Preparation method and use of TCR gene modified CD8+T memory stem cell

A gene modification and memory technology, which is applied in the fields of immunology and tumor therapy, can solve problems such as the complexity of the formation process, achieve long-lasting effects, avoid serious adverse reactions, and have better anti-tumor effects

Inactive Publication Date: 2018-01-05
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

Method used

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  • Preparation method and use of TCR gene modified CD8+T memory stem cell
  • Preparation method and use of TCR gene modified CD8+T memory stem cell
  • Preparation method and use of TCR gene modified CD8+T memory stem cell

Examples

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Embodiment 1

[0063] A method for preparing TCR gene-modified CD8+T memory stem cells (TCR-Tscm) capable of recognizing multiple tumor-specific antigens in vitro, the method comprising the following steps:

[0064](A) The mononuclear cells isolated from the peripheral blood of tumor patients or healthy donors were cultured to collect the mononuclear cells of adherent growth and suspension growth respectively; -CSF) and interleukin-4 (IL-4), the adherent monocytes obtained in the culture step (C) were induced to form immature dendritic cells; (D) tumor antigens were obtained from fresh tumor cells Under the action of combined use of 1 to 4 immunogenic cell death (ICD) inducers, tumor antigens or artificially synthesized specific tumor antigen epitope peptides and adjuvant recombinant human calreticulin complexes are released, which are easily taken up by DC cells , or tumor-specific antigen peptides derived from: NY-ESO-1, AFP, CEA, CA-125, MUC-1, ETA, MAGE, mutant Ras, Raf and mutant p53, i...

Embodiment 2

[0066] Example 2 Tumor cells are induced by ICD inducers to appear ICD characteristic CRT translocation phenomenon

[0067] 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:

[0068] 1. Surgical resection of tumor specimens or core-needle biopsy of tumor tissue, under sterile conditions, remove necrotic tissue and adjacent non-tumor tissue (using colon cancer CT26 tumor as a model);

[0069] 2. Wash with sterile normal saline for 3 times; use sterile tissue scissors to cut up the tumor tissue, add RPMI 1640 medium, and grind thoroughly;

[0070] 3. Collect the single-cell suspension after filtering through a 200-mesh sterile net;

[0071] 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 part to detect the translocation of ICD ...

Embodiment 3

[0084] Example 3 Preparation and identification of CD8+Tscm cells

[0085] 1. Culture PBMC with serum-free medium, collect suspended cells, and adjust the cell concentration to 1-2x10 with serum-free medium 6 / ml;

[0086] 2. Positive selection of CD8+ naive T lymphocytes by immunomagnetic bead method;

[0087] 3. Antigen-loaded DC cells and CD8+ initial T cells were co-cultured according to a certain number ratio, and recombinant human IL-2 (300U / ml) and recombinant human IL-7 (300U / ml) were added to the serum-free culture medium. IL-15 (300U / ml);

[0088] 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) in the culture medium, every 3 days The medium was changed once a day and a half.

[0089] 5. Collect the cells on the 7th day, the number of cells should reach 1×10 6 more than one;

[0090] 6. Identification of CD8+Tscm cells: The expression of CD8, CD62L,...

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Abstract

The invention belongs to the technical fields of immunology and tumor therapy, and relates to a preparation method and a use of a TCR gene modified CD8+T memory stem cell. The method comprises the following steps: co-incubating a tumor antigen and an immature dendritic cell to obtain a specific tumor antigen-supported mature dendritic cell, co-culturing the specific tumor antigen-supported maturedendritic cell and a CD8+ naive T cell, and adding a stem cell differentiation inhibitor to promote the generation of a CD8+ stem cell-like memory T lymphocyte; separating the CD8+ stem cell-like memory T lymphocyte; and cloning a T cell receptor gene (TCR) for specifically identifying a specific antigenic epitope, and carrying out lentivirus packaging or retroviral vector cotransfection on an autologous CD8+ Tscm cell to in-vitro prepare a CD8+ TCR-Tscm cell specific for different tumor specific antigens. The CD8+ TCR-Tscm cell prepared by the method has the advantages of overcoming of the tumor heterogeneity, high specificity, few adverse reactions, and highly-efficient and lasting tumor preventing and treating effects.

Description

technical field [0001] The invention belongs to the technical field of immunology and tumor therapy, and relates to a method for preparing CD8+T memory stem cells in vitro by using genetic engineering technology, in particular to a CD8+T memory stem cell (T cellReceptor-T memory stem) modified by TCR gene cell, CD8+TCR-Tscm) preparation method and application thereof; especially the in vitro preparation method of TCR gene-modified CD8+T memory stem cells (TCR-Tscm) that can recognize multiple tumor-specific antigens and its use in Use in the preparation of anti-tumor preparations. Background technique [0002] The prior art discloses that malignant tumor is a systemic disease, which is the malignant change of normal human organ tissue cells under the action of various pathogenic factors, evading immune surveillance, breaking the immune balance, resulting in immune tolerance, and then showing unlimited growth of new organisms. In view of the fact that malignant tumors are a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A61K35/17A61P35/00
Inventor 吴向华邢凯琳胡爱群
Owner FUDAN UNIV SHANGHAI CANCER CENT
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