CAR-T cell, preparation method thereof and medicine

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...

Examples

preparation example Construction

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

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

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

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

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

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

[0045] In the past studies, CAR-T cells cultured for 8...

Embodiment 1

[0056] Example 1 (d8pCAR-T)

[0057] Obtain 10mL peripheral whole blood from healthy donors or leukocyte apheresis to obtain leukocyte collection, use the kit RosetteSep Human T Cells enrichment cocktail from STEMCELL Technologies, operate according to the instructions, separate from peripheral whole blood or leukocyte collection with a purity of More than 99% of CD3+ T cells are frozen in liquid nitrogen or used directly.

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

[0059] Day D1: Use TAKARA's fibronectin solution to coat a six-well culture plate at room...

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...

Examples

preparation example Construction

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

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

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

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

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

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

[0045] In the past studies, CAR-T cells cultured for 8...

Embodiment 1

[0056] Example 1 (d8pCAR-T)

[0057] Obtain 10mL peripheral whole blood from healthy donors or leukocyte apheresis to obtain leukocyte collection, use the kit RosetteSep Human T Cells enrichment cocktail from STEMCELL Technologies, operate according to the instructions, separate from peripheral whole blood or leukocyte collection with a purity of More than 99% of CD3+ T cells are frozen in liquid nitrogen or used directly.

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

[0059] Day D1: Use TAKARA's fibronectin solution to coat a six-well culture plate at room...

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