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Method and combination for polarizing and amplifying CD4+T cells and application in healing tumor expressing specific antigen

A technology of cells, blood cells, applied in the field of biological cells

Inactive Publication Date: 2019-01-04
田野 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, few clinical trials have focused in depth on the specific composition of CD8+ and / or CD4+ T cells

Method used

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  • Method and combination for polarizing and amplifying CD4+T cells and application in healing tumor expressing specific antigen
  • Method and combination for polarizing and amplifying CD4+T cells and application in healing tumor expressing specific antigen
  • Method and combination for polarizing and amplifying CD4+T cells and application in healing tumor expressing specific antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] (1) Purified human-derived CD4+ T cells were obtained: Peripheral blood was subjected to Ficoll density gradient treatment to obtain surviving cells and red blood cells were removed. Purified CD4+ T cells were obtained by using the Pan Human CD4+ T cell magnetization sorting system produced by Miltenyi.

[0049] (2) Activation of T cells: For the T cells obtained in step 1, add 100ng / ml TGFβ, 100ng / mL IL-4, 100ng / mL IL- 21 and 100ng / mL anti-IFNγ; the same below) was activated with Dynabeads produced by Thermal Fisher.

[0050] (3) Culture and cell proliferation: On the third day after activation, the cells were centrifuged, the supernatant was removed, and cultured in RPMI medium supplemented with 10% fetal bovine serum, Antibiotic-Antimycotic and 100U / mL IL-2 ; stimulate proliferation and induce differentiation;

[0051] The activated T cells will proliferate rapidly, and the cell number doubling time is less than 24 hours. The overall expansion time will last appro...

Embodiment 2

[0054] Other contents are as in Example 1, the method for polarizing and expanding CD4+T cells. Add a transduction step for T cells.

[0055] (1) As in Example 1, purified human CD4+ T cells were obtained from the peripheral blood of type B leukemia patients. For a better final result, CD4+, CD62+ T cells can be sorted, because the higher the purity of the initial cells, the higher the purity of the induced cells.

[0056] (2) As in Example 1, CD4+ T cells are activated with Dynabeads in the culture medium

[0057] (3) Using the methods described in other literatures, package retroviruses capable of transducing human T cells, whose transduction sequence is a chimeric antigen receptor molecule of CAR019. [US Patent US 9,328,156B2Seq8]

[0058] (4) Culture and cell proliferation: On the third day after activation, the cells were centrifuged, the supernatant was removed, and cultured in RPMI medium supplemented with 10% fetal bovine serum, antibiotics and 100 U / mL IL-2. Keep ...

Embodiment 3

[0061] (1) Extract CD4+, CD62L+ cells from splenocytes of C57B6 mice. The mouse spleen or lymph node is used, after harvesting, it is ground and passed through a 40 micron sieve to obtain a cell suspension. Treat the cell suspension or mouse blood with common erythrocyte lysate and wash twice with phosphate buffer. The cells were counted, and the purified CD4+ T cells were obtained by using the Pan Mouse CD4+ T cell magnetization sorting system produced by Miltenyi. CD4+, CD62+ T cells can be sorted, because the higher the purity of the initial cells, the higher the purity of the induced cells.

[0062] (2) According to the methods described above and published methods, induce differentiation into Th1, Th17 and Thscm. Groups were compared.) Cell RNA was extracted using Qiagen's RNeasy Kit. And the RNA was reverse transcribed into a cDNA template using Thermo Fisher Scientific's SuperScript III. And use the primers provided in the table below to do real-time fluorescent qua...

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Abstract

The invention belongs to the field of biological cells and relates to a method for polarizing and amplifying CD4+T cells and application. The method includes that human-source or rat-source CD4+ undifferentiated T cells are adopted, and a series of cell factors like T cell activation and conversion growth factor beta, interleukin-4, interlenkin-21 and gamma-interferon neutralizing antibody are adopted for co-stimulation. By the method, destructiveness and self-differentiation multiplication ability for cancer cells are maintained while T cells polarized by the method are multiplied massively such as by 500-1000 times within 14 days. The T cells are different from anticancer subgroup like Th1, Th17 and Th9 reported before in character and exceed the same in cancer killing efficacy to some extent. CD4+T cell subgroup polarized by the method has strong treatment effect on various cancers like solid and blood tumor expressing specific antigen.

Description

technical field [0001] The invention belongs to the field of biological cells, and in particular relates to a method and composition for polarizing and expanding CD4+ T cells and its application in curing tumors expressing specific antigens. Background technique [0002] Immune cell reinfusion is a cancer treatment method invented in recent years. Its basic principle is: the reinfused cells will have a specific attack on the host's cancer cells and cancer tissues, or stimulate other immune cells in the host to attack the cancer . [0003] Researchers have used a variety of cells for reinfusion, including T lymphocytes, macrophages (Macrophage), natural killer cells (NK) and natural killer T cells (NKT). At the same time, many experiments have adopted: the processing and modification of the cells listed above (such as chimeric antigen receptor T cell therapy, CAR-T), or the combined use of multiple cells, or the combination of cells and other drugs / cytokines / vaccine (For ex...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12Q1/6888A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0636C12N2500/84C12N2501/2302C12Q1/6888
Inventor 鲁勇田野孙涛
Owner 田野
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