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Method for in-vitro preparation of tumor antigen-specific CD8+ T memory stem cells

A tumor antigen and lymphocyte technology, applied in the field of immunology and tumor treatment, can solve the problems of complex formation process, and achieve the effect of reducing immune tolerance, better effect and increasing yield

Active Publication Date: 2016-07-27
FUDAN UNIV SHANGHAI CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the formation process of autologous tumor antigen immune memory is very complicated, and it must be processed, processed and presented by antigen-presenting cells. Under the stimulation of an appropriate amount of antigen and an appropriate time, activated T cells undergo an expansion period , T cells enter systolic and memory-forming phases after antigen clearance
Therefore, there is currently no mature method to prepare memory T cells with tumor antigen specificity and stem cell characteristics in vitro

Method used

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  • Method for in-vitro preparation of tumor antigen-specific CD8+ T memory stem cells
  • Method for in-vitro preparation of tumor antigen-specific CD8+ T memory stem cells
  • Method for in-vitro preparation of tumor antigen-specific CD8+ T memory stem cells

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Embodiment 1

[0049] Example 1 The tumor cells are induced by ICD inducer to appear the characteristic CRT translocation phenomenon of ICD.

[0050] In the present invention, tumor cells that induce immunogenic cell death using ICD inducers are used as the source of whole cell antigens, and the specific steps are as follows:

[0051] 1.1 Surgical resection of tumor specimens or crude needle biopsy of tumor tissue, under aseptic conditions, remove necrotic tissue and adjacent non-tumor tissue (using colon cancer CT26 tumor as a model);

[0052] 1.2 Wash with sterile saline for 3 times; cut the tumor tissue into pieces with sterile tissue scissors, add RPMI1640 medium, and grind thoroughly;

[0053] 1. Collect the single cell suspension after filtering with a 3200-mesh sterile net;

[0054] 1.4 Resuspend cells to 1-2x10 in serum-free medium 7 / ml, add ICD inducers such as oxaliplatin or doxorubicin to act for 24 hours, if 8% rose bengal is used, it only takes 30 minutes; then extract the pa...

Embodiment 2D

[0056] The cultivation and identification of embodiment 2DC cells

[0057] Dendritic cells (Dendritic Cells, DC) are the most powerful antigen-presenting cells (Antigen Presenting Cells, APCs) found so far. It has been confirmed that DC is the only APC that can significantly stimulate the proliferation of naive T cells (Naive T cells), while other APCs (such as mononuclear macrophages, B cells, etc.) can only stimulate activated or memory T cells. DC is the initiator of the body's adaptive T cell immune response and plays an extremely important role in tumor immunity. If tumor antigen-loaded DCs are co-cultured with sorted CD8+ naive T lymphocytes, under the action of specific cytokines, the body can be stimulated to produce tumor antigen-specific Tscm cells.

[0058] The steps of culturing and identification of DC cells are as follows:

[0059] 2.1 Collect 50ml-60ml of anticoagulated peripheral blood intravenously;

[0060] 2.2 Separation and purification of mononuclear ce...

Embodiment 3

[0067] Preparation and identification of embodiment 3CD8+Tscm cells

[0068] 3.1 Culture PBMC with serum-free medium, collect suspended cells, and adjust the cell concentration to 1-2x10 with serum-free medium 6 / ml;

[0069] 3.2 Positive selection of CD8+ initial T lymphocytes by immunomagnetic bead method;

[0070] 3.3 Antigen-loaded DC cells and CD8+ initial T cells were co-cultured at a ratio of 1:10 (number ratio), and recombinant human IL-2 (300 U / ml) and recombinant human IL-7 (300 U / ml) were added to the serum-free culture medium ) and IL-15 (300U / ml);

[0071] 3.4 After 3 days of co-culture, isolate T lymphocytes and continue to add recombinant human IL-2 (300U / ml) and recombinant human IL-7 (300U / ml) and IL-15 (300U / ml) to culture medium, every 3 days Change medium once in half volume.

[0072] 3.5 Collect cells on the 7th day, the number of cells should reach 1×10 6 more than one;

[0073] 3.6 Identification of CD8+Tscm cells: The expression of CD8, CD62L, CCR...

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Abstract

The invention relates to a method for in-vitro preparation of tumor antigen-specific CD8+ T memory stem cells and application of the tumor antigen-specific CD8+ T memory stem cells in antitumor immune response. The method comprises the following steps: subjecting tumor antigen and immature dendritic cells to co-incubation so as to obtain mature dendritic cells loaded with specific tumor antigen; subjecting the mature dendritic cells and CD8+ initial T cells to co-culture so as to promote generation of tumor antigen-specific CD8+ T memory stem cells; and separating out the CD8+ T memory stem cells capable of specifically recognizing specific tumor cells. The tumor antigen-specific CD8+ T memory stem cells prepared by using the method has good antineoplastic effect, high specificity and few adverse reaction.

Description

technical field [0001] The invention relates to the fields of immunology and tumor treatment. Specifically, the present invention relates to a method for preparing tumor antigen-specific CD8+ memory stem cell-like T lymphocytes (Tmemorystemcell, Tscm) in vitro. The invention also relates to the use of the prepared tumor-specific Tscm to prevent and treat tumors. technical background [0002] The current tumor T lymphocyte therapy, that is, by preparing tumor antigen-specific T cells, and then isolating and expanding to a certain number (~10 10 1) and then infuse it back to the patient, which is a very promising tumor treatment strategy. The key to this technology is the quantity, activity, phenotype and survival time of antigen-specific T cells after reinfusion, so many laboratories in the world are trying to find better ways to prepare and reinfuse tumor antigen-specific T cells. T cells in order to produce better anti-tumor efficacy. Tumor antigen-specific memory T cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0784A61K39/00A61P35/00
Inventor 吴向华胡爱群邢凯琳
Owner FUDAN UNIV SHANGHAI CANCER CENT
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