Dendritic cell vaccine sensitized by A2B5 + glioma cells

A technology of dendritic cells and glioma stem cells, applied in the fields of biotechnology and medicine, can solve the problem of limited ability of sensitizing T cells

Inactive Publication Date: 2022-03-22
上海尚泰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immature DCs have limited ability to prime

Method used

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  • Dendritic cell vaccine sensitized by A2B5 + glioma cells
  • Dendritic cell vaccine sensitized by A2B5 + glioma cells
  • Dendritic cell vaccine sensitized by A2B5 + glioma cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0277] Example 1. Preparation of A2B5+ glioma stem cell antigen

[0278] Take out the fresh human glioma tissue block and wash it 2-3 times with PBS containing double antibody, cut into 1mm 3 size. Wash 2-3 times with DMEM / F12 culture medium, add 5-10 times the tissue volume of type IV collagenase, shake and digest at 200rpm in a 37°C incubator for 15min, centrifuge at 1000rpm / min for 5min, filter with a 100μm cell sieve, and prepare a single cell suspension.

[0279] Add 5 mL of erythrocyte lysate to the cell pellet of the above single-cell suspension, incubate in a 37°C incubator for 5-10 min, wash with PBS three times; use serum-free culture medium (containing 20 ng / mL bFGF, 20 ng / mL EGF and DMEM / F12 culture medium of B27) to resuspend the cell pellet, count and press 2~4×10 5 Cells / mL were inoculated in an ultra-low adsorption six-well plate.

[0280] The cell pellet was collected, and A2B5+ cells were sorted by magnetic beads. Gamma ray irradiation, the irradiated ...

Embodiment 2

[0281] Example 2. Transfection, maturation induction and specificity of DC CTL preparation of

[0282] 1) DC transfection and maturation induction: the patient's own peripheral blood was collected, and autologous plasma (inactivated at 56°C for 30 minutes, stored at 4°C) and peripheral blood were separated by Ficoll-Hypaque (polysucrose-diatrizoate) density gradient separation method Mononuclear cell PBMC, this PBMC was treated with serum-free x-vivo15 at 5-7×10 7 The amount of cells was plated in a T75 culture flask, and the suspension cells were collected after 1 hour for the separation of CD3+ T lymphocytes, and the adherent cells were replaced with IL-4 (100ng / mL), GM-CSF (100ng / mL) and autologous plasma (2 %) of 1640 medium stimulation induced monocytes to differentiate into DC.

[0283] On day 5, immature DCs suspended in a 24-well plate were sensitized with the A2B5+ stem cell antigen prepared as in Example 1. After 24 hours of sensitization, replace with fresh DC...

Embodiment 3

[0288] Example 3. Antigen-specific CTL tumor cell killing experiment

[0289] In order to verify the specific killing effect of antigen-specific CTLs on glioma stem cells, the target cells of this example were sorted A2B5+ cells, and an in vitro killing verification experiment of antigen-specific CTLs was carried out. On the 14th day of specific expansion of T lymphocytes, CTL and tumor target cells were added to 96-well plates at 1:1, 5:1 and 10:1, respectively, and incubated for 16 hours, then added CCK8 kit reagents and incubated for 1 hour. The OD value of cells in each group was detected by a microplate reader, and finally the specific killing efficiency of CTL on cell lines was calculated.

[0290] Such as image 3 The results shown show that the A2B5-targeting CTL prepared in Example 2 has a good killing effect and strong specificity on A2B5+ cells, and the tumor killing rate of conventional CTL is 30% of the tumor killing rate of the present invention. -60%. This ...

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Abstract

The invention provides a dendritic cell vaccine sensitized by an A2B5 < + > brain glioma stem cell-like antigen. Specifically, the invention also provides a dendritic cell targeting A2B5 positive tumor, a cytotoxic T lymphocyte, a cell preparation and a preparation method and application thereof. The dendritic cell vaccine can be used for tumor prevention and active immunotherapy, has good safety, high purity, efficient killing ability and strong specificity, and can enhance the immunocompetence of patients, improve prognosis and prevent tumor recurrence to a certain extent.

Description

technical field [0001] The present invention relates to the fields of biotechnology and medicine, in particular, the present invention relates to a dendritic cell vaccine sensitized by A2B5+ glioma stem cell-like antigen and its preparation method and application. Background technique [0002] Dendritic cells (DC) are the most functional professional antigen-presenting cells in the human body, which can activate CD8+ cytotoxic T cells (CTL) and CD4+ helper T cells (Th), and play an important role in anti-tumor and anti-virus important role in the immune response. Most studies have shown that tumors in vivo inhibit DC maturation rather than directly inhibit DC function. Studies have found that the number and function of dendritic cells in malignant tumor tissues are negatively correlated with the degree of infiltration of tumor cells into the surrounding tissues in the primary tumor tissue and metastases, and the clinical stage of the patient, while the infiltration dendriti...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K35/17A61K35/15A61P35/00C12N5/0784C12N5/0783C07K14/47
CPCA61K39/0011A61P35/00C12N5/0639C12N5/0638C12N2502/1121C12N2501/2304C12N2501/22C12N2501/24C12N2501/90C12N2501/2307C12N2501/2315C12N2501/2302
Inventor 李晨蔚周慧斌邓婷婷周冰逸胡雪
Owner 上海尚泰生物技术有限公司
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