CAR-T cell, preparation method thereof and medicine

A cell and dead cell technology, applied in the field of cell biology, can solve the problems of large side effects and weak tumor cell killing ability.

Active Publication Date: 2020-08-11
湖南光琇医院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, CAR-T cells still have serious side effects in the application, such as causing sever

Method used

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  • CAR-T cell, preparation method thereof and medicine
  • CAR-T cell, preparation method thereof and medicine
  • CAR-T cell, preparation method thereof and medicine

Examples

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Example Embodiment

[0039] The present invention relates to a preparation method of CAR-T cells, including the following steps:

[0040] (1) Inoculate T cells in CAR-T medium and culture for 18-24 hours;

[0041] (2) Introduce the vector carrying the chimeric antigen receptor sequence and the puromycin resistance gene into the cells obtained in step (1), and culture for 18-24 hours;

[0042] (3) Collect the cells obtained in step (2) to 0.5~1×10 6 Cell density per ml is inoculated in CAR-T medium and cultured for 36 to 48 hours;

[0043] (4) Collect the cells obtained in step (3) to 0.5~1×10 6 Cell density per ml is inoculated in CAR-T medium, and puromycin is added, and cultured for 36 to 48 hours;

[0044] (5) Collect the cells obtained in step (4) and remove the dead cells from them, to 0.5~1×10 6 Cell density per ml is inoculated into CAR-T medium, and puromycin is added, and cultured for 36 to 48 hours to obtain CAR-T cells.

[0045] In the past studies, CAR-T cells cultured for 8-12 days in vitro are ...

Example Embodiment

[0056] Example 1 (d8pCAR-T)

[0057] Obtain 10 mL of peripheral whole blood or leukocyte apheresis from healthy donors to obtain leukocyte collections. Use the RosetteSep Human T Cells enrichment cocktail from STEMCELL Technologies. The operation is carried out in accordance with the instructions. The purity is More than 99% of CD3+ T cells are stored in liquid nitrogen or used directly.

[0058] Day D0: Take a certain amount of CD3+ T cells and use CAR-T medium (serum-free medium + 3% CTS TM ImmuneCell SR + 1% non-essential amino acids + 1% HEPES + 1% sodium pyruvate + 1% streptomycin-penicillin + 1% GlutaMAX + 100Unit / ml IL-2) for culture, and add Invitrogen's magnetic beads Dynabeads Human T-Activator CD3 / CD28, the addition ratio is the number of cells: the number of magnetic beads = 2:1. At 37℃, 5% CO 2 Incubate for 18h-24h in the incubator.

[0059] Day D1: Use TAKARA’s fibronectin solution to coat a six-well culture plate at room temperature for 2h or 4°C overnight. After the...

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Abstract

The invention relates to a CAR-T cell, a preparation method thereof and a medicine. Negative T cells which fail to transduce CAR genes are removed through culture and purification operation of specific steps, T cells with puromycin resistance genes are reserved, and the proportion of CAR positive T cells is greatly increased. More importantly, experimental comparison shows that although the culture time of the CAR-T cells obtained through culture and purification operation of the specific steps is prolonged, the side effects of the CAR T cells in application are remarkably reduced, for example, the secretion level of inflammatory cytokines IL-6 and other proinflammatory related cytokines is reduced; Th17 cell subgroup components are reduced, and IL-17/ IL-17A secretion is reduced; anti-inflammatory related cytokine secretion is increased; and the killing capacity on tumor cells in vitro and in mice is greatly enhanced, and the survival time of the mice is prolonged.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a CAR-T cell, a preparation method and a medicine thereof. Background technique [0002] Chimeric Antigen Receptor T cell (CAR-T) therapy, which is a new type of precise targeted therapy for tumor treatment, has achieved good results in clinical tumor treatment through optimization and improvement in recent years. It is a very promising new tumor immunotherapy method that is accurate, fast, efficient, and has the potential to cure cancer. T cells, also called T lymphocytes, are a type of human white blood cells, derived from bone marrow hematopoietic stem cells, mature in the thymus, and then migrate to human blood, lymph and surrounding tissues and organs to exert immune functions. It can resist pathogenic infection, remove tumors, foreign bodies, etc. The chimeric antigen receptor (CAR) is introduced into human T cells through genetic engineering technology to obtain CAR...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K39/00A61P35/00
CPCC12N15/86C12N5/0636A61K39/0011A61P35/00C12N2740/15043C12N2510/00A61K2039/5156A61K2039/5158
Inventor 卢光琇林戈陈濂生易辉君郝建军周英邵梦思罗孝宇吴静
Owner 湖南光琇医院有限公司
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