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Immunomodulatory specific chimeric antigen receptor cells as well as preparation method and application thereof

A chimeric antigen receptor and specific technology, applied in the direction of blood/immune system cells, receptors/cell surface antigens/cell surface determinants, botanical equipment and methods, etc., can solve the problem of low killing efficiency, tumor cell The problem of poor specificity

Active Publication Date: 2020-12-29
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, Provide immune checkpoint regulatory molecules combined with chimeric antigen receptors specifically targeting human NKG2DL, and their recombinant expression vectors, engineered immune checkpoint regulation combined with specific targeting of human NKG2DL chimeric antigen receptor modified immune response cells and its application

Method used

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  • Immunomodulatory specific chimeric antigen receptor cells as well as preparation method and application thereof
  • Immunomodulatory specific chimeric antigen receptor cells as well as preparation method and application thereof
  • Immunomodulatory specific chimeric antigen receptor cells as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1 Preparation of expression plasmids for immune checkpoint regulation combined with chimeric antigen receptors specifically targeting human NKG2DL

[0134] The overall design is as follows:

[0135] 1. Determination of the amino acid sequence of a chimeric antigen receptor specifically targeting human NKG2DL through immune checkpoint regulation

[0136] First, the full-length amino acid sequence of human NKG2D (NP_031386.2), the full-length amino acid sequence of PD1 protein (NP_005009.2), and the full-length amino acid sequence of TIGIT protein were searched from the Genbank database of the National Library of Medicine (NCBI). (NP_776160.2).

[0137] Secondly, construct immune checkpoint regulation (PD1 or TIGIT) combined with a chimeric antigen receptor specifically targeting human NKG2DL, that is, determine the amino acid sequence of the chimeric antigen receptor molecule:

[0138]From the amino terminal to the carboxyl terminal, the amino acid sequence of ...

Embodiment 2

[0162] Example 2: Preparation of virus liquid of lentiviral vector

[0163] The recombinant plasmid (KD-593 / 323 expression plasmid) obtained in Example 1 expressing immune checkpoint regulation combined with the chimeric antigen receptor 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 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 expression of immune checkpoint regulation joint specificity The virus liquid of the len...

Embodiment 3

[0164] Example 3: Isolation and culture of T cells

[0165] 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 6pcs / ml, finally at 37°C, 5...

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Abstract

The invention relates to an immunomodulatory specific chimeric antigen receptor cell as well as a preparation method and application thereof. According to the invention, a recombinant vector of a chimeric antigen receptor of immune checkpoint regulation (PD1 or TIGIT) combined with specifically targeting human NKG2DL, and is used for modifying immune response cells; the obtained novel functionalized immune response cell can effectively target and attack various tumors, and a preparation for treating malignant tumors is prepared. Engineering immune cells modified by the chimeric antigen receptors of immune checkpoint regulation (PD1 or TIGIT) combined with specifically targeting human NKG2DL is adopted, inactivation of immune cells induced by inhibition factors is inhibited, immune escape of tumor cells is prevented, and antitumor activity can be obviously improved.

Description

technical field [0001] The invention belongs to the technical field of immunotherapy and biomedicine, and relates to an amino acid coding sequence of a chimeric antigen receptor that regulates immune checkpoints and specifically targets human NKG2DL, and modified immune response cells, as well as their preparation methods and Application 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] Immunotherapy represented by CAR-T (Chimeric Antigen Receptor T-Cell, Chimeric Antigen Receptor T-Cell) is based on the principle of modifying...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/10C12N15/867C12N7/01A61K39/00A61P35/00A61P35/02A61P37/02A61P31/12A61P31/04C12R1/93
CPCA61K2039/5154A61K2039/5158A61P31/04A61P31/12A61P35/00A61P35/02A61P37/02A61K39/001102A61K39/001111C07K14/70503C07K14/7051C07K14/70521C07K14/7056C07K2319/03C07K2319/33C12N5/0636C12N7/00C12N15/86C12N2510/00C12N2740/15021C12N2740/15043C12N2800/107
Inventor 代红久徐慧李晓宇朱靓婧
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
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