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Preparing method for high-toxicity human Vgamma9Vdelta2 T cells induced by PD-1 antibody

A PD-1 and cell technology, applied in the field of cellular immunology, can solve the problems of limited application and promotion, low number of γδT cells, etc., and achieve the effect of enhancing secretion capacity, improving cytotoxicity, and simple preparation method

Inactive Publication Date: 2016-04-06
SHENZHEN HORNETCORN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The low number of γδT cells in the body, the diversity of their functions (including positive and negative anti-tumor effects), and the incompetence of γδT cells due to PD-1-mediated tumor immune escape limit their clinical application. app promotion for

Method used

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  • Preparing method for high-toxicity human Vgamma9Vdelta2 T cells induced by PD-1 antibody
  • Preparing method for high-toxicity human Vgamma9Vdelta2 T cells induced by PD-1 antibody
  • Preparing method for high-toxicity human Vgamma9Vdelta2 T cells induced by PD-1 antibody

Examples

Experimental program
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Effect test

example 1

[0028] Example 1 is to stimulate PBMCs with IL-12 and PD-1 antibodies to obtain a large number of Vγ9Vδ2T cells with high purity and high cytotoxic activity, which specifically includes the following steps:

[0029] 1. Collect the peripheral venous blood of the patient with a 50ml syringe under sterile conditions, and obtain mononuclear cells by centrifugation of polysucrose-diatrizoate according to the density gradient. The specific steps are: centrifuge at 500g / min for 7 minutes, absorb the upper plasma layer, inactivate at 56°C for 30 minutes, then inactivate at 900g / min for 10 minutes, and prepare the supernatant plasma for later use; double-dilute the lower layer of blood cells with normal saline, human lymphocyte separation medium Add the diluted blood to the centrifuge tube at a ratio of 1:2, centrifuge at 680g / min for 20 minutes, absorb the buffy coat, wash twice with normal saline, and centrifuge at 500g / min and 410g / min for 7 minutes respectively. Peripheral blood mo...

example 2

[0032] In Example 2, the morphology, purity, immunophenotype, cytokine secretion and cytotoxic activity of the above-mentioned cultured Vγ9Vδ2T cells were detected. The specific operation includes the following steps:

[0033] 1. After 24 hours of cell culture, it can be seen that Vγ9Vδ2T cells sink to the bottom of the culture flask and form small colonies. After 48 hours, the colonies become larger. After 6-11 days of culture, the cell colonies can be seen to increase, increase, and have irregularities. of monocytes. After 11 days of culture, most of the cells increased in volume and were oval or irregular (see appendix figure 1 ), stained with 4% trypan blue, and counted under a microscope, the results showed that the viability of the cells prepared by the present invention was 97.3% (n=10).

[0034] 2. Take the cells on day 0, 7, 14 and 21 respectively, wash the cells twice with flow cytometry buffer, and adjust the cell density to 1×10 6 cell / ml, add 50ul of cell suspe...

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Abstract

The invention discloses a preparing method for high-toxicity human Vgamma9Vdelta2 T cells induced by a PD-1 antibody. The preparing method comprises the following steps that firstly, human peripheral blood mononuclear cells are obtained through saccharosan-glucosamine diatrizoate density gradient centrifugation; secondly, separated PBMCs are put into an RPMI1640 culture medium containing an irritant mycobacterium tuberculosis heat-resistant antigen, cell factors rhIL-2 and autologous plasma accounting for 5-10%; rhIL-12 and the PD-1 antibody are added at the same time to culture PBMCs; thirdly, the fresh RPMI1649 culture medium is supplemented every other 2-3 days according to the growing condition of the cells; fourthly, the cells are collected on the 10th day to the 16th day according to the growing condition of the cells, and the high-toxicity human Vgamma9Vdelta2 T cells are obtained. The preparing method for the Vgamma9Vdelta2 T cells which are fast in cell proliferation, high in cell purity, high in cell toxicity, strong in tumor damage capability and low in cost is provided, and a basis for clinical application of the Vgamma9Vdelta2 T cells on tumor treatment medicine is laid.

Description

technical field [0001] The invention belongs to the technical field of cellular immunity, in particular to a preparation method of highly toxic human Vγ9Vδ2T cells. Background technique [0002] γδT cells have the characteristics of both innate immunity and acquired immunity, as well as their specific recognition and killing effect on tumor cells, making them have a very broad application prospect in tumor immunotherapy. In recent years, adoptive cellular immunotherapy based on γδT cells has made great progress in clinical practice. There are two strategies for γδT cells to be used in tumor immunotherapy: 1. Inducing the activation and proliferation of γδT cells in vivo; 2. Activating and modifying γδT cells in vitro and directly infusing adoptive immunotherapy. A series of clinical studies have shown that both adoptive immunotherapy and in vivo expansion of Vγ9Vδ2 T cells are safe treatments that can induce objective clinical responses in tumor therapy. [0003] However, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2501/2302C12N2501/2312C12N2501/998
Inventor 付凡饶秀茸崔博靖马飞王宇环
Owner SHENZHEN HORNETCORN BIOTECH
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