Genetically modified immune cell as well as preparation method and application thereof
A technology of immune cells and coding genes, which is applied in the field of cellular immunotherapy, can solve the problems of inability to add functional components and further enrich the functions of CAR-T or CAR-NK cells, and achieve high-efficiency transfection, enhanced clearance, and enriched functions Effect
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Embodiment 1
[0066] Example 1 Design and Construction of CAR Molecular Carrier
[0067] In this example, the fragments with the complete DAP10 molecule and intracellular signal transduction domains of 4-1BB and CD3ζ were first synthesized, and connected to the pWPXLD-EGFP vector to construct the expression plasmid DAP10BB3 (abbreviated as DAP10), such as Figure 1A shown;
[0068] Subsequently, the scFv targeting GPC3 and Meothelin (including the IgG-CH3 region) were specifically amplified by PCR, and cloned into the above-mentioned expression plasmid DAP10, respectively, to obtain chimeric DAP10 cells targeting GPC3 (phosphatidylinositol protein 3). The CAR molecule antiGPC3-DAP10ec+tm+cd-BB3 (GPC3-DAP10 for short) of the outer and transmembrane region, 4-1BB and CD3ζ signal transduction domain, and the chimeric DAP10 targeting Mesothelin (mesothelin) extracellular and The CAR molecule antiMSLN-DAP10ec+tm+cd-BB3 (MSLN-DAP10 for short) of the transmembrane region, 4-1BB and CD3ζ signal tra...
Embodiment 2
[0069] Example 2 Overexpression of CAR virus packaging
[0070] Transform the constructed plasmid into Escherichia coli, select a single clone with the target plasmid for overnight culture, use a plasmid extraction kit to extract the plasmid, and carry out virus packaging;
[0071] Use 293T cells for virus packaging, transfect the helper plasmid and the expression plasmid with CAR molecules into 293T cells at the same time, harvest the first and second viruses after 48h and 72h respectively, filter and freeze at -80°C for T used for cell transduction.
Embodiment 3
[0072] Example 3 Construction of Genetically Modified T Cells Expressing CAR
[0073] First, mononuclear cells (PBMC) were isolated from adult peripheral blood, and the total T cells were sorted out using a T cell sorting kit. After being stimulated by CD3 and CD28 antibodies in vitro for 24 hours, the overexpressed CAR molecule prepared in Example 2 was added. 12 hours after transduction, the T cells were centrifuged to change the medium; three days after the transduction, the content of GFP-positive cells was measured by flow cytometry, and the ratio of CAR-T was evaluated; 2×10 cells were cultured per ml of medium. 6The density culture of 1 cell expands CAR-T cell, obtains DAP10-CAR-T (the amino acid sequence of DAP10-CAR is shown in SEQ ID NO:2), antiGPC3-DAP10-CAR-T (the amino acid sequence of antiGPC3-DAP10-CAR The sequence is shown in SEQ ID NO: 3), antiMSLN-DAP10-CAR-T (the amino acid sequence of antiMSLN-DAP10-CAR is shown in SEQ ID NO: 4) cells, and the cells were fr...
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