Preparation method of CAR (Cheimeric Antigen Receptors)-T cells for co-expressing IL18 (Interleukin 18) and CCL19 (Chemokine C-C motif ligand 19) protein and targeting MUC1 gene

A co-expression and cell technology, applied in the field of immune cell preparation, can solve problems such as unsatisfactory effects, achieve the effect of releasing inhibition, promoting proliferation and stability, and increasing safety

Inactive Publication Date: 2018-12-21
杭州荣泽生物科技集团有限公司
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Problems solved by technology

However, due to the suppression of the tumor microenvironment in some hematological tumors and most solid tumors, the effect is not very satisfactory. The significant role played by the fourth-generation CAR-T in the tumor microenvironment has important clinical application value

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  • Preparation method of CAR (Cheimeric Antigen Receptors)-T cells for co-expressing IL18 (Interleukin 18) and CCL19 (Chemokine C-C motif ligand 19) protein and targeting MUC1 gene
  • Preparation method of CAR (Cheimeric Antigen Receptors)-T cells for co-expressing IL18 (Interleukin 18) and CCL19 (Chemokine C-C motif ligand 19) protein and targeting MUC1 gene
  • Preparation method of CAR (Cheimeric Antigen Receptors)-T cells for co-expressing IL18 (Interleukin 18) and CCL19 (Chemokine C-C motif ligand 19) protein and targeting MUC1 gene

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Embodiment Construction

[0031] The present invention provides a method for preparing CAR-T cells that co-express IL18 and CCL19 and target MUC1, which includes preparation of CD4+ / CD8+ T cells, co-expression of IL18 and CCL19 proteins, and construction of CAR-T cells targeting MUC1 , lentivirus preparation co-expressing 18×19 CAR molecules, transfection of T cells with lentivirus, activation of anti-CD3+CD28 antibody magnetic beads, and MUC1 CAR-T cells were obtained by screening and culturing.

[0032] Preparation of CD4+ / CD8+ T

[0033] Use an EDTA anticoagulant tube to collect peripheral blood from healthy people, add an equal volume of phosphate buffer PBS to dilute, add the diluted peripheral blood to a 50ml centrifuge tube with an equal volume of lymphocyte separation medium (Ficoll), and centrifuge at 400g for 30min; The buffy coat cells on the solution were transferred to a new 50ml centrifuge tube, and washed 3 times with PBS centrifugation at 300g for 10min to obtain PBMC cells; TMHuman ...

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Abstract

The invention relates to a preparation method of CAR (Cheimeric Antigen Receptors)-T cells for co-expressing IL18 (Interleukin 18) and CCL19 (Chemokine C-C motif ligand 19) protein and a targeting MUC1 gene. The preparation method comprises the following steps: transfecting DC<4+> / CD<+8> positive cells by lentivirus containing IL18 and CCL19 protein coding sequences and targeting MUC1 CAR; screening and culturing amplified and transfected T cells to obtain the CAR-T cells for co-expressing IL18 and CCL19 protein and the targeting MUC1 gene. According to the preparation method provided by the invention, the proliferation capability and the life cycle of the CAR-T cells are enhanced through expressing third cell signals IL18 and CCL19, improving the osmosis effect of the CAR-T cells in solidtumors and maintaining the killing effect of the CAR-T cells on the solid tumors. Furthermore, the co-expressed IL18 and CCL19 are connected through a 2A short peptide and can be expressed respectively.

Description

technical field [0001] The present invention relates to the field of immune cell preparation, in particular, the present invention relates to a preparation method of co-expressing IL18 and CCL19 proteins and targeting MUC1 gene CAR-T cells. Background technique [0002] After surgery, radiotherapy, and chemotherapy, tumor immunotherapy has become an effective treatment method and has recently received widespread attention. Among them, Chimeric Antigen Receptors-T cell technology (Cheimeric Antigen Receptors-T cell, CAR-T) is a newly emerging adoptive cell therapy (Adoptive cell transfer therapy, ACT), which modifies the patient's T cells in vitro, After activation and proliferation, it is infused back into the patient's body, through the specific receptors expressed by T cells, targeted recognition of tumor cells, and shows strong killing activity and persistence. [0003] Chimeric antigen receptor (CAR) is composed of extracellular antibody binding region, hinge region, tr...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12N15/62
CPCC07K14/52C07K14/54C07K14/7051C07K14/70517C07K14/70575C07K14/70578C07K16/3092C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 陈相波冯煜雷鸣田朋飞
Owner 杭州荣泽生物科技集团有限公司
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