Preparation method and application of CAR-T cell targeting B7H3

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

Active Publication Date: 2019-06-14
XUZHOU MEDICAL UNIV
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  • Abstract
  • Description
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Problems solved by technology

Its mRNA expression is extensive, bu

Method used

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  • Preparation method and application of CAR-T cell targeting B7H3
  • Preparation method and application of CAR-T cell targeting B7H3
  • Preparation method and application of CAR-T cell targeting B7H3

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Embodiment 1

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

[0033] (1) prepare PBMC cells;

[0034] Take 20ml of peripheral blood from healthy volunteers, centrifuge (1300g, 10 minutes) and save the autologous plasma for later use. The remaining blood cells are diluted with an equal volume of normal saline, added to the upper layer of the lymphocyte separation medium, centrifuged (600g, 25 minutes), and the intermediate white blood The membrane layer 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-B7H3 3.75 μg containing the CAR structure, the helper plasmid psPAX2 3.75 μg, and the envelope plasmid VSV-G 2.5 μg in 300 μl opti-MEM medium, and in another 300 μl opti-MEM medium Add 20 μl PEI reagent dropwise, shake and mix, and let stand at room temperature for 5 minutes, add the mixture containing PEI reagent to the plasmid mixture...

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Abstract

The invention relates to a preparation method of a CAR-T cell targeting B7H3. The preparation method includes first preparing a PBMC cell; then co-transfecting a 293T cell with a shuttle plasmid LV-B7H3 containing the CAR structure, a helper plasmid psPAX2 and an envelope plasmid VSV-G to obtain a packaged B7H3-CAR virus; then taking a PBMC cell, using anti-human CD3 and anti-human CD28 as activators, culturing and activating for 48 hours and adding the B7H3-CAR virus for infection. By means of the preparation scheme, the expression of IFN-gamma in the CAR-T cell is increased, and the cell killing activity is high. The CAR-T cell targeting B7H3 has a killing effect on various solid tumor cells, has high killing activity, is safe and effective, and can be used for immunotherapy of kidney cancer, lung cancer, liver cancer, glioma, ovarian cancer, breast cancer and the like.

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 kind of genetically modified T cell, which contains tumor antigen-specific recognition single-chain antibody (scFv) and T cell activation gene 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 the surface antigens of cancer cells, and then kill cancer cells. It is precisely because CAR-T cells do not need 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 leukemia patient Emily Whitehead. In 2017, the US FDA made a breakthrough approval of two CA...

Claims

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Application Information

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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 XUZHOU MEDICAL UNIV
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