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Macrophages capable of targeting tumor cells and preparation method thereof

A macrophage and tumor cell technology, applied in the field of macrophage and its preparation, can solve the problems of poor recognition ability and weak killing effect, and achieve the effect of easy entry

Active Publication Date: 2019-01-25
赛元生物科技(杭州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first purpose of the present invention is to provide a macrophage capable of targeting tumor cells, so as to alleviate the poor recognition and killing ability of CAR-T cells on tumor cells, especially solid tumor cells, in CAR-T cell therapy in the prior art weak technical issues

Method used

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  • Macrophages capable of targeting tumor cells and preparation method thereof
  • Macrophages capable of targeting tumor cells and preparation method thereof
  • Macrophages capable of targeting tumor cells and preparation method thereof

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preparation example Construction

[0102] A method for preparing macrophages capable of targeting tumor cells, expressing genes encoding chimeric antigen receptors in macrophages to obtain macrophages capable of targeting tumor cells. This method provides a new idea for immuno-oncology therapy.

[0103] In a preferred embodiment of the present invention, the preparation method further includes the step of preparing HLA-I gene-deficient macrophages.

[0104] In a preferred embodiment of the present invention, the preparation method further includes the step of preparing B2M gene-deficient macrophages.

[0105] In a preferred embodiment of the present invention, the preparation method includes directional differentiation of pluripotent stem cells to obtain macrophages capable of targeting tumor cells, and the pluripotent stem cells contain genes encoding chimeric antigen receptors.

[0106] In some embodiments, the pluripotent stem cells are HLA-I deficient pluripotent stem cells.

[0107] In some embodiments, ...

Embodiment 1

[0163] Example 1 Preparation of induced pluripotent stem cells

[0164]On day -1, 10ml of peripheral blood was drawn from the patient or volunteer, and PBMC (peripheral blood mononuclear cells) were separated with lymphocyte separation medium, cultured in H3000+CC100, and MEF cells (fibroblasts) were revived.

[0165] On day 0, take 1-2 million PBMCs and electroporate plasmids containing reprogramming factors OCT4, SOX2, KLF4, LIN28, and L-MYC. After electroporation, the cells are cultured in H3000+CC100 medium, centrifuged at 250 rcf for 5 min after 4 h, and discarded. The supernatant was resuspended with H3000+CC100 medium, and cultured in MEF cell plates.

[0166] On day 2, recover MEF cells.

[0167] On day 3, take the cell supernatant into a 15ml centrifuge tube, add 200ul Tryple to digest the adherent cells for 5min, stop the digestion with 1ml H3000, pipette and transfer to the corresponding centrifuge tube, centrifuge at 250rcf for 5min, discard the supernatant, and u...

Embodiment 2

[0172] Example 2 Recovery, culture and passage of 293T cells

[0173] (1) Resuscitation: Take out the frozen cells from the liquid nitrogen tank and quickly place them in a 37°C water bath, shake them quickly to melt them. Prepare a 15ml centrifuge tube in an ultra-clean bench, add 5ml of complete medium and the cells in the frozen tube, mix well, and centrifuge at 250rcf / min for 5min. Discard the supernatant, resuspend with 5ml of complete medium and transfer to T25 culture flask, 37°C, 5% CO 2 cultured in an incubator. Observe the cell survival rate the next day, discard the old medium, and add 5ml of fresh medium.

[0174] (2) Subculture: when the cells grow to 80%-90%, subculture. Discard the supernatant, add 5ml of PBS and shake gently, discard the PBS, add 1ml of 0.25% trypsin, digest for 10s to 20s until the cells become round and the intercellular space becomes larger, add 3ml of complete medium, mix well, transfer to a 15ml centrifuge tube, and centrifuge 250rcf / m...

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Abstract

The invention relates to the field of biotechnology, in particular to a macrophage capable of targeting tumor cells and a preparation method thereof. Macrophages contain chimeric antigen receptors. The inventors have found that due to the limitations of the microenvironment of solid tumors, CAR-T cell is very difficult to enter the tumor, even if they do, the killing effect on tumor cells will beweakened because of the inhibition in the microenvironment. Aiming at the above technical defects, the inventors proposed another idea of tumor immunotherapy, in which the chimeric antigen receptor isexpressed in macrophages. At that same time, the inventor found through the experiment the application of chimeric antigen receptors in CAR-T cell therapy to macrophages can realize the expression ofchimeric antigen receptors on the surface of macrophages, target tumor cells and activate macrophages to phagocytize tumor cells. At the same time, the invention provides a preparation method of themacrophage, which provides a new idea and technical means for tumor immunotherapy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a macrophage capable of targeting tumor cells and a preparation method thereof. Background technique [0002] With the development of immunology, genome editing and synthetic biology, the research on tumor immunotherapy has advanced by leaps and bounds, especially adoptive immunotherapy has broad prospects. Adoptive immunotherapy is a method of adoptively infusing in vitro stimulated and cultured lymphocytes into tumor patients to treat tumors. Chimeric antigen receptor (CAR)-modified T cells is a new method of adoptive immunotherapy for tumors that has developed rapidly in recent years. The modification of CAR enables T cells to have better tumor targeting, stronger killing activity and long-lasting vitality, improving the therapeutic effect. [0003] However, on the one hand, the engineering transformation in CAR-T cells is faced with the problems of low vector transformation eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/62
CPCC12N5/0645C12N5/0696C12N15/85C07K14/7051C07K2319/02C07K2319/03C12N2510/00A61P35/02C12N2506/11C12N2506/45A61K35/15C07K14/705C07K16/2803C07K2317/622A61K39/001112A61K2039/5156A61K2039/5154A61P35/00
Inventor 张进张丽罗涛
Owner 赛元生物科技(杭州)有限公司
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