Preparation method of CAR-T cells

A cell and nuclear cell technology, applied in the field of CAR-T cell preparation, can solve the problems of short survival time of CAR-T cells, lack of continuity of therapy, low positive rate, etc., achieve good survival in vivo, prolong survival, and facilitate The effect of the operation

Pending Publication Date: 2019-05-17
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, due to the limitations of the preparation method of the CAR-T cells used for therapeutic doses, the prepared CAR-T cells are in the terminal stage of differentiation, and the positive rate is low, resulting i...

Method used

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  • Preparation method of CAR-T cells
  • Preparation method of CAR-T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Proliferation of T cells cultured with MK2206 in vitro

[0040] The purpose of this example is to determine the effect of MK2206 on the proliferation of T cells, and evaluate the effect of MK2206 on the proliferation of T cells by cell counting.

[0041] On the 0th day, after adding MK2206 to the T cell culture, the drug was withdrawn on the 5th day, and the effect of MK2206 on cell proliferation was evaluated by cell counting, and the counting was continued until the end of the expansion on the 10th day. The results are shown in figure 1 ;

[0042] Depend on figure 1 It can be seen that MK2206 treatment did not weaken the proliferation of T cells, which was not statistically significant compared with the untreated group.

Embodiment 2

[0043] Example 2 Delayed terminal differentiation of CAR-T cells treated with MK2206 in vitro

[0044] CAR-T cells were cultured with MK2206. In the experimental group, CD3, CD28 antibody-coupled magnetic beads were used to activate T cells by adding medium containing IL-2 and 2 μM K2206. After two days of activation, slow cells carrying CAR sequences were added. Viruses were co-cultured for 10 days.

[0045] At the end of the culture, the expression of CD45RA and CCR-7 in CAR-T cells was evaluated by flow cytometry, and the differentiation of the cells was determined by staining with specific antibodies against CD45RA and CCR-7. The results are shown in figure 2 ;

[0046] Depend on figure 2 It can be seen that the proportion of central memory cell population cells of CAR-T cells cultured with IL-2 and MK2206 was significantly increased compared with cultures cultured with IL-2 alone.

Embodiment 3

[0047] Example 3 CAR-T cells treated with MK2206 in vitro significantly increased the expression of CAR positive rate

[0048] For the CAR-T cells prepared in Example 2, continue to culture until the 10th day after virus transduction, and evaluate the expression of the CAR sequence of the CAR-T cells by flow cytometry. staining to determine the expression of CAR, the results are shown in image 3 ;

[0049] Depend on image 3 It can be seen that compared with the culture cultured with IL-2 alone, the CAR-positive rate expression of CAR-T cells cultured with IL-2 and MK2206 was significantly increased.

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Abstract

The invention provides a preparation method of CAR-T cells. According to the method, an Akt inhibitor MK2206 is added to both T lymphocyte activation and lentivirus infection, the CAR positive rate ofthe obtained CAR-T cells is significantly improved, and the ratio of memory cells is greatly improved, so that the terminal differentiation is delayed, and the survival condition in vivo is significantly improved. Under the condition that the killing capability is detected undamaged in vitro, the differentiation is delayed, and the CAR expression positive rate is increased. The CAR-T cells are better in the survival state in vivo. The treatment effect is better, and broad application prospects and great market value are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a preparation method of CAR-T cells. Background technique [0002] Cancer is composed of normal cells that have undergone epigenetic transformation to become abnormal cancer cells. Cancer cells are able to express proteins and other antigens differently than normal cells. These abnormal tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells such as T and B lymphocytes from successfully targeting cancer cells. [0003] Human T cell therapy relies on enriched or modified human T cells in vitro to target and kill cancer cells in a subject. Various techniques have been developed in the medical field to enrich the concentration of naturally occurring T cells capable of targeting tumor antigens or to genetically modify T cells to specifically target known cancer ant...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867
Inventor 张青郑骏年丁佳戈
Owner XUZHOU MEDICAL UNIV
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