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Preparation method of universal heterologous CAR-T cells and application

A general-purpose, cell-based technology, applied in the fields of medicine, immunology, cell biology and molecular biology, can solve problems such as delaying the best treatment time for patients, and achieve the benefit of whole-process quality control, saving time and quality of waiting for cells easy to unify effect

Inactive Publication Date: 2017-04-26
GUANGDONG PANGUARD CELL BIOLOGICAL TECH CO LTD
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Problems solved by technology

[0017] The current immune cell therapy is mainly adoptive cell therapy (ACT). This therapy is to obtain T lymphocytes from the patient's own body, and then inject them into the patient's body after gene modification and expansion in vitro. This adoptive cell therapy has MHC non-restricted The target cell recognition function has achieved certain curative effect in clinical application research, but this kind of therapy requires precise cooperation between production equipment and clinical use, and it takes a long time to produce CAR-T cells with therapeutic efficacy, which may delay the treatment of patients. optimal treatment time

Method used

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  • Preparation method of universal heterologous CAR-T cells and application

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Embodiment 1

[0043] (a) drawing blood from healthy volunteers, and isolating T cells;

[0044] (b) construct scFv-CD28-CD137-CD19-CD3 full-length gene;

[0045] (c) scFv-CD28-CD137-CD19-CD3 full-length gene into T cells;

[0046] (d) in vitro culture and expansion of the CAR-T cell population;

[0047] (e) depleting the patient's lymphocytes with PNAs;

[0048] (f) CAR-T cells are reinfused into the patient for anti-tumor therapy;

[0049] (a), Isolation and activation of peripheral blood mononuclear cells

[0050] Healthy volunteers with normal liver and kidney function and good response of PBMC to CD3 / CD28 stimulation were selected.

[0051] 1. Extract 10ml of peripheral blood from healthy volunteers with anticoagulant blood vessels;

[0052] 2. Add erythrocyte lysate and an equal volume of PBS to the tube, and gently pipette to form a cell suspension of 20ml;

[0053] 3. Take another two 50ml centrifuge tubes and add 10ml LTS1077 lymphocyte stratification solution. Use a pipette ...

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Abstract

The invention provides a preparation method of universal heterologous CAR-T cells and an application. Constructed CD28-CD137-CD19-CD3 full-length gene is introduced into allogeneic T cells of healthy persons with CRISPR / Cas9 technology, in order to produce CAR-T cells with targeting cytotoxicity; after amplification in vitro, the cells are fed back to patients for carrying out antineoplastic treatment, and in the treatment process, a PNAs method is used for removing lymphocyte in order to avoid antigraft reaction of hosts. The chimeric antigen receptors can be prepared in large scale with T lymphocyte of healthy human, and antineoplastic treatment of heterologous cancer patients.

Description

technical field [0001] The present invention relates to the fields of medicine, immunology, cell biology and molecular biology, and in particular to a method for preparing a general allogeneic CAR-T cell and its application. Background technique [0002] As a new cancer treatment method, immune cell therapy has been clinically proven more and more. CAR-T cell (Chimeric Antigen Receptor T cell) technology uses the patient's own immune cells to eliminate cancer cells. Cellular immune cell therapy may cure blood cancers such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL). It also has obvious effects in the treatment of solid tumors. While curing tumors, it does not have the therapeutic toxicity of traditional radiotherapy and chemotherapy, so immune cell therapy has broad prospects in the treatment of tumors. [0003] Adoptive cell therapy (ACT) has attracted much attention in clinical research due to its short-term ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10A61K35/17A61P35/00
CPCC12N15/85A61K35/17C12N5/0636C12N2510/00C12N2800/107C12N2810/10
Inventor 张严冬李相鲁谢海涛
Owner GUANGDONG PANGUARD CELL BIOLOGICAL TECH CO LTD
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