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Preparation method of CAR-[gamma][delta]T cells

A cell and cell culture technology, applied in the field of CAR-γδT cell preparation, can solve the problems of unsatisfactory expansion and modification efficiency, and few reports on the preparation and culture of γδT cells

Active Publication Date: 2021-11-19
GUANGZHOU BIO GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are few reports on the preparation and culture of γδT cells modified for chimeric antigen receptors, and the efficiency of expansion and modification is not satisfactory

Method used

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  • Preparation method of CAR-[gamma][delta]T cells
  • Preparation method of CAR-[gamma][delta]T cells
  • Preparation method of CAR-[gamma][delta]T cells

Examples

Experimental program
Comparison scheme
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preparation example Construction

[0040] The present invention relates to a preparation method of CAR-γδT cells, which comprises:

[0041] 1) Cultivate PBMC in the culture medium for 2-4 days, and define the 0th day of initial culture as D0;

[0042] 2) transfer the cells to a culture container coated with RetroNectin and loaded with lentiviruses for expressing chimeric antigen receptor nucleic acid and culture the cells for 8h to 24h;

[0043] 3) Repeat step 2);

[0044] 4) Change the medium and inoculate into a new culture container to cultivate cells;

[0045] 5) Separating and purifying γδT cells from D7 to D9 to continue culturing;

[0046] 6) Harvest the γδT cells from D10 to D24.

[0047] In the present invention, by selecting a specific time point to transduce the CAR gene, the proportion of γδT cells is increased and the number of cells is small, so that the CAR can be modified as much as possible into the γδT cells; while the selection of the time point for separating and purifying γδT cells is in...

Embodiment 1

[0083] Example 1: Chimeric Antigen Receptor Design

[0084] In this example, the single-chain antibody (scFv) of the anti-CD19 antibody FMC63 was used as the antigen-binding domain, combined with the CD8α signal peptide, CD8α hinge region and transmembrane region, DAP10 co-stimulatory domain and CD3ζ signal transduction domain to construct a CD19-targeting Second-generation CARs.

[0085] FMC63-DAP10-CD3Z (468aa)

[0086] / / MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR / /

Embodiment 2

[0087] Example 2: Lentiviral Packaging

[0088] 2.1 Plasmid transfection: Put the plasmid, PEI, and Opti-MEM medium at room temperature for 5 minutes; take 36 μl of Opti-MEM4 in a 1.5ml EP tube, then add 64 μl of PEI to mix, and let stand at room temperature for 5 minutes; according to 6:9:9: 16 Add pLP1, pLP2, pLP-BaEVTR, expression plasmids, and add Opti-MEM to 500 μl, and let stand at room temperature for 5 minutes; add the prepared PEI-Opti-MEM solution to Opti-MEM containing plasmids, let stand at room temperature for 20 minutes ; Slowly drop 1ml of DNA / PEI mixture into 293T, mix gently, incubate in a 37°C incubator, replace with fresh medium after 16-18h, put in 5% CO 2 , and continue to incubate in a 37°C incubator.

[0089] 2.2 Virus collection and concentration: 48 hours after plasmid transfection, collect the supernatant, centrifuge at 2820g for 20min at 4°C to remove cell debris, and filter the supernatant with a 0.45μm filter; transfer the filtered virus supernata...

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Abstract

The invention relates to a preparation method of CAR-[gamma][delta]T cells. The method comprises the following steps: 1) culturing PBMC (peripheral blood mononuclear cells) in a culture medium for 2-4 days, and defining the 0th day of initial culture as D0; 2) transferring the cells into a culture container coated with RetroNectin and loaded with lentivirus for expressing nucleic acid of a chimeric antigen receptor, and culturing the cells for 8-24 hours; 3) repeating the step 2); 4) changing the liquid, and inoculating the cells into a new culture container to culture cells; 5) separating and purifying [gamma][delta]T cells at D7-D9, and continuously conducting culturing; and (6) harvesting [gamma][delta]T cells at D10-D24. In the step 1), the culture medium comprises a basic culture medium and additional components; and the additional components comprise 5%-15% (v / v) of FBS, 200 IU / mL to 800 IU / mL of IL2, 1 ng / mL to 20 ng / mL of IL21 and 1 [mu]M to 10 [mu]M of bisphosphonate.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for preparing CAR-γδT cells. Background technique [0002] At present, CAR T cells have made significant progress in the treatment of tumors, especially CD19 CAR T cells have achieved encouraging results in the treatment of B-cell malignant hematomas. Most of the current CAR T clinical trials are modified using the patient's own T cells. Therefore, the decline in the quality and quantity of T cells and the increase in the time and cost of preparing autologous CAR T cell products have become important factors hindering the development of CAR T cell therapy. . Therefore, general-purpose CAR T cell products can not only improve the quality and quantity of CAR T cells, but also save production time and cost. In addition, traditional CAR T is modified by using αβT cells, which rely on HLA to recognize tumor-associated antigens. If αβT cells are used as the cell type of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2509/00
Inventor 罗敏李光超丁雯周兆
Owner GUANGZHOU BIO GENE TECH CO LTD