Anti-tumor NK cell, and preparation method and application thereof

A NK cell, anti-tumor technology, applied in anti-tumor drugs, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of NK cells unable to respond to activation, application effect to be improved, and unsatisfactory response performance. , to achieve the effect of improving NK cell tumor killing, realizing specific recognition and killing, and easy to achieve

Active Publication Date: 2020-04-07
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of intracellular signal transduction molecules, this kind of anti-tumor NK cells is likely to cause NK cells to fail to respond to activation or to have weak activation. Moreover, the activation process of this anti-tumor NK cells is inhibited by tumor cell PD-L1 signaling , the specificity is weak, the response performance is unsatisfactory, and the application effect needs to be improved

Method used

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  • Anti-tumor NK cell, and preparation method and application thereof
  • Anti-tumor NK cell, and preparation method and application thereof
  • Anti-tumor NK cell, and preparation method and application thereof

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preparation example Construction

[0064] A preparation method of anti-tumor NK cells of the present invention comprises the following steps:

[0065] 1) Synthetic chimeric antigen receptor coding gene

[0066] Design specific primers, respectively, for the gene encoding the extracellular domain fragment of PD-1, the gene encoding the F2A peptide fragment, the gene encoding the extracellular region of the CD8a signal peptide, the gene encoding the extracellular variable region of the single-chain antibody that binds to the HER2 protein, The gene encoding the CD8αLinker region, the gene encoding the CD28 transmembrane and intracellular co-stimulatory region, and the gene encoding the CD3ζ fragment that transmits signals in the cell were amplified by PCR, and the amplified products were sequentially connected to obtain the chimeric antigen receptor encoding gene ;

[0067] 2) Construction of recombinant lentiviral vector

[0068] The chimeric antigen receptor HER2-CAR and the enhanced sPD1-HER2-CAR coding gene we...

Embodiment 1

[0079] (1) Construction of HER2-CAR

[0080] According to the sequence of the target fragment of the chimeric antigen receptor, the primers required for PCR are designed and synthesized.

[0081] First, use PCR to amplify the Anti-HER2 ScFv fragment using the pACgp67B-Her2 plasmid as a template; use overlap extension PCR to connect the Anti-HER2 ScFv fragment to the leader fragment; use PCR to amplify the human cDNA as a template to obtain CD28 and CD3ζ region; use overlap extension PCR to connect CD28 fragment with CD3ζ; use overlap extension PCR to connect CD28+CD3ζ fragment with linker.

[0082] Next, overlap extension PCR was performed using CD8a leader+Anti-HER2 ScFv and linker+CD28+CD3ζ as templates to obtain a complete HER2-CAR fragment. Using human cDNA as a template, PCR amplification was performed to obtain the extracellular segment of PD-1.

[0083] (2) Construction of sPD1-HER2-CAR

[0084] Based on the construction of HER2-CAR, overlap extension PCR was used to...

Embodiment 2

[0116] Western-blot detection of the expression of sPD-1 in the supernatant of transformed cells

[0117] 1) Supernatant sample preparation: collect the culture supernatant of NK92, HER2-NK92 and sPD1-HER2-NK92 cells, and centrifuge to remove cell debris;

[0118] 2) Configure 8% lower layer separating gel and 4% lower layer stacking gel;

[0119] 3) Add 20 μL of loading buffer and supernatant premix to each well, use the pre-stained protein marker as a control, perform electrophoresis detection, transfer the protein to the NC membrane, use 5% skimmed milk to block at room temperature for 2 hours, and incubate with the primary antibody at 4°C Overnight, wash 4 times with TBST, 5 min each time, incubate with secondary antibody at room temperature for 1 h, wash 4 times with TBST, 5 min each time;

[0120] 4) Visualization, according to the results of western blot, the expression level of sPD-1 in the culture supernatant of cells transfected with different lentiviruses and the c...

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Abstract

The invention provides an anti-tumor NK cell, and a preparation method and application thereof. The anti-tumor NK cell is an NK cell edited by a chimeric antigen receptor. The chimeric antigen receptor comprises an extracellular domain fragment of PD-1, an F2A peptide fragment, the extracellular region of a CD8a signal peptide, the extracellular variable region of a single-chain antibody capable of recognizing and binding to HER2 protein, a CD8 alpha Linker region, a CD28 transmembrane and intracellular synergistic co-stimulation region, and a CD3 zeta fragment for intracellular signal transmission. The NK cell disclosed by the invention can express a CAR element targeting HER2 on a cell membrane and express secretory PD-1 protein at the same time, so the binding of PD-1 and PD-L1 is blocked, and the killing function of the NK cell is increased; and the anti-tumor NK cell is used for preparing anti-tumor drugs, can target tumor cells and enhance cytotoxic effect, is low in immunogenicity, easily realizes response and activation and can achieve allogeneic reinfusion, and the tumor killing effect of the NK cell is remarkably improved.

Description

technical field [0001] The invention relates to the technical field of cell therapy drugs, in particular to an anti-tumor NK cell and its preparation method and application. Background technique [0002] In recent years, cell therapy has emerged as a powerful treatment for advanced cancers. Because it can not only target the tumor itself, but also improve the patient's immune system, it is considered to be one of the most promising treatments for cancer. Among them, chimeric antigen receptor-modified T cells (CAR-T) have become a typical representative of tumor adoptive immunotherapy due to their remarkable clinical efficacy in some hematological tumors. However, CAR-T still has many limitations that affect its wide clinical application, such as the in vitro transformation of autologous T cells, the generation of cytokine storm, and the poor clinical treatment effect of solid tumors. Therefore, there is an urgent need to find new immune cell therapy methods to improve its ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K39/00A61P35/00
CPCC07K16/32C07K14/7051C07K14/70521C12N5/0646C12N15/86A61K39/001106A61P35/00C07K2317/622C07K2319/03C07K2319/02C07K2319/33C12N2740/15043A61K2039/5156Y02A50/30
Inventor 李静夏文姣戚欣陈俊生李红岩耿美玉
Owner OCEAN UNIV OF CHINA
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