CIK (Cytokine-Induced Killer Cell) capable of knocking down expression of endogenous PD-1 (programmed death 1) and preparation method and application thereof

An endogenous, PD-1 technology, applied in the field of cell therapy, can solve the problems of affecting PD-1 binding, high cost, and large amount of antibodies

Active Publication Date: 2017-11-03
BEIJING SHUNLEI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the amount of antibody required for intravenous infusion of PD-1 antibody is large and the cost is high; at the same time, bloo...

Method used

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  • CIK (Cytokine-Induced Killer Cell) capable of knocking down expression of endogenous PD-1 (programmed death 1) and preparation method and application thereof
  • CIK (Cytokine-Induced Killer Cell) capable of knocking down expression of endogenous PD-1 (programmed death 1) and preparation method and application thereof
  • CIK (Cytokine-Induced Killer Cell) capable of knocking down expression of endogenous PD-1 (programmed death 1) and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1. Preparation and identification of CIK cells capable of knocking down endogenous PD-1 expression

[0069] 1. Preparation of CIK cells capable of knocking down endogenous PD-1 expression

[0070] 1. Preparation of lymphocyte medium: serum-free medium containing IL-2 with a final concentration of 300IU / ml and IFN-γ with a final concentration of 1000IU / ml. Wherein, the serum-free medium is the product of Lonza Company, the product number is 04-418Q; the IL-2 is the product of peprotech company, the product number is 200-02; the IFN-γ is the product of peprotech company, the product number is 300-02 .

[0071] 2. Collect the peripheral blood of healthy people (of course, if the peripheral blood of most tumor patients can also be used), after testing that there is no pollution, dilute it with an equal volume of normal saline, and use Ficoll lymphocyte separation fluid (the Ficoll lymphocyte separation fluid) Density is 1.077g / ml) the peripheral blood of dilution ...

Embodiment 2

[0089] Detection of the killing effect of the CIK cells capable of knocking down endogenous PD-1 expression prepared in Example 2 and Example 1 on tumor cells

[0090] First, the human tumor cell line SKOV3 was inoculated on the cell culture plate of the Eisen Live Cell Detection Station. After 12 hours, when the cells adhered to the wall, a certain amount of CIK, CIK-pCDH-miRNA-PD1, CIK-pCDH-miRNA was added -PD1-GFP cells, forming different effector-target ratios. Continue culturing for 48 hours to detect the killing effect of various CIK cells on tumor cells.

[0091] The result is as Figure 5 As shown, it can be seen from the figure that the tumor killing activity of CIK cells transfected with pCDH-miRNA-PD1 or pCDH-miRNA-PD1-GFP plasmid is significantly improved.

[0092] Beijing Ruijian Technology Co., Ltd.

[0093] CIK capable of knocking down endogenous PD-1 expression and its preparation method and application

[0094] GNCLN171462

[0095] 2

[0096] Patent...

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Abstract

The invention discloses a CIK (Cytokine-Induced Killer Cell) capable of knocking down endogenous PD-1 (programmed death 1) expression and a preparation method and application thereof. The invention provides a method for preparing a CIK preparation capable of knocking down endogenous PD-1 expression, comprising the following steps: A) inducing the differentiation of PBMC to CIK in vitro by using cytokines; B) transferring a vector capable of knocking down endogenous PD-1 expression into the CIK obtained in step A), culturing for 13 to 16 days and harvesting a cell, thereby obtaining the CIK preparation capable of knocking down the endogenous PD-1 expression, wherein the vector capable of knocking down endogenous PD-1 expression contains a plurality of tandem encoded genes targeting miRNAs of PD-1, and the number of the encoded genes is 2 to 30. Experimental results show that the vector can significantly reduce the expression level of endogenous PD 1 in the CIK, thereby significantly enhancing the tumor-killing activity of a CIK cell.

Description

technical field [0001] The invention belongs to the field of cell therapy, and relates to a CIK capable of knocking down endogenous PD-1 expression, a preparation method and application thereof. Background technique [0002] CIK (Cytokine-Induced Killer Cells) is a group of heterogeneous cells obtained by co-culturing human peripheral blood mononuclear cells with various cytokines for a period of time in Chinese. It has not only the strong anti-tumor activity of T lymphocytes, but also the non-MHC (major histocompatibility antigen) restricted tumor killing ability of NK cells (natural killer cells). CIK cells have the characteristics of high tumor killing activity, broad tumor killing spectrum, low toxicity to normal tissues, and high expansion in vitro. A large number of clinical cases have confirmed their tumor killing activity. [0003] PD-1 (programmed death receptor) is a protein molecule expressed on the surface of T cells. When PD-1 binds to its ligand PD-L1 or PD-L2...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C12N5/0638C12N15/86C12N2510/00C12N2740/15043
Inventor 袁绍鹏占顺利高磊
Owner BEIJING SHUNLEI BIOTECHNOLOGY CO LTD
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