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Method for in-vitro expansion of CD8<+>T cells

An in vitro expansion and cell technology, which is applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of T cell proliferation and killing function inhibition, so as to promote the anti-tumor effect, increase the expansion multiple, Inhibition of negative regulatory effects

Pending Publication Date: 2016-06-29
杭州特马赛生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] To address existing CD8 + For the problem of inhibited T cell proliferation and killing function, the present invention provides an effective and simple way to amplify CD8 + T cell method, using the source of cancerous pleural effusion to extract TIL cells, stimulated by various cytokines in vitro, obtain a large number of TIL cells with strong killing ability, improve the proliferation and killing effect of CD8+ T cells, and inhibit CD4 + CD25 + Treg cells proliferate, weaken their immunosuppressive effect, and enhance the anti-tumor effect of TIL cells

Method used

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  • Method for in-vitro expansion of CD8&lt;+&gt;T cells
  • Method for in-vitro expansion of CD8&lt;+&gt;T cells
  • Method for in-vitro expansion of CD8&lt;+&gt;T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 (experimental group):

[0028] (1) Obtain a mixture of tumor cells and TIL cells:

[0029] Take 500ml of pleural fluid from the patient and inject it into an anticoagulant bottle containing heparin sodium, shake it gently to mix it evenly, divide it evenly into centrifuge tubes, and centrifuge at 2000r / min for 10min. After centrifugation, discard the supernatant. Wash 2 times with saline.

[0030] (2) Separation of TIL cells:

[0031] Adhesion method: Take 15ml of the cells obtained in step (1) and add an equal volume of normal saline to 30ml (this is right), mix well, add 15ml of 100% lymphocyte separation solution to the centrifuge tube, the lymph separation solution and the cells The volume ratio of the suspension is 1:2, slowly superimpose 30ml of the cell suspension on the lymph separation medium along the tube wall, centrifuge at 2000r / min for 20min, collect the buffy coat after centrifugation, which is the mixture of tumor cells and TIL cells, 1500...

Embodiment 2

[0036] Example 2: No anti-PD-1 monoclonal antibody added (control group)

[0037] Except for (3) cell expansion, no anti-PD-1 monoclonal antibody was added, and other specific operation steps were as in Example 1.

[0038] The TIL cells obtained from the experimental group of Example 1 and the control group of Example 2 were detected by amplification factor

[0039] On the 1st, 7th, 14th, and 21st days, 200 μl of cells were taken, blown into single cells, and counted by a Counterstar automatic blood cell counter. Cell expansion factor = total number of cells counted on that day / total number of cells before culture on the first day. The experimental results are shown in Table 1.

[0040] Table 1 The change of cell number of TIL cells at different times (×10 8 / L)

[0041]

[0042] Treg phenotype detection was performed on the TIL cells obtained from the experimental group of Example 1 and the control group of Example 2

[0043] Collect 2×10 on days 1, 7, 14, 21 6 / ml cell...

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Abstract

The invention provides a method for in-vitro expansion of CD8<+>T cells. The method comprises steps as follows: TIL (tumor infiltrating lymphocyte) cells are extracted from a malignant pleural and peritoneal effusion source, cytokines IL-2, CD3McAb and PD-1 monoclonal antibodies are added to a TIL cell culture medium, the concentration of the IL-2 is 300-1,000 U / ml, the concentration of the CD3McAb is 10-50 ng / ml, and the concentration of the PD-1 monoclonal antibodies is 50-200 ng / ml. The cell factor combination adopted by the method can increase the TIL cell expansion multiple, effectively inhibit the negative regulation effect of Treg cells on the CD8<+>T cells and promote the anti-tumor effect.

Description

technical field [0001] The invention belongs to the technical field of tumor adoptive immune cell therapy, and in particular relates to a tumor-infiltrating lymphocyte (Tumor Infiltrating Lymphocyte, TIL) that can be specifically expanded. + T cell approach. Background technique [0002] Tumor-infiltrating lymphocytes (TILs) were originally discovered and isolated from mouse tumor tissues by Rosenberg et al. + , CD8 + T cells are predominant, but also contain a small amount of NK and NKT cells. TIL cells have been used in the research of anti-tumor therapy because of their specific tumor cell killing function. The main effector cells that perform the killing function are composed of CD8 + T cell composition. Although TIL cell therapy technology has entered clinical application and has certain curative effect on advanced cancers such as nasopharyngeal carcinoma, liver cancer, breast cancer, lymphoma, and melanoma, it is difficult to expand large-scale TIL cells and the pr...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2501/2302C12N2501/998
Inventor 吴晓星杨军刘广平
Owner 杭州特马赛生物技术有限公司
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