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Method for reversing failure of anti-tumor infiltrating lymphocytes and application of method

A lymphocyte and tumor infiltration technology, applied in the field of cell culture, can solve the problems of aging and excessive differentiation of anti-tumor T cells, and achieve the effect of low cost, good effect and broad clinical application prospects.

Inactive Publication Date: 2016-11-09
杨世成 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In our previously reported method, large amounts (>1×10 12 ) anti-tumor TILs cells, but under the culture conditions of IL-2, the gene-modified anti-tumor T cells still undergo excessive differentiation and aging process

Method used

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  • Method for reversing failure of anti-tumor infiltrating lymphocytes and application of method
  • Method for reversing failure of anti-tumor infiltrating lymphocytes and application of method
  • Method for reversing failure of anti-tumor infiltrating lymphocytes and application of method

Examples

Experimental program
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Embodiment 1

[0033]This example specifically studies the effect of the cytokine composition of the present invention on the level of CD62L expression membrane of TILs cultured in vitro, and the specific steps of cell culture are as follows:

[0034] Step 1: The tumor-infiltrating lymphocytes are cultured in a serum-free medium, counting from inoculation, and adding a cytokine composition for culture in the first 3 days of culture;

[0035] Step 2: After the third day of culture, the cytokines in the cytokine composition remove IL-12, continue to culture until the sixth day, and collect tumor infiltrating lymphocytes;

[0036] The cytokine composition is selected from any one of composition 1, composition 2 or composition 3; the composition 1 is IL-2 and IL-12; composition 2 is IL-7 and IL- 12; Composition 1 is IL-12 and IL-21; cytokines except IL-2 which is 100 IU / ml, the concentration range of other cytokines is 0.01-1000ng / ml.

[0037] In the control group, only IL-2 factor was added, a...

Embodiment 2

[0039] In this example, the changes of other membrane molecular markers were further analyzed. The experimental procedure is as shown in Example 1, and the control group is also only added with IL-2 factor. Such as image 3 As shown, 6 cases of TILs derived from different tumor patients were cultured by cytokines and their combinations for 6 days, and the cell phenotype was checked by flow cytometry for the phenotype of the differentiated molecules on its surface. Among them, there was no significant difference in the expression of CD8 molecules in TILs, but there were significant differences in the expression of CD27, CD28 and CD127 molecules, which are all cell surface markers used to evaluate poorly differentiated anti-tumor T cells cultured in vitro.

[0040] The statistical data of 6 cases such as image 3 Shown: TILs cultured in IL-2 group expressed lower levels of CD27, CD28 and CD127 membrane molecules. *: Indicates that compared with other groups, P<0.01. In addit...

Embodiment 3

[0042] In this example, the gene expression of molecules such as CD62L and CD27 was further analyzed, and the experimental steps were the same as those in Example 1. as shown Figure 4 As shown, the present invention analyzes the effects of cytokines and combinations on the expression of related differentiation marker genes in TILs cells cultured in vitro. As shown in the figure, 6 cases of TILs derived from different tumor patients were cultured with cytokines and their combinations for 6 days, then the total RNA was isolated and purified, and the expression of related differentiation genes was detected by fluorescent quantitative PCR. Figure A shows the copy number of mRNA expression of four stem cell factors SOX2, NANOG, OCT4 and Lin8. *: Indicates that the group is compared with the IL-2 group, P<0.01. Panel B shows copy number changes of IFNg. Figure C shows the copy number of expression of CD27, CD28 and other related genes, *: indicates comparison with other groups, ...

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Abstract

The invention relates to the field of cell culture, and discloses a method for reversing failure of anti-tumor infiltrating lymphocytes and an application of the method, wherein the method, by virtue of a means of adding a cell factor composition, can achieve the purposes of better preserving tumor infiltrating lymphocytes and promoting the poorly differentiated phenotype of T cells of the tumor infiltrating lymphocytes. Compared with a conventional anti-tumor T cell in-vitro culture method, the method disclosed by the invention can creatively delay a process of in-vitro tumor infiltrating lymphocyte failure, and the anti-tumor T lymphocytes, at a low concentration, can show an efficient tumor suppression activity in an in-vitro pre-clinical experimental model. The method disclosed by the invention is simple and easy to implement, low in cost, better in effect and broad in clinical application prospect.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for reversing exhaustion of anti-tumor infiltrating lymphocytes and its application. Background technique [0002] Tumor-infiltrating lymphocytes (TILs) are T cells with strong anti-tumor activity that infiltrate into tumor tissues. A very powerful anti-tumor preparation with long-lasting anti-tumor effects has been widely used in the treatment of tumors and achieved remarkable therapeutic effects. However, in the process of in vitro expansion of TIL isolated and cultured in vitro, there is an irreversible aging process of progressive differentiation and exhaustion, which leads to the inability of long-term survival of TIL expanded in vitro into the tumor-bearing body, thus greatly affecting the immune system. tumor effect. In order to enhance the long-term survival of TIL in the tumor-bearing body and achieve a durable and efficient anti-tumor effect, the present invention...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCC12N5/0636A61K35/17C12N2501/2302C12N2501/2307C12N2501/2312C12N2501/2321
Inventor 杨世成郑北平
Owner 杨世成
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