Preparation method of genetically modified T cells expressing chimeric antigen receptors
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A chimeric antigen receptor, gene modification technology, applied in receptor/cell surface antigen/cell surface determinant, fusion cells, cells modified by introducing foreign genetic material, etc., can solve the problem of cell survival rate, cell proliferation rate Reduced, low gene transfer efficiency, cell damage, etc.
Active Publication Date: 2022-05-13
NAT UNIV CORP TOKAI NAT HIGHER EDUCATION & RES SYST
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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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preparation example Construction
[0095] The preparation method of this mode includes the following steps (i) to (iv). In addition, for matters not mentioned (for example, the preparation method of the T cell-containing cell population, the basic manipulation based on stimulation with anti-CD3 antibody and anti-CD28 antibody, the method for the treatment to incapacitate the proliferation ability, the method based on the transposon) The operation of gene introduction, the basic operation of co-cultivation, the recovery method of cells, etc.) are the same as the above-mentioned first preparation method, therefore, the overlapping description is omitted, and the corresponding description is cited.
[0096] (i) Step of preparing non-proliferative cells holding viral peptide antigens by stimulating the T cell-containing cell population with anti-CD3 antibody and anti-CD28 antibody , obtained by culturing in the presence of viral peptide antigens and treatment to incapacitate the ability to proliferate
[0097] (ii...
Embodiment
[0115]
[0116] Compared with the case of using viral vectors, CAR therapy using the transposon method is particularly advantageous in terms of safety. On the other hand, the efficiency of gene introduction is low, the cells are easily damaged during the operation (for example, electroporation) at the time of gene introduction, and the number of obtained cells is small. In order to overcome these problems, the following studies were conducted.
[0117] 1. Materials
[0118] (1) Antibodies
[0119] Anti-CD3 antibody (Miltenyi Biotec)
[0120] Anti-CD28 antibody (Miltenyi Biotec)
[0121] (2) Culture medium
[0122] TexMACS (Miltenyi Biotec)
[0123] (3) Cytokine
[0124] Recombinant human IL-7 (Miltenyi Biotec)
[0125] Recombinant human IL-15 (Miltenyi Biotec)
[0126] (4) Viral peptide mixture
[0127] PepTivator (registered trademark) CMV pp65-premium grade, human (Miltenyi Biotec)
[0128] PepTivator (registered trademark) AdV5Hexon-premium grade, human (Milteny...
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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...
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