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Conditional suicidal anti-tumor targeting immune cell and preparation method thereof

An immune cell and suicidal technology, applied in the field of immune cells, can solve problems such as poor controllability, high cost, and short survival time in vivo, and achieve the effect of avoiding potential side effects and solving large side effects

Inactive Publication Date: 2017-06-20
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 suicidal anti-tumor targeting immune cell and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Preparation of lentiviral particles

[0055] First, select the lentiviral vector pLVX-IRES-ZsGreen1 from Clontech, which not only contains multiple cloning sites, but also has a green fluorescent marker gene ZsGreen, and clones the SV40LT gene into the carrier gene between the BamHI and EcoRI sites by means of DNA recombination. In between, obtain the plasmid pLVX-SV40LT-ZsGreen that can be packaged into lentiviral particles, and use the lentiviral packaging system to package the plasmid pLVX-SV40LT-ZsGreen to a titer of not less than 10 7 / ml of lentiviral particles.

Embodiment 2

[0057] Preparation of immortalized T cells

[0058] Collect 10mL of human peripheral blood and obtain the mononuclear cells (PMBC) in human peripheral blood by the 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 the two The upper layer of Ficoll in the centrifuge tube, to avoid mixing the two solutions together, centrifuge at 2000rpm for 20min, white stratified cells appear, then use a pipette to absorb this layer of cells into another clean 15mL centrifuge tube, and add PBS to 15mL, 1500rpm After centrifuging 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 RPMI1640 containing 10% FBS, 1% P / S for culture Cells were resus...

Embodiment 3

[0062] Preparation of immortalized NK cells

[0063] First, 10 mL of human peripheral blood was collected, and the mononuclear cells (PMBC) in human peripheral blood were obtained by the Ficoll method, and the specific operations were 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, then gently add 10mL diluted blood to the two The upper layer of Ficoll in the centrifuge tube, avoid mixing the two solutions together, centrifuge at 2000rpm for 20min, white layered cells appear, then use a pipette to absorb the layer of cells into another clean 15mL centrifuge tube, and add PBS to 15mL, 1500rpm After centrifuging 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 RPMI1640 containing 10% FBS, 1% P / S for cultur...

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Abstract

The invention discloses a conditional suicidal anti-tumor targeting immune cell and a preparation method thereof. The preparation method comprises the following steps of A, using a DNA (deoxyribonucleic acid) reconstruction technique to insert a cell immortalized gene into a cloning site of a first cloning carrier, and preparing a virus particle; B, utilizing magnetic beads to specifically select one type of immune cell from human peripheral blood, and utilizing the virus particle prepared in step A to infect, so as to obtain an immobilized immune cell; C, utilizing the DNA reconstruction technique to insert a suicidal gene into a second cloning carrier, and transferring into the prepared immobilized immune cell, so as to obtain a conditional suicidal immune cell; D, utilizing the DNA reconstruction technique to insert a single-chain antibody gene into a cloning site of a third cloning carrier, and preparing into a virus particle; transfecting the conditional suicidal immune cell, so as to obtain the conditional suicidal anti-tumor targeting immune cell. The preparation method can solve the problems of great side effect, failure to widely apply in clinic, short surviving time in human body, higher cost and poor controllability in the tumor cell targeting treatment process of the prior art.

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, in the treatment of acute lymphoblastic leukemia and lymphoma, the single-chain antibody T lymphocyte technology (CAR-T) carrying tumor-specific antigen presentation has a remission rate of more than 80% in tumor patients. 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 individual The chemical pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/63
CPCC12N5/0636C12N5/0646C12N15/63C12N15/86C12N2510/04C12N2501/51C12N2501/515C12N2501/2302C12N2740/15043A61K39/464499A61K39/4611A61K2239/38A61K39/4613A61K2239/31
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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