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Preparation method of high-efficiency killer cell preparation adopting immunodetection point dual-block CTL (cytotoxic lymphocyte)

A technology of immune detection points and killing cells, which is applied in the direction of blood/immune system cells, cell culture active agents, biochemical equipment and methods, etc., can solve the problems of low clinical objective effective rate and low tumor killing efficiency, and achieve slowing down of T Effect of cell depletion, improvement of tumor killing effect, and improvement of clinical objective efficiency

Active Publication Date: 2016-12-07
浓孚雨医药河北有限公司
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  • Application Information

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Problems solved by technology

[0006] The main purpose of the present invention is to solve the problems of low tumor killing efficiency and low clinical objective effective rate caused by the tumor microenvironment in the current clinical application of cellular immunotherapy. The technical method of the present invention can significantly improve the tumor killing efficiency and clinical efficacy. Objective efficiency

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  • Preparation method of high-efficiency killer cell preparation adopting immunodetection point dual-block CTL (cytotoxic lymphocyte)
  • Preparation method of high-efficiency killer cell preparation adopting immunodetection point dual-block CTL (cytotoxic lymphocyte)
  • Preparation method of high-efficiency killer cell preparation adopting immunodetection point dual-block CTL (cytotoxic lymphocyte)

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Experimental program
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Embodiment 1

[0031] In this example 1, PD-1 and CTLA-4 antibodies were used to block the inhibitory signal of CTL preparations to prepare high-efficiency double-deleted CTL preparations. At the same time, the tumor cell surface PD-1 ligands PD-L1 and CTLA-4 were detected. The expression characteristics of ligands B7-1 and 2 (CD80, CD86), the specific steps are as follows:

[0032] 1) Acquisition of monocytes:

[0033] Intravenous extraction or blood component separator collects peripheral blood mononuclear cells of tumor patients (or collects cord blood hematopoietic stem cells to prepare allogeneic DC) 100ml, collects whole blood cells by centrifugation, transfers the lymphocyte separation solution, and centrifuges 2000rpm×15 with a horizontal rotor Minutes, aspirate the middle albuginea layer, and collect mononuclear cells.

[0034] 2) DC and T cell sorting:

[0035] The monocytes collected above were transferred to a culture flask containing serum-free medium and placed at 37°C, 5% CO 2 Incuba...

Embodiment 2

[0047] This example 2 is to use Dual-block CTL effector cell preparations to conduct laboratory killing experiments (in vitro) and animal experiments (in vivo) on target cells (breast cancer). At the same time, it is combined with DC-CIK and antigen-specific CTL The effector cells were subjected to a control study to verify the advantages of Dual-block CTL in killing efficiency.

[0048] 1. In vitro killing experiment research:

[0049] 1) Effector cells: Dual-block CTL (experimental group), T cells (control 1), DC-CIK (control 2), CTL (control 3).

[0050] 2) Target cell: breast cancer cell line (MDA-SB435S).

[0051] 3) Experimental grouping: Dual-block CTL (experimental group), T cell (control 1), DC-CIK (control 2), CTL (control 3), NS (negative control).

[0052] 4) In vitro killing and cytokine secretion experiments:

[0053] Using cytotoxicity analysis, inoculate MDA-SB435S target cells in 96-well plates, and add effector cells Dual-blockCTL, CTL, DC-CIK, T and NS, 3 wells per gr...

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Abstract

The invention discloses a preparation method of a high-efficiency killer cell preparation adopting an immunodetection point dual-block CTL (cytotoxic lymphocyte). The preparation method comprises steps as follows: step 1), collecting mononuclear cells in peripheral blood; step 2), performing DC (dendritic cell) separation and T cell separation on the separated mononuclear cells with an adherence method; step 3), adding adhering DCs to a serum-free culture medium containing GM-CSF (granulocyte-macrophage colony-stimulating factor) and IL-4 (interleukin-4) for preparation of a DC vaccine; step 4), adding suspending T cells to CD3 and IL-2 for T cell culture and amplification; step 5), adding antigen-supported DCs to the amplified T cells for preparation of the CTL, and adding PD-1 (Programmed death 1) and CTLA-4 (cytotoxic t lymphocyte associated antigen-4) antibodies for preparation of the cell preparation with the dual-block CTL effect. According to the novel technology and the novel method provided by the invention, an immune brake effect is effectively regulated and T cell depletion is retarded by blocking inhibitory molecules on the tumor specific CTL surfaces, so that the tumor cytotoxicity of CTL effector cells is substantially improved.

Description

Technical field [0001] The invention relates to a new anti-tumor cellular immunotherapy technology and a new preparation, in particular to a new, clinical type, tumor-specific immune detection point double blocking CTL high-efficiency killer cell preparation and a preparation method thereof. Background technique [0002] Cellular immunotherapy has gone through the course of LAK, DC-CIK, CTL and TIL. Although the killing efficiency has gradually improved and clinical trials have achieved good clinical effects, the objective effectiveness of low efficiency is still the main clinical problem that needs to be resolved. The key to high-efficiency tumor killing technology lies in the core issues of how to break tumor immune tolerance, improve the tumor-specific recognition function of T cell preparations, enhance the killing efficiency of effector T cells in vivo, and reduce clinical side effects. [0003] In recent years, immune checkpoint blocking has become a research hotspot in cellu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61P35/00C12N5/0784C12N5/0783
CPCA61K39/0011A61K2039/5154C12N5/0636C12N5/0639C12N2501/22C12N2501/2304
Inventor 蔡建辉张伟崔红娟李冬斌蔡颖
Owner 浓孚雨医药河北有限公司
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