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Preparation method of CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cells, prepared CAR-T cells and application thereof

A cell and lymphocyte technology, applied in the field of immunology, can solve the problems of different proliferation efficiency and survival persistence of CAR-T cells, achieve good therapeutic or preventive effects, broad application prospects, and promote proliferation.

Inactive Publication Date: 2018-06-01
SHENZHEN WINGOR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Nevertheless, there are still differences in the effects of CAR-T therapy in multiple tumor treatment studies. It is speculated that one of the reasons is due to the different proliferation efficiency and survival persistence of CAR-T cells prepared in the prior art.

Method used

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  • Preparation method of CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cells, prepared CAR-T cells and application thereof
  • Preparation method of CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cells, prepared CAR-T cells and application thereof
  • Preparation method of CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cells, prepared CAR-T cells and application thereof

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preparation example Construction

[0014] A preparation method of CAR-T cells, comprising the following steps:

[0015] S1. Isolation, activation and expansion of T lymphocytes: the isolated CD3+CD8+ T lymphocytes are activated and then cultured and expanded;

[0016] S2. The CAR-carrying recombinant lentivirus infects the T lymphocytes treated in step S1 to prepare the CAR-T cells, wherein the CAR gene carried in the CAR-carrying recombinant lentivirus comprises CD28 intracellular region and In the 4-1BB intracellular region, the nucleotide sequences of the CD28 intracellular region and the 4-1BB intracellular region are shown in SEQ ID7 and SEQ ID9, respectively.

[0017] It can be seen from the above description that the beneficial effects of the present invention are: the optimally designed CAR gene structure and sequence are conducive to the expression and proliferation in the later stage, and the use of two co-stimulatory molecule intracellular regions helps to promote the proliferation of CAR-T cells; C...

Embodiment 1

[0036] Embodiment 1 of the present invention is: a preparation method of CAR-T cells, comprising the following steps:

[0037] Isolation, activation and expansion of S1 and T lymphocytes

[0038] 50 mL of peripheral blood was collected under sterile conditions, and the blood samples were sent to the laboratory for the isolation of peripheral blood mononuclear cells. The peripheral blood was centrifuged at 2000 rpm for 10 min, and the upper autologous plasma was collected. The remaining blood was diluted 1:1 with 0.01mol / L PBS solution by pipetting evenly, the mononuclear cells were separated by lymphocyte separation medium, the cells were washed with PBS, and the cell pellet was resuspended in GTT551 serum-free medium containing 10% autologous plasma, and the Instructions for the Pan T Cell Isolation Kit II (available directly from Miltenyi Company) to isolate T lymphocytes from peripheral blood mononuclear cells. Using anti-CD3 and anti-CD8 antibodies, CD3+CD8+ T lymphocyte...

Embodiment 2

[0051] The second embodiment of the present invention is: a preparation method of CAR-T cells, comprising the following steps:

[0052] Isolation, activation and expansion of S1 and T lymphocytes

[0053] 50 mL of peripheral blood was collected under sterile conditions, and the blood samples were sent to the laboratory for the isolation of peripheral blood mononuclear cells. The peripheral blood was centrifuged at 2000 rpm for 10 min, and the upper autologous plasma was collected. The remaining blood was diluted 1:1 with 0.01mol / L PBS solution by pipetting evenly, the mononuclear cells were separated by lymphocyte separation medium, the cells were washed with PBS, and the cell pellet was resuspended in GTT551 serum-free medium containing 10% autologous plasma, and the Instructions for the Pan T Cell Isolation Kit II (Miltenyi Company) to isolate T lymphocytes from peripheral blood mononuclear cells. Using anti-CD3 and anti-CD8 antibodies, CD3+CD8+ T lymphocytes were sorted b...

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Abstract

The invention discloses a preparation method of CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cells, the prepared CAR-T cells and application thereof. The preparation method comprises the following steps: S1, separating, activating and amplifying T lymphocytes: after activating separated CD3+CD8+T lymphocytes, culturing and amplifying; S2, infecting the T lymphocytes treated in step S1 bya recombinant slow virus carrying a CAR, so as to prepare the CAR-T cells, wherein a CAR gene carried in the recombinant slow virus carrying the CAR comprises a CD28 intracellular domain and a 4-1BBintracellular domain; nucleotide sequences of the CD28 intracellular domain and the 4-1BB intracellular domain are shown as SEQ ID7 and SEQ ID9 respectively. The CAR-T cells prepared by the method canbe widely applied to treatment of various cancers. Compared with the prior art, the proliferation rate of the prepared CAR-T cells is remarkably improved when compared with CAR-T cells prepared by aconventional method.

Description

technical field [0001] The present invention relates to the field of immunology, in particular to a preparation method of chimeric antigen receptor T-cell (Chimeric Antigen Receptor T-cell, CAR-T), the prepared CAR-T cell and application thereof. Background technique [0002] In the past 10 years, immunotherapy has shown great potential and prospects in cancer treatment, and the emergence of chimeric antigen receptor T cell therapy is a major breakthrough in the field of immunotherapy. In 1989, Gross et al first proposed the construction of specific chimeric antigen receptor modified T lymphocytes (CAR-T). CAR-T therapy is to use the patient's own immune cells to kill tumor cells. It is a method to specifically kill tumor cells. This treatment method extracts T lymphocytes in the peripheral blood of the patient, and then uses the extracted T lymphocytes through genetic engineering. After transformation, the transformed T lymphocytes have a strong ability to specifically rec...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 姜舒张芸纪惜銮杨顺罗朝霞
Owner SHENZHEN WINGOR BIO TECH
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