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A kind of preparation method and application of car-t cell targeting b7h3

A technology of B7H3-CAR and LV-B7H3, applied in the field of immunology, can solve the problems of limited protein expression and achieve high killing activity

Active Publication Date: 2022-07-15
江苏集萃崛创生物科技研究所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its mRNA expression is extensive, but protein expression is very limited

Method used

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  • A kind of preparation method and application of car-t cell targeting b7h3
  • A kind of preparation method and application of car-t cell targeting b7h3
  • A kind of preparation method and application of car-t cell targeting b7h3

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Embodiment 1

[0032] A preparation method of CAR-T cells targeting B7H3, comprising the following steps:

[0033] (1) Prepare PBMC cells;

[0034] Take 20ml of peripheral blood from healthy volunteers, centrifuge (1300g, 10 minutes) and reserve autologous plasma for later use. Dilute the remaining blood cells with an equal volume of normal saline, add them to the upper layer of the lymphocyte separation solution, centrifuge (600g, 25 minutes), and absorb the intermediate white blood cells. Membrane cells were washed with physiological saline, centrifuged (400 g, 10 minutes), and the supernatant was discarded to obtain PBMC cells.

[0035] (2) Mix the shuttle plasmid LV-B7H33.75 μg containing the CAR structure, the helper plasmid psPAX 23.75 μg, and the envelope plasmid VSV-G 2.5 μg in 300 μl opti-MEM medium, in another 300 μl opti-MEM medium Add 20 μl PEI reagent dropwise, shake and mix well, let stand for 5 minutes at room temperature, add the mixture containing PEI reagent dropwise to the ...

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Abstract

The present invention relates to a method for preparing CAR-T cells targeting B7H3. First, PBMC cells are prepared; then a shuttle plasmid LV-B7H3 containing a CAR structure, an auxiliary plasmid psPAX2 and an envelope plasmid VSV-G are co-transfected into 293T cells. , to obtain the packaged B7H3-CAR virus; then take PBMC cells, use anti-human CD3 and anti-human CD28 as activators, culture and activate for 48h, add B7H3-CAR virus for infection, and obtain. The preparation scheme increases the expression of IFN-γ in CAR-T cells and has high cell killing activity. The CAR-T cell targeting B7H3 of the present invention has killing effect on various solid tumor cells, has high killing activity, is safe and effective, and can be used for kidney cancer, lung cancer, liver cancer, glioma, ovarian cancer, breast cancer and the like. immune cell therapy.

Description

technical field [0001] The invention relates to a preparation method of CAR-T cells targeting B7H3, belonging to the technical field of immunology. Background technique [0002] Chimeric Antigen Receptor Modified T cell (CAR-T) is a genetically modified T cell that contains tumor antigen-specific recognition single-chain antibody (scFv) and T cell activator by gene transduction technology. The sequenced CAR is introduced into the patient's T cells, so that these CAR-transduced T cells can directly recognize and activate cancer cell surface antigens, thereby killing cancer cells. It is precisely because CAR-T cells do not require antigen presentation to kill cancer cells, which greatly improves the killing efficiency of cancer cells. In 2012, Professor Carl June of the University of Pennsylvania used chimeric antigen receptor-modified T cells targeting CD19 to cure a child with leukemia, Emily Whitehead. In 2017, the U.S. FDA breakthrough approved two CAR-T cell drugs for B-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/62A61K35/17A61P35/00
Inventor 王刚郑骏年方琳柴大飞李慧忠杜红伟田卉
Owner 江苏集萃崛创生物科技研究所有限公司