Construction method of chimeric antigen receptor double-negative T cell

Abstract

The invention discloses a construction method of a chimeric antigen receptor double-negative T cell. The construction method comprises the following steps: according to characteristics of CD3<+> / CD4<-> / CD8<->double-negative T cell, selecting the purified CD3<+> / CD4<-> / CD8<->double-negative T cell in vitro, and utilizing the synergistic principle of multiple cell factor signal channels and a TCR signal channel to screen out an in-vitro culture scheme suitable for activation and virus transfection of the double-negative T cell. The in-vitro culture scheme can quickly and controllably activate the double-negative T cell, expresses the specific chimeric antigen receptor on the double-negative T cell through a lentiviral transfection technology at high efficiency, preparing a CAR-DN-T cell, enables the CAR-DN-T cell to specifically identify the tumor cell corresponding to the chimeric antigen receptor, promotes the CAR-DN-T cell to be further activated and proliferated, and quickly kills and wounds the tumor cell. According to the construction method disclosed by the invention, the prepared CAR-DN-T cell is from homologous variants of other healthy donators, so that the preparation of the cell gets rid of limitation of the current illness situation of a patient, the cell can be prepared on a large scale in advance, and the patient can be subjected to low-dosage re-transfusion treatment for multiple times according to the clinical diagnosis result at any time.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a method for constructing chimeric antigen receptor double-negative T cells, in particular to a chimeric antigen receptor CD3 + / CD4 － / CD8 － Construction method of double-negative T cells. Background technique [0002] Chimeric antigen receptor T cell (CAR-T cell) technology is to use genetic engineering and virus transfection technology to construct an exogenous chimeric antigen receptor for killer T cells, so that it can specifically recognize tumor markers , and promote the proliferation and activation of CAR-T cells, and continuously exert the tumor-killing function. CD19 + The excellent clinical trial results of acute B-lymphoblastic leukemia have attracted widespread attention in the medical field and are considered to be the most promising therapy for curing tumors. [0003] However, the current CAR-T clinical trials are all isolated T cells from the patient's own ...

AI Technical Summary

Problems solved by technology

However, there is a big technical bottleneck in practical application. The main reason is that the molecular weight of CRISPR-Cas9 and other vectors used in gene knockout technology is relatively large, it is difficult to introduce T cells, and the efficiency of gene knockout is low.

Moreover, if virus transfection is used to introduce CRISPR-Cas9 and other vectors, chimeric antigen receptors will resist secondary virus transfection, so the yield of TCR-KO-CAR-T cells is extremely low, and large-scale transfection cannot be performed. Scale preparation, poor applicability

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