Quasi-chimeric antigen receptor-modified CIK (cytokine induced killer) as well as preparation method and application thereof

A technology of chimeric antigen receptors and cell preparations, which is applied in the field of cell therapy and can solve problems such as high cost, large amount of antibodies, and affecting binding

Active Publication Date: 2018-02-09
BEIJING SHUNLEI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, intravenous infusion of CTLA4 antibody requires a large amount of antibody and high cost; at the same time, blood components, lymphoid tissue microenvironment and other components will affect the binding of CTLA4 antibody to B7 on the surface of T cells

Method used

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  • Quasi-chimeric antigen receptor-modified CIK (cytokine induced killer) as well as preparation method and application thereof
  • Quasi-chimeric antigen receptor-modified CIK (cytokine induced killer) as well as preparation method and application thereof
  • Quasi-chimeric antigen receptor-modified CIK (cytokine induced killer) as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1. Preparation and Identification of CIK Cells Similar to Chimeric Antigen Receptor Modification

[0081] The chimeric antigen receptor-modified CIK cells prepared in this example can knock down endogenous CTLA4 expression, and can simultaneously express CTLA4 extracellular region-CD28-41BB-CD3 fusion protein.

[0082] 1. Preparation of CIK cells similar to chimeric antigen receptor modification

[0083] 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 .

[0084] 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 ...

Embodiment 2

[0103] Detection of the killing effect of CIK cells modified by similar chimeric antigen receptors prepared in Example 2 and Example 1 on tumor cells

[0104] 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-CTLA4ECD- CD28-41BB-CD3-IRES-miRNA-CTLA4-GFP, CIK-pLV-CTLA4ECD-CD28-41BB-CD3-IRES-miRNA-CTLA4 cells formed different effect-to-target ratios. Continue culturing for 48 hours to detect the killing effect of various CIK cells on tumor cells.

[0105] The result is as Figure 4 As shown, it can be seen from the figure that CIK cells transfected with pLV-CTLA4ECD-CD28-41BB-CD3-IRES-miRNA-CTLA4-GFP or pLV-CTLA4ECD-CD28-41BB-CD3-IRES-miRNA-CTLA4 plasmids have a positive effect on the tumor The killing activity was significantly improved.

[0106] Beijing Ruijian Technology Co., Ltd.

...

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Abstract

The invention discloses a quasi-chimeric antigen receptor-modified CIK (cytokine induced killer) as well as a preparation method and the application thereof. The method for preparing a quasi-chimericantigen receptor-modified CIK cell preparation comprises the following steps: silencing 5'-UTR, 3'-UTR and / or an intracellular region of a CTLA4 mRNA in the CIK by using multiple serially-connected miRNAs, and expressing a CTLA4 extracellular region-CD28-41BB-CD3 fusion protein, so as to prepare the quasi-chimeric antigen receptor-modified CIK cell preparation. An experiment shows that the carriercan greatly reduce the expression of endogenesis CTLA4 in a CIK cell, and after the expressed CTLA4 extracellular region-CD28-41BB-CD3 fusion protein is combined with B7 on the surface of a cell T, the CIK cell is activated, thereby obviously improving the tumoricidal activity of the CIK cell.

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 the expression of endogenous CTLA4 and simultaneously expressing the extracellular region of CTLA4-CD28-41BB -CIK of 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 高磊袁绍鹏占顺利
Owner BEIJING SHUNLEI BIOTECHNOLOGY CO LTD
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