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Armed specific chimeric antigen receptor cells targeting TGF-β and its preparation method and application

A chimeric antigen receptor, TGF- 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. problems, to achieve the effect of improving killing efficiency, high killing rate, and good industrial application prospects

Active Publication Date: 2021-04-30
NANJING KAEDI BIOTHERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] 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, Provide TGF-β subtypes TGF-β1, TGF-β2, TGF-β3 cytokines targeting tumor microenvironment and chimeric antigen receptors specifically targeting human NKG2DL, and their genes and recombinant expression vectors, engineered Armed and targeted TGF-β specific targeting human NKG2DL chimeric antigen receptor modified immune response cells and application thereof

Method used

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  • Armed specific chimeric antigen receptor cells targeting TGF-β and its preparation method and application
  • Armed specific chimeric antigen receptor cells targeting TGF-β and its preparation method and application
  • Armed specific chimeric antigen receptor cells targeting TGF-β and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1 Preparation of an expression plasmid that is armed with a chimeric antigen receptor that targets TGF-β and specifically targets human NKG2DL

[0131] The overall design is as follows:

[0132] 1. Determination of the amino acid sequence of the chimeric antigen receptor armed with TGF-β and specifically targeting human NKG2DL

[0133] First, the full-length amino acid sequence of human NKG2D (NP_031386.2) and the full-length amino acid sequence of human TGF-βRⅡ protein (NP_003233.4) were searched from the Genbank database of the National Library of Medicine (NCBI).

[0134] Secondly, construct a chimeric antigen receptor specifically targeting human NKG2DL that targets TGF-β, that is, determine the amino acid sequence of the armed chimeric antigen receptor molecule:

[0135] From the amino terminal to the carboxyl terminal, the amino acid sequence of the guide peptide (as shown in SEQ ID No.1), the human NKG2D amino acid sequence (such as SEQ ID No.2, SEQ ID N...

Embodiment 2

[0157] Example 2: Preparation of virus liquid of lentiviral vector

[0158] The recombinant plasmid (KD-275 expression plasmid) obtained in Example 1 expressing the chimeric antigen receptor targeting TGF-β and specifically targeting human NKG2DL and the packaging vectors psPAX2 and VSVG, according to the ratio of 10:8:5 Ratio, with Lipofectamine TM 6000 transfection reagent (purchased from ThermoFisher, product model 11668019) co-transfected 293T cells (see ThermoFisher transfection manual for specific transfection operation process), and replaced with complete medium 6 hours after transfection (purchased from Life Technologies , the product model is 11995-065), after culturing for 48 hours and 72 hours, the cell supernatants rich in lentiviral particles were collected respectively, and the virus supernatants were concentrated by ultracentrifugation to obtain cells carrying TGF-β and expressing armed targets. The virus liquid of the lentiviral vector specifically targeting ...

Embodiment 3

[0159] Example 3: Isolation and culture of T cells

[0160] 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), adjust the cell solubility to 1×10 6 pcs / ml, finally at 37°C, 5% CO ...

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Abstract

The present invention relates to a coding amino acid sequence of a chimeric antigen receptor armed with targeting TGF-β and specifically targeting human NKG2DL, and a modified immune response cell thereof, its preparation method and application. The present invention is a method of arming the recombinant vector of the chimeric antigen receptor specifically targeting human NKG2DL targeting TGF-β, and using it to modify immune response cells; the obtained novel functional immune response cells can effectively target and attack multiple A tumor, and the prepared preparation for treating malignant tumors. The chimeric antigen receptor-modified engineered immune cells armed with targeting TGF-β and specifically targeting human NKG2DL of the present invention can suppress immunosuppression in the tumor microenvironment and prevent immune escape of tumor cells, and can significantly improve the killing efficiency of tumor cells .

Description

technical field [0001] The invention belongs to the technical field of immunotherapy biomedicine, and relates to an amino acid coding sequence of a chimeric antigen receptor armed with targeting TGF-β and specifically targeting human NKG2DL, immune response cells modified therefor, and their preparation method and applications in pharmaceutical preparation. 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] CAR-T (Chimeric Antigen Receptor T-Cell, Chimeric Antigen Receptor T-Cell) immunotherapy, its principle is mainly to take the patient's own T cells for g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00A61P37/02A61P31/04A61P31/10A61P39/06A61P35/02C12R1/93
CPCA61K2039/5154A61K2039/5156A61K2039/5158A61P31/04A61P31/10A61P35/00A61P35/02A61P37/02A61P39/06A61K39/001102A61K39/001103A61K39/001111C07K14/7051C07K14/7056C07K14/71C07K2319/03C07K2319/33C12N5/0636C12N7/00C12N15/86C12N2510/00C12N2740/15021C12N2740/15043C12N2800/107
Inventor 代红久徐慧王梦瑶朱靓婧
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
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