Preparation method of high-efficiency killer cell preparation with double blocking CTL of immune checkpoint

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 Effects of cell depletion, improvement of tumor killing effect, and improvement of clinical objective effectiveness

Active Publication Date: 2018-02-02
浓孚雨医药河北有限公司
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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 with double blocking CTL of immune checkpoint
  • Preparation method of high-efficiency killer cell preparation with double blocking CTL of immune checkpoint
  • Preparation method of high-efficiency killer cell preparation with double blocking CTL of immune checkpoint

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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, and a high-efficiency double-deleted CTL preparation was prepared. At the same time, the ligands PD-L1 and CTLA-4 of PD-1 on the surface of tumor cells were detected. The expression characteristics of ligands B7-1 and 2 (CD80, CD86), the specific steps are as follows:

[0032] 1) Obtaining monocytes:

[0033] Collect 100ml of peripheral blood mononuclear cells (or collect hematopoietic stem cells from umbilical cord blood to prepare allogeneic DC) from tumor patients by venous extraction or blood component separation machine, collect whole blood cells by centrifugation, transfer into lymphocyte separation medium, and centrifuge at 2000rpm×15 with a horizontal rotor Minutes, aspirate the middle buffy coat to collect monocytic cells.

[0034] 2) DC and T cell sorting:

[0035] The monocytes collected above were transferred into culture flasks containing serum-free...

Embodiment 2

[0047] Example 2 is the application of 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, DC-CIK and antigen-specific CTL Effector cells were controlled to verify the advantage 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 cells: breast cancer cell line (MDA-SB435S).

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

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

[0053] For cytotoxicity analysis, inoculate MDA-SB435S target cells in a 96-well plate, add effector cells Dual-block CTL, CTL, DC-CIK, T and NS, respectively, 3 wells in each group, co-c...

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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 technology and a new preparation of anti-tumor cell immunotherapy, in particular to a novel, clinical, 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 history of LAK, DC-CIK, CTL and TIL. Although the killing efficiency has gradually improved and clinical trials have achieved good clinical efficacy, the low objective effective rate is still the main problem to be solved in clinical practice. The key to high-efficiency tumor killing technology lies in core issues such as how to break tumor immune tolerance, improve the tumor-specific recognition function of T cell preparations, enhance the in vivo killing efficiency of effector T cells, and reduce clinical side effects. [0003] In recent years, immune checkpoint blockade has become a research hotspot in cellular immu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/00A61P35/00C12N5/0784C12N5/0783
CPCA61K39/0011A61K2039/5154C12N5/0636C12N5/0639C12N2501/22C12N2501/2304
Inventor 蔡守锋张伟高飞李冬斌蔡子琪
Owner 浓孚雨医药河北有限公司
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