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EGFR-targeting chimeric antigen receptor and CAR-NK cell, preparation method for EGFR-targeting CAR-NK cell, and applications for EGFR-targeting chimeric antigen receptor and CAR-NK cell

A chimeric antigen receptor and targeting technology, applied in the fields of biology and medicine, can solve problems such as inability to maintain long-term adaptive immunity

Inactive Publication Date: 2020-05-12
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the first-generation CAR-NK does not contain other co-stimulatory signaling domains, it cannot maintain long-term adaptive immunity

Method used

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  • EGFR-targeting chimeric antigen receptor and CAR-NK cell, preparation method for EGFR-targeting CAR-NK cell, and applications for EGFR-targeting chimeric antigen receptor and CAR-NK cell
  • EGFR-targeting chimeric antigen receptor and CAR-NK cell, preparation method for EGFR-targeting CAR-NK cell, and applications for EGFR-targeting chimeric antigen receptor and CAR-NK cell
  • EGFR-targeting chimeric antigen receptor and CAR-NK cell, preparation method for EGFR-targeting CAR-NK cell, and applications for EGFR-targeting chimeric antigen receptor and CAR-NK cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] To isolate and obtain the mononuclear cells from the peripheral blood of healthy adults, the specific steps are as follows:

[0074] Collect peripheral blood from healthy adults via vein, centrifuge at 250g for 10min, discard the supernatant, add a volume of PBS to the lower sediment obtained by centrifugation and mix well, and add the mixed solution to the upper layer of the lymphocyte separation solution with a pipette . After centrifugation at 800g for 30min, the uppermost layer is the plasma and buffer layer, the second layer is the white mist layer, the third layer is the relatively clear lymphocyte separation liquid layer, and the lowermost layer is the red blood cell and granulocyte layer, which is rich in mononuclear cells. Set on the second floor. Transfer the obtained second-layer liquid to another centrifuge tube, add three times the volume of PBS solution, mix well, centrifuge at 400 g for 10 min, remove the supernatant, and obtain peripheral blood mononucl...

Embodiment 2

[0077] Carry out NK cell expansion culture and identification, the specific steps are as follows:

[0078] The mononuclear cell population obtained in Example 1 was activated and expanded in 10 ml NK cell feeder-free medium containing 10% autologous serum at 37°C containing 5% CO 2 cultured in an incubator for 1 week, and then in 10 ml NK cell feeder-free medium containing 5% autologous serum at 37°C with 5% CO 2 cultured in an incubator for a total of two weeks. Then centrifuge at 4°C and 400g for 10min to collect the cells, resuspend the cells with 1ml of PBS containing 0.5% BSA, centrifuge briefly twice for about 2 seconds, and discard the supernatant. The cells obtained after centrifugation were resuspended in 95 ul of PBS containing 0.5% BSA, and 5 ul of Fc blocker was added, and kept on ice for 30 min. Add 300ul of 0.5% BSA PBS to the cell solution obtained after treatment with Fc blocker, divide into two tubes of 100ul; add 5ul CD3-PE-Cy7, 5ul CD56-PE, 5ul CD69-APC-Cy...

Embodiment 3

[0082] To construct EGFR-CAR-NK cells, the specific steps are as follows:

[0083] The nucleic acid sequence of the EGFR extracellular domain-binding antibody was constructed into the third-generation CAR plasmid scFv, named EGFR-CAR. Among them, the third-generation CAR plasmid model diagram is as follows: figure 1 As shown, the nucleic acid sequence of the highly specific EGFR extracellular domain-binding antibody is SEQ ID NO: 2, and the structure of the EGFR-CAR plasmid after construction is shown in figure 2 Shown, the nucleotide sequence of the gene encoding EGFR-CAR plasmid is SEQ ID NO:1. The EGFR-CAR plasmid was synthesized by conventional biosynthesis methods and synthesized by Aikangde Biomedical Technology Co., Ltd.;

[0084] Take the 293T / 17 cells in the exponential growth phase and transfer them to a large dish with a cell density of 70%-80%. Add CAR, pMD2.G, psPAX2 lentiviral plasmids and 50ul Lipo2000 to 200ul Opti-MEM (serum-reduced medium) and mix well, p...

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Abstract

The invention provides an EGFR-targeting chimeric antigen receptor. The EGFR-targeting chimeric antigen receptor comprises an antigen binding domain of an anti-EGFR antibody, wherein the antigen binding domain of the anti-EGFR antibody is expressed as EGFRscFv; and the EGFRscFv has a nucleic acid sequence as shown in SEQ ID NO: 2. The invention also provides a CAR-NK cell and a preparation methodthereof. The NK cell expresses EGFRscFv of the EGFR-targeting chimeric antigen receptor to obtain the EGFR-targeting CAR-NK cell. The invention also provides a composition, an anticancer agent and applications. The EGFR-targeting CAR-NK cell provided by the invention has specific recognition on an EGFR target of triple negative breast cancer, kills EGFR positive triple negative breast cancer cellsin a targeting manner and promotes release of cytokines. When the CAR-NK cells are used as effector cells to recognize tumors, leukocyte antigens of the CAR-NK cells do not need to match the tumors,so graft-versus-host reaction cannot be generated. The CAR-NK therapy method does not cause cytokine storm and is safer. The CAR-NK cell is wide in sources like peripheral blood mononuclear cells, induced pluripotent stem cells, umbilical cord blood, human embryonic stem cells and NK-92 cell lines.

Description

technical field [0001] The present invention relates to the technical fields of biology and medicine, and in particular to an EGFR-targeted chimeric antigen receptor, CAR-NK cells and their preparation methods and applications. Background technique [0002] According to the 2018 Global Cancer Annual Report, breast cancer is still the most common type of cancer among women. In 2018 alone, there were 2,088,849 new cases of breast cancer worldwide, and 626,679 female patients died of breast cancer. According to the "2019 National Cancer Report" and "2018 China Cancer Registration Annual Report", there are about 279,000 new cases of breast cancer in my country, and about 66,000 deaths. It is estimated that by 2021, there will be 2.5 million breast cancer patients in my country. Breast cancer seriously endangers the life and quality of life of patients, while the cost of treatment and related social and economic problems bring a heavy burden to individuals and the country. Howe...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/13C12N15/62C12N5/10C12N15/867A61K39/00A61P35/00
CPCA61K39/0011A61P35/00C07K14/7051C07K16/2863C07K16/32C07K16/40C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 刘岩张京钟余爽王飞飞房旭婕安黎
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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