Method for preparing genetically-modified t cells which express chimeric antigen receptor

Abstract

In order to improve the efficiency of gene introduction in CAR therapy employing a transposon technique, provided is a method for preparing genetically-modified T cells which express a chimeric antigen receptor, comprising: (1) a step for preparing non-proliferative cells obtained by stimulating a group of cells including T cells using an anti-CD3 antibody and an anti-CD28 antibody, and thereafter, subjecting the cells to a treatment for causing the cells to lose their proliferation capability; (2) a step for obtaining genetically-modified T cells into which a target antigen-specific chimericantigen receptor gene has been introduced using a transposon technique; (3) mixing the non-proliferative cells prepared at step (1) with the genetically-modified T cells obtained at step (2), and co-culturing the mixed cells while stimulating the mixed cells using the anti-CD3 antibody and the anti-CD28 antibody; and (4) a step for collecting the cultured cells. Also, provided is a method for preparing genetically-modified T cells which express a chimeric antigen receptor, comprising: (i) a step for preparing non-proliferative cells holding a viral peptide antigen, which cells are obtained bystimulating a group of cells including T cells using an anti-CD3 antibody and an anti-CD28 antibody, and thereafter, subjecting the cells to culturing in the presence of the viral peptide antigen anda treatment for causing the cells to lose their proliferation capability; (ii) a step for obtaining genetically-modified T cells into which a target antigen-specific chimeric antigen receptor gene hasbeen introduced using a transposon technique; (iii) mixing the non-proliferative cells prepared at step (i) with the genetically-modified T cells obtained at step (ii), and co-culturing the mixed cells; and (iv) a step for collecting the cultured cells.

Description

technical field [0001] The invention relates to a preparation method and application of genetically modified T cells expressing chimeric antigen receptors. This application claims priority based on Japanese Patent Application No. 2015-200458 for which it applied on October 8, 2015, All the content of this patent application is incorporated in this specification 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 clinically used. A CAR typically has a structure in which the single-chain variable region of an antibody is used as an extracellular domain, and a transmembrane region, CD3ζ, and an intracellular domain of a molecule that transmits costimulatory signals are connected to the extracellular domain. By binding to the antigen according to the specificity of the antibody, CAR-T cells are activated to damage target cells (cancer ...

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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