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Chimeric antigen receptor cell targeting human claudin18.2 and nkg2dl and its preparation method and application

A chimeric antigen receptor, targeting technology, applied in genetically modified cells, microorganism-based methods, cells modified by introducing foreign genetic material, etc., can solve the problem of low killing efficiency and low tumor cell specificity, etc. problem, to achieve the effect of improving killing efficiency, high killing rate and simple steps

Active Publication Date: 2022-05-17
NANJING KAEDI BIOTHERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the above-mentioned problems and / or other problems in related technologies, the purpose of the present invention is to overcome the problems of low specificity and low killing efficiency of effector cells in the intra-tumor environment in killing tumor cells faced in the existing tumor clinical technology, Provides bispecific targeting binding polypeptide domains targeting human Claudin18.2 and NKG2DL or functional variants thereof, bispecific chimeric antigen receptors targeting human Claudin18.2 and NKG2DL or functional variants thereof, Its encoding nucleotide sequence and its expression vector, engineered immune response cells modified by bispecific chimeric antigen receptors targeting human Claudin18.2 and NKG2DL, and applications thereof

Method used

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  • Chimeric antigen receptor cell targeting human claudin18.2 and nkg2dl and its preparation method and application
  • Chimeric antigen receptor cell targeting human claudin18.2 and nkg2dl and its preparation method and application
  • Chimeric antigen receptor cell targeting human claudin18.2 and nkg2dl and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1 Expression plasmids of chimeric antigen receptors bispecifically targeting human Claudin18.2 and NKG2DL

[0128] The overall design is as follows:

[0129] 1. Determination of the amino acid sequence of the bispecific chimeric antigen receptor targeting Claudin18.2 and NKG2DL

[0130] First, the full-length amino acid sequence of human NKG2D (NP_031386.2) was searched from the Genbank database of the National Library of Medicine (NCBI), and the full-length human amino acid sequence number of Claudin18.2 is: NP_001002026.1. After immunizing mice with human Claudin18.2 protein, the antibody fragment was purified and humanized to obtain a humanized single-chain antibody (scFv) against human Claudin18.2; the single-chain antibody was composed of a heavy chain and a light chain. The connecting segment between chain and light chain is (G 4 S) 3 Finally, 4 scFv antibody fragments with high affinity to the target antigen Claudin18.2 were obtained through effect scr...

Embodiment 2

[0155] Example 2: Preparation of virus liquid of lentiviral vector

[0156] The recombinant plasmid (KD-496 expression plasmid) obtained in Example 1 and the packaging vector pol / gag, Rev and VSVG of the chimeric antigen receptor targeting human Claudin18.2 and NKG2DL bispecifically, according to 12:10:5 :6 ratio, with Lipofectamine TM 6000 transfection reagents (purchased from Beyontien, product model C0526) were used to co-transfect 293T cells (see the transfection instructions for specific transfection procedures), and replaced with complete medium 6 hours after transfection (purchased from hyclone, The product model is SH30243.01), after 48 hours and 72 hours of culture, the cell supernatants rich in lentiviral particles were collected respectively, and the virus supernatants were concentrated by ultracentrifugation to obtain the bispecific targeting human Claudin18.2 The virus liquid of the lentiviral vector of the chimeric antigen receptor of NKG2DL (hereinafter referr...

Embodiment 3

[0157] Example 3: Isolation and culture of T cells

[0158] Fresh peripheral blood from healthy donors was taken, and fresh peripheral blood mononuclear cells were separated by density gradient centrifugation; paramagnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody (purchased from Invitrogen, USA, product information for Human T-Activator CD3 / CD28) to enrich CD3+ T cells, specifically, dilute peripheral blood mononuclear cells to a concentration of (10-30)×10 6 single cells / ml, and then mixed the magnetic beads and cells in a culture dish at a ratio of 3:1, incubated at room temperature for 2-3 hours, and used a Magnetic particles concentrator (MPC for short, purchased from Invitrogen, USA) company) to enrich CD3+ T cells. Finally, the enriched CD3+T cells were resuspended in culture medium (purchased from Life Technologies, USA, product information is OpTmizer TM T-Cell Expansion SFM), adjusted to a cell concentration of 1×10 6 pcs / ml, finally at 37°C, ...

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Abstract

The invention belongs to the field of chimeric antigen receptor cells, and relates to a chimeric antigen receptor cell targeting human Claudin18.2 and NKG2DL and its preparation method and application. The chimeric antigen receptor cell contains targeting human Claudin18.2 and target-binding amino acid sequence of human NKG2DL. The immune response cells modified by the bispecific chimeric antigen receptor targeting Claudin18.2 and NKG2DL of the present invention can enhance the combination with tumor cells and obviously improve the anti-tumor activity.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptor cells, and relates to an amino acid coding sequence of a bispecific chimeric antigen receptor targeting human Claudin18.2 (CLDN18.2) and NKG2DL, and its modified immune response Cells, methods of their preparation and use in the manufacture of medicaments. Background technique [0002] With the rapid development of biotechnology, immune cell therapy has become the fourth largest therapy in the field of cancer treatment. [0003] Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, and immune junction blockade therapy. Among them, in the field of cell therapy, CAR-T therapy has undoubtedly become a star that research institutions and pharmaceutical companies are vying for. [0004] Immunotherapy represented by CAR-T (Chimeric Antigen Receptor T-Cell, Chimeric Antigen Receptor T-Cell) is based on the principle of modifying chimeric antigen receptors...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N7/01C12N15/867A61K39/00A61P31/04A61P31/12A61P35/00A61P35/02A61P37/02C12R1/93
CPCC07K16/28C07K14/7056C07K14/7051C12N5/0636C12N7/00C12N15/86A61K39/001102A61K39/001111A61P35/00A61P35/02A61P37/02A61P31/12A61P31/04C07K2319/03C07K2319/33C07K2317/622C12N2510/00C12N2740/15021C12N2740/15043C12N2800/107
Inventor 代红久徐慧朱靓婧
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
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