and CD3z ; according to the construction and application, the structure uses both 41BB co-stimulatory molecules and NKG2D natural co-stimulatorymolecules DAP10, and NKG2D is an NK cell surface-activated receptor, and can bind to eight ligands (MICA, MICB, ULBP1-6) of NKG2D; the NKG2D ligand (NKG2DL) is not expressed on the surface of a normalcell or the expression quantity is very small, but when the cell is infected by pathogens or cancerated or aged, the expression quantity of the ligands can be greatly increased, so that the ligands become ideal immunotherapy targets.

Description

technical field [0001] The invention belongs to the field of biotechnology engineering, and specifically relates to the construction and application of third-generation NKG2D CAR-T or CAR-NK cells (taking multiple myeloma and non-small cell lung cancer as examples). Background technique [0002] CAR-T or CAR-NK therapy is a kind of cellular immunotherapy based on chimeric antigen receptor (CAR). Through in vitro gene transfer technology, the gene sequence encoding chimeric antigen receptor (CAR) is transfected into T cells or NK cells to generate tumor-specific T cells or NK cells that can bind to the target antigen. Ordinary CAR-T or CAR-NK cells include single-chain recognition antibodies outside the membrane, transmembrane segments, costimulatory molecules (41BB, CD28, etc.) and CD3z in the intracellular segment, NKG2D CAR-T cells or CAR-NK cells are different The extracellular segment is composed of NKG2D receptors, which directly recognize and kill tumor cells. [000...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/10
CPCC07K14/7051C07K14/705C07K2319/00
Inventor 高基民魏成王春玲赵爱
Owner 浙江启新生物技术有限公司
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