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CIK modified by similar chimeric antigen receptor, as well as preparation method and application thereof

A Chimeric Antigen Receptor, a technology of No. 1, applied in the field of cell therapy, can solve the problems of large amount of antibodies, affecting PD-1 binding, and high cost

Active Publication Date: 2018-01-23
BEIJING SHUNLEI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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, blood components, lymphoid tissue microenvironment and other components will affect the binding of PD-1 antibody to PD-1 on the surface of T cells

Method used

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  • CIK modified by similar chimeric antigen receptor, as well as preparation method and application thereof
  • CIK modified by similar chimeric antigen receptor, as well as preparation method and application thereof
  • CIK modified by similar chimeric antigen receptor, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Preparation and Identification of CIK Cells Similar to Chimeric Antigen Receptor Modification

[0062] The chimeric antigen receptor-modified CIK cells prepared in this example can knock down endogenous PD-1 expression, and can simultaneously express PD1 extracellular region-CD28-41BB-CD3 fusion protein.

[0063] 1. Preparation of CIK cells similar to chimeric antigen receptor modification

[0064] 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 .

[0065] 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 the...

Embodiment 2

[0084] Detection of the killing effect of CIK cells modified by similar chimeric antigen receptors prepared in Example 2 and Example 1 on tumor cells

[0085] 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 grew adherently, a certain amount of CIK (without transfection of any plasmid), CIK-pLV-PD1ECD- CD28-41BB-CD3-IRES-miRNA-PD1-GFP, CIK-pLV-PD1ECD-CD28-41BB-CD3-IRES-miRNA-PD1 cells formed different effect-to-target ratios. Continue culturing for 48 hours to detect the killing effect of various CIK cells on tumor cells.

[0086] The result is as Figure 4 As shown, it can be seen from the figure that the CIK cells transfected with pLV-PD1ECD-CD28-41BB-CD3-IRES-miRNA-PD1-GFP or pLV-PD1ECD-CD28-41BB-CD3-IRES-miRNA-PD1 plasmid have a significant effect on the tumor The killing activity was significantly improved.

[0087] Beijing Ruijian Technology Co., Ltd.

[0088] CI...

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Abstract

The invention discloses a CIK modified by similar chimeric antigen receptor, as well as a preparation method and application thereof. The method comprises the following steps: transferring a recombinant vector which can knock down endogenous PD-1 expression and can further express PD1 extracellular region-CD28-41BB-CD3 fusion protein into CIK, and harvesting cells after culturing is performed, soas to obtain a CIK cell preparation; ensuring that the recombinant vector contains 5'-UTR and 3'-UTR of a plurality of targeted PD-1mRNA in series connection and / or encoding gene of intracellular region miRNA. Experiments prove that the vector can greatly reduce the endogenous PD-1 expression in a CIK cell, and the expressed PD1 extracellular region-CD28-41BB-CD3 fusion protein activates the CIK cell after being combined with PD-L1 / L2 on the surface of tumor cell, so that the tumoricidal activity of the CIK cell is obviously improved.

Description

technical field [0001] The invention belongs to the field of cell therapy, and relates to a chimeric antigen receptor-like modified CIK and its preparation method and application, in particular to a method capable of knocking down endogenous PD-1 expression and simultaneously expressing PD1 extracellular region-CD28 CIK of -41BB-CD3 fusion protein and its preparation method and application. 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 clinic...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/62A61K35/17A61P35/00
Inventor 占顺利高磊袁绍鹏
Owner BEIJING SHUNLEI BIOTECHNOLOGY CO LTD
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