Conditional suicide anti-tumor targeted immune cell and preparation method thereof

An immune cell, suicidal technology, applied in the field of immune cells, can solve the problems of poor controllability, high cost, short survival time in the body, etc., to avoid potential side effects and solve the effects of large side effects

Inactive Publication Date: 2020-01-07
苏州博棠再生医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a conditional suicide anti-tumor targeted immune cell and its preparation method, aiming to solve the problem of large side effects of tumor cell-targeted therapy in the prior art, which cannot be widely used clinically, Problems of short survival time in vivo, high cost, and poor controllability

Method used

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  • Conditional suicide anti-tumor targeted immune cell and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Preparation of lentiviral particles

[0055] Firstly, use Clontech's lentiviral vector pLVX-IRES-ZsGreen1, which not only contains multiple cloning sites, but also carries the green fluorescent marker gene ZsGreen. The SV40LT gene is cloned into the vector gene at BamHI and EcoRI sites by DNA recombination. At the same time, the plasmid pLVX-SV40LT-ZsGreen that can be packaged into lentiviral particles was obtained, and the plasmid pLVX-SV40LT-ZsGreen was packaged to a titer of not less than 10 with a lentivirus packaging system. 7 / ml lentiviral particles.

Embodiment 2

[0057] Preparation of immortalized T cells

[0058] Collect 10 mL of human peripheral blood, and obtain monocytes (PMBC) in human peripheral blood by Ficoll method. The specific operation is as follows:

[0059] Transfer 10mL whole blood into a 50mL centrifuge tube, add 10mL PBS solution to dilute and mix gently; Take two 15mL centrifuge tubes, first add 5mL 9% Ficoll solution, and then gently add 10mL diluted blood to two tubes The upper layer of the Ficoll of the centrifuge tube, avoid mixing the two solutions, centrifuge at 2000rpm for 20min, white layered cells appear, then use a pipette to pipette the layer of cells into another clean 15mL centrifuge tube, and add PBS to 15mL, 1500rpm After centrifugation for 10 minutes, remove the supernatant, then add RPMI1640 medium containing 10% FBS, 1% P / S to 15 mL, centrifuge at 1500 rpm for 10 minutes, remove the supernatant, and then add 10 mL of RPMI1640 containing 10% FBS, 1% P / S to culture Mononuclear cells (PMBC) are obtained by ...

Embodiment 3

[0062] Preparation of immortalized NK cells

[0063] First, collect 10 mL of human peripheral blood and obtain the monocytes (PMBC) in the human peripheral blood by the Ficoll method. The specific operations are as follows:

[0064] Transfer 10mL whole blood into a 50mL centrifuge tube, add 10mL PBS solution to dilute and mix gently; Take two 15mL centrifuge tubes, first add 5mL 9% Ficoll solution, and then gently add 10mL diluted blood to two tubes The upper layer of the Ficoll of the centrifuge tube, avoid mixing the two solutions, centrifuge at 2000rpm for 20min, white layered cells appear, then use a pipette to pipette the layer of cells into another clean 15mL centrifuge tube, and add PBS to 15mL, 1500rpm After centrifugation for 10 minutes, remove the supernatant, then add RPMI1640 medium containing 10% FBS, 1% P / S to 15 mL, centrifuge at 1500 rpm for 10 minutes, remove the supernatant, and then add 10 mL of RPMI1640 containing 10% FBS, 1% P / S to culture Mononuclear cells (P...

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Abstract

Provided are a conditional suicide anti-tumour-targeting immune cell and a preparation method therefor. The preparation method comprises the steps of: A. using a recombinant DNA technique to insert a cell-immortalized gene into a cloning site of a first cloning vector and preparing same into a virion; B. selecting specifically an immune cell in human peripheral blood by using magnetic beads, and then infecting same with the virion prepared in step A to obtain an immortalized immune cell; C. using the recombinant DNA technique to insert a suicide gene into a second cloning vector, and then transferring same into the obtained immortalized immune cell to obtain a conditional suicide immune cell; D. using the recombinant DNA technique to insert a single-chain antibody gene into a cloning site of a third cloning vector and preparing same into a virion, and then transfecting the conditional suicide immune cell to obtain a conditional suicide anti-tumour-targeting immune cell.

Description

Technical field [0001] The invention relates to the field of immune cells, in particular to a conditional suicide anti-tumor targeted immune cell and a preparation method thereof. Background technique [0002] Immune cells have immune surveillance and killing effects on tumor cells, especially immune cells carrying tumor-specific antigen-presenting single-chain antibodies (ScFV) can efficiently and specifically recognize, bind and kill related tumor cells. For example, single-chain antibody T lymphocyte technology (CAR-T) with tumor-specific antigen presentation in the treatment of acute lymphoblastic leukemia and lymphoma, the remission rate of tumor patients is as high as 80%. However, traditional CAR-T cell technology has potential side effects such as cytokine storm. If this side effect cannot be effectively controlled, the probability of death of tumor patients is 5-10%; on the other hand, conventional CAR-T cell technology requires individuality The chemical preparation of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/63
CPCC12N5/0636C12N5/0646C12N15/63C12N15/86C12N2501/2302C12N2501/51C12N2501/515C12N2510/04C12N2740/15043
Inventor 刘小青陈光风史秀娟朱良
Owner 苏州博棠再生医学科技有限公司
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