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Specific chimeric antigen receptor cell armed with targeted CXCR2 ligand as well as preparation method and application thereof

A chimeric antigen receptor and armed target technology, applied in the field of drug preparation, amino acid coding sequence, and modified immune response cells, can solve the problems of low tumor cell specificity and low killing efficiency

Active Publication Date: 2020-09-01
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 low specificity and low killing efficiency of effector cells in the tumor environment that tend to bind and kill tumor cells in the existing tumor clinical technology To solve the problem, provide chemokines targeting CXCR2 ligands IL-8, CXCL1, and CXCL2 in the tumor microenvironment and chimeric antigen receptors that specifically target human NKG2DL, as well as their genes and recombinant expression vectors, and engineered arms Immune response cells modified specifically targeting human NKG2DL chimeric antigen receptor targeting CXCR2 ligand and application thereof

Method used

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  • Specific chimeric antigen receptor cell armed with targeted CXCR2 ligand as well as preparation method and application thereof
  • Specific chimeric antigen receptor cell armed with targeted CXCR2 ligand as well as preparation method and application thereof
  • Specific chimeric antigen receptor cell armed with targeted CXCR2 ligand as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Example 1 Preparation of an expression plasmid (KD-207 lentiviral vector) armed with a chimeric antigen receptor targeting CXCR2 ligand and specifically targeting human NKG2DL

[0142] The overall design is as follows:

[0143] 1. Determination of the amino acid sequence of a chimeric antigen receptor that specifically targets human NKG2DL and is armed with a CXCR2 ligand

[0144] First, the full-length amino acid sequence of human NKG2D (NP_031386.2) and the full-length amino acid sequence of human CXCR2 protein (NP_001548.1) were searched from the Genbank database of the National Library of Medicine (NCBI).

[0145] Secondly, construct a chimeric antigen receptor specifically targeting human NKG2DL that is armed with a CXCR2 ligand, that is, determine the amino acid sequence of the armed chimeric antigen receptor molecule:

[0146] From the amino terminal to the carboxyl terminal, the amino acid sequence of the guide peptide (as shown in SEQ ID No.1), the amino acid ...

Embodiment 2

[0165] Example 2: Preparation of virus liquid (KD-207 virus liquid) of lentiviral vector

[0166] The recombinant plasmid (KD-207 lentiviral vector) obtained in Example 1 expressing chimeric antigen receptors armed with targeting CXCR2 ligands 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 reagents (purchased from ThermoFisher, product model 11668019) were used to co-transfect 293T cells (see the ThermoFisher transfection manual for specific transfection procedures), 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 cells carrying the expression armed targeting CXCR2 ligand A viral fl...

Embodiment 3

[0167] Example 3: Isolation and culture of T cells

[0168] Take fresh peripheral blood from healthy donors, and separate fresh peripheral blood mononuclear cells by density gradient centrifugation; then use paramagnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody (purchased from Invitrogen, USA, product information To enrich CD3+ T cells for Dynabeads® Human T-Activator CD3 / CD28), 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 particle 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™ T-Cell Expansion SFM), and the cell solubility was adjusted to 1×10 6 pcs / ml, finally at...

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Abstract

The invention relates to a specific chimeric antigen receptor cell armed with a targeted CXCR2 ligand as well as a preparation method and application thereof. Through a method, which uses a recombinant carrier of a chimeric antigen receptor that specifically targets human NKG2DL armed with a targeting CXCR2 ligand to modify a immune response cell, the obtained novel functionalized immune responsecell can effectively attack multiple tumors in a targeted mode; and the functionalized immune response cell can be used to prepare preparations for treating malignant tumors. The engineering immune cells modified by a chimeric antigen receptor that is armed with a targeting CXCR2 ligand and specifically targets human NKG2DL can more easily migrate to a tumor position and continuously kill the tumors in a targeted mode.

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 a CXCR2 ligand and specifically targeting human NKG2DL, immune response cells modified therefor, and their preparation methods 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, the principle is mainly to take the patient's own T cells for...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K39/00A61K35/17A61P35/00A61P35/02A61P39/06A61P37/02A61P31/00
CPCA61K35/17A61K39/0011A61P31/00A61P35/00A61P35/02A61P37/02A61P39/06A61K2039/80A61K2039/804A61K2039/812A61K2039/828A61K2039/836A61K2039/844A61K2039/852A61K2039/86A61K2039/884C07K14/7051C07K16/24C07K16/2851C07K2319/02C07K2319/03C07K2319/33
Inventor 代红久徐慧朱靓婧
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
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