Preparation method of genetically modified T cells expressing chimeric antigen receptors

Abstract

The present invention realizes the improvement of the gene introduction efficiency of CAR therapy using the transposon method. The present invention provides a method for preparing genetically modified T cells expressing chimeric antigen receptors, comprising the following steps: (1) preparing non-proliferative cells, wherein the non-proliferative cells are obtained by using anti-CD3 antibodies and It is obtained by stimulating the cell population containing T cells with an anti-CD28 antibody, and then treating the proliferation ability; (2) obtaining the genetically modified T cells with the target antigen-specific chimeric antigen receptor gene introduced by the transposon method The step of cells; (3) mixing the non-proliferative cells prepared in step (1) and the genetically modified T cells obtained in step (2), and co-cultivating them while stimulating with anti-CD3 antibody and anti-CD28 antibody step; (4) the step of recovering the cultured cells. In addition, the present invention provides a method for preparing a genetically modified T cell expressing a chimeric antigen receptor, comprising the following steps: (i) preparing a non-proliferative cell retaining a viral peptide antigen, said viral peptide retaining Antigen non-proliferative cells are obtained by culturing in the presence of viral peptide antigens and incapacitating the proliferative ability after stimulating a T-cell-containing cell population with an anti-CD3 antibody and an anti-CD28 antibody; (ii) A step of obtaining a genetically modified T cell introduced with a target antigen-specific chimeric antigen receptor gene by a transposon method; (iii) combining the non-proliferative cells prepared in step (i) and the gene obtained in step (ii) The step of modifying T cell mixing and co-cultivation; (iv) the step of recovering the cultured cells.

Description

technical field [0001] The present invention relates to a preparation method of gene-modified T cells expressing chimeric antigen receptor and use thereof. This application claims priority based on Japanese Patent Application No. 2015-200458 for which it applied on October 8, 2015, the entire content of which is incorporated herein by reference. Background technique [0002] Gene-modified T-cell therapy (CAR therapy) using a chimeric antigen receptor (Chimeric Antigen Receptor. hereinafter also referred to as "CAR") is being used clinically. A CAR typically has the following structure: a single-chain variable region of an antibody as an extracellular domain, to which a transmembrane region, CD3ζ, and an intracellular domain of a molecule that transmits costimulatory signals are linked. By binding to the antigen according to the specificity of the antibody, CAR-T cells are activated and damage target cells (cancer cells, etc.). CAR therapy has the advantages of relatively e...

AI Technical Summary

Problems solved by technology

Although the transposon method can carry out long-lasting gene transfer similarly to the viral vector method, there are the following problems: compared with the viral vector method, the gene transfer efficiency is low, and in addition, the cells follow the gene transfer operation (electroporation and its improved method, etc.) ) was damaged, cell survival rate, cell proliferation rate decreased

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