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Method for enhancing the activity of CIK cells, CIK cells, and preparation method and application thereof

A cell activity and cell technology, applied in the field of cell engineering, can solve problems such as large side effects, increased risk of CIK cell therapy, lack of tumor antigen specificity, etc., achieving the effect of no toxic side effects, considerable effect, and enhanced killing activity

Active Publication Date: 2016-09-07
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some chemically modified CIK cells (such as CART, etc.) perfected the lack of tumor antigen specificity of CIK cells, the introduction of a virus infection system increased the risk of CIK cell therapy
Studies have found that α-galactosylceramide (α-GalCer) has the ability to directly activate NKT cells, but this powerful NKT cell activator will cause large side effects

Method used

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  • Method for enhancing the activity of CIK cells, CIK cells, and preparation method and application thereof
  • Method for enhancing the activity of CIK cells, CIK cells, and preparation method and application thereof
  • Method for enhancing the activity of CIK cells, CIK cells, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This example is used to illustrate the preparation of CIK cells of the present invention.

[0039] (1) Collect venous blood from patients with cancer (ovarian cancer, lung cancer, colon cancer, and gastric cancer) in a vacuum tube containing heparin, and use lymphocyte separation medium and density gradient centrifugation to separate mononuclear cells (PBMCs).

[0040] (2) After PBMCs were washed three times with normal saline, they were placed in CIK cell culture medium GT-T551 containing 0.6 vol% autologous serum, and the final cell concentration was adjusted to 2×10 6 Cells / ml; Cells were inoculated into 75 cm cell culture flasks coated with CD3 monoclonal antibody at a final concentration of 5 μg / ml and RetroNectin at a final concentration of 10 μg / ml. Add human protein interferon-γ and 1000U / ml human interleukin-2 to the medium at a final concentration of 1000U / ml, at 37°C, 5% CO 2 cultured in an incubator.

[0041] (3) On the fourth day, 100 ml of CIK cell cultu...

Embodiment 2

[0046] This example is used to illustrate the phenotype analysis of CIK cells of the present invention.

[0047] CIK cells treated with different doses of decitabine in Example 1 were phenotype detected by flow cytometry, and CD3 + CD56 + , CD8 + CD56 + and CD3 + CD8 + cell ratio.

[0048] like figure 1 A and figure 1 As shown in B, as the dose of decitabine increased, CD3 + CD56 + The proportion of cells increased accordingly, being 4.52%, 4.81%, 8.04% and 19.45% respectively; CD8 + CD56 + The proportion of cells also increased, respectively 3.55%, 4.08%, 6.89% and 16.4%; while CD3 + CD8 + The proportion of cells was not significantly different. In addition, the proportion of CD3 / CD56 double-positive effector cells in CIK cells in patients with various tumor types such as lung cancer, colon cancer, and gastric cancer has a certain increase (respectively as figure 1 C. figure 1 D and figure 1 E shown).

Embodiment 3

[0050] This example is used to illustrate the effect of DNA demethylation drugs on the proliferation activity of CIK cells.

[0051] CIK cells were prepared according to the method of Example 1, and different groups of CIK cells were counted by a cell counter, and the statistical results were as follows: figure 2 shown. from figure 2 It can be seen that DNA demethylation drugs can enhance the proliferation activity of CIK cells.

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Abstract

The present invention relates to the field of cell engineering, and discloses a method for enhancing the activity of CIK cells. The method includes the culture of CIK cells in the presence of a DNA demethylation drug. The invention also discloses CIK cells and a preparation method thereof. The preparation method includes culturing peripheral blood mononuclear cells in the presence of cytokines for 9 to 11 days, and then culturing in the presence of a DNA demethylation drug. The invention also discloses the application of the CIK cells in the preparation of the preparation for the treatment of tumors. In addition, the invention also discloses the application of the DNA demethylation drug as a stimulating factor in enhancing the activity of CIK cells. The invention by the use of DNA demethylation drug significantly enhances the ratio of utility cells in CIK cells, significantly increase the killing activity of CIK cells on tumor cells, also can promote the proliferation activity of CIK cells, and has good clinical safety, no toxic or side effects, considerable overall anti-tumor effect, and great value in clinical application.

Description

technical field [0001] The invention relates to the field of cell engineering, in particular to a method for enhancing the activity of CIK cells, CIK cells and their preparation method and application. Background technique [0002] CIK (cytokine induced killer) cells, that is, cytokine-induced killer cells, are a type of heterogeneous cell population obtained after co-cultivation and stimulation with various cytokines, and express NK cell surface markers (CD56 molecules) and T cell surface markers at the same time. Marker (CD3 molecule), so it is also called NKT cells (natural killer T cells); it has both the strong anti-tumor activity of T cells and the non-MHC-restricted tumor killing characteristics of NK cells, and has become an important program for adoptive immunotherapy of tumors . The CIK cells currently prepared for adoptive immunotherapy are actually expanded in vitro with CD3 + CD56 + and CD3 + CD8 + Mainly heterogeneous cell population, the main utility cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
Inventor 聂晶韩为东吕海燕刘传杰李祥陈美霞张燕
Owner GENERAL HOSPITAL OF PLA
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