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Preparation method and application of high-purity universal CAR-T

A kind of CAR-T, general-purpose technology, applied in the field of biological cells, can solve the problem that the killing ability of tumor cells cannot be implemented, and achieve the effect of high knockout efficiency, reduction of unknown risks, and high T cell survival rate

Inactive Publication Date: 2020-12-18
GUANGDONG XIANKANGDA BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, after the TCR of T cells is knocked out, the TCR-dependent killing ability of tumor cells cannot be carried out. At this time, the killing of T cells can only be achieved by the activation of the transferred CAR, so there is no TCR transferred into CAR. (-)T cells play very little role in the body, which means that the general-purpose CAR-T preparations infused to patients contain some useless CAR(-)TCR(-)T

Method used

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  • Preparation method and application of high-purity universal CAR-T
  • Preparation method and application of high-purity universal CAR-T
  • Preparation method and application of high-purity universal CAR-T

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Experimental program
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Embodiment

[0042] The preparation method and functional verification of the universal CAR-T in this example include the following steps:

[0043] Step 1: Isolation of PBMC from peripheral blood and expansion of T cells

[0044] Mononuclear cells were isolated from donor peripheral blood, density gradient centrifugation was performed using ficol, and T cells were enriched with a T cell sorting kit (CD3 MicroBeads, human-lyophilized, 130-097-043), using conjugated anti-CD3 / anti-CD28 magnetic beads activate culture and expand T cells;

[0045] The medium used TexMACS GMP Medium (Miltenyi Biotec, 170-076-309), containing 10% FBS, 2mM L-glutamine, 100IU / ml rhIL2, all cells were placed at 37°C, 5% CO 2 cultured in a constant temperature incubator.

[0046] Step 2: Cell Line Culture

[0047] Cell line expressing GPC3: Huh-7 (human liver cancer cell), purchased from ATCC.

[0048] Cell line not expressing GPC3: A549 (human non-small cell lung cancer cells), purchased from ATCC.

[0049] Cell...

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Abstract

The invention relates to the technical field of biological cells, in particular to a preparation method and application of high-purity universal CART. According to the preparation method, expression rate of CAR of the universal CART is 100%. The preparation method utilizes crispr-cas9 technology to knock out TRAC and HLA-I B2M genes of T cell TCR to prepare the universal CAR-T, enables expressionrate of CAR of the universal CAR-T to reach 100%, and reduces amount of CAR(-)TCR(-)T cells infused into a human body, thereby reducing unknown risk.

Description

technical field [0001] The invention relates to the technical field of biological cells, in particular to a preparation method and application of a high-purity universal CAR-T. Background technique [0002] In recent years, tumor immunotherapy has developed rapidly, especially adoptive cell therapy (ACT), which means that immune cells such as T cells and NK cells are isolated from patients, modified and expanded in vitro, and then reintroduced into patients for tumor treatment. method of treatment. In 2013, the immunotherapy of tumors was rated as the first among the "Top Ten Breakthroughs" by Science Impurities. [0003] CAR-T and TCR-T are important components of adoptive cell therapy, especially CAR-T therapy, which has made remarkable achievements in the treatment of blood tumors and achieved a high remission rate. A typical CAR structure consists of three parts Composition, extracellular recognition of tumor antigen scFv, hinge and transmembrane domain, intracellular ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/62C12N15/55C12N9/22C12N5/10A61K39/00A61P35/00A61P1/16
CPCA61K2039/5156A61P1/16A61P35/00A61K39/001102A61K2039/844C07K14/7051C07K16/303C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N9/22C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 马丽雅谢海涛薛卫巍
Owner GUANGDONG XIANKANGDA BIOTECH CO LTD
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