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
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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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