Preparation method of CAR-T cell with CD133 specificity, based on non-viral vector and capable of automatically secreting PD1 scFv
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Embodiment 1
[0031] Example 1: Construction of minicircle CAR plasmid:
[0032] By searching the CD133 antibody and PD1 antibody sequences, the scFv segments in the CD133 and PD1 antibody sequences were selected, and the second-generation CAR-T technology was used to design the CAR-T sequence, and the PD1 scFv was connected through P2A. The terminal repeat sequence required by the seat system is constructed by the company and verified by sequencing.
[0033] Construct the above sequence into the multifunctional cloning site in the parent plasmid pMC.BESPX-MCS for preparing the minicircle; purchase the transposase plasmid pCMV(CAT)-T7-SB100, and CMV-SB100×-SV40 polyA on the plasmid The segment was integrated into the minicircle parent plasmid pMC.BESPX-MCS through homologous recombination; the above two plasmids were sequenced and verified (see figure 1 Figure a).
[0034] The above plasmid was transformed into competent cells ZYCY10P3S2T by heat shock method, then 200 μl of SOC medium wa...
Embodiment 2
[0035] Example 2: CAR-T cell preparation stage
[0036] Take blood from a healthy donor, centrifuge at 500g for 10min, discard the upper plasma, add an equal volume of PBS, resuspend, transfer to a 50ml centrifuge tube reserved for 20ml of lymphocyte separation medium, make up to 50ml with normal saline; centrifuge at 800g for 20min (liter 9, down 6), divided into 4 layers, take the white floc (white film) into a new 15ml centrifuge tube, wash the white film with elution buffer, count; every 10 7 For each T cell, add 5ul Pan T Cell Biotin antibody to 60μl elution buffer, incubate at 4°C for 5min; then add 10ul Pan T cell magnetic beads and incubate at 4°C for 10min; after the incubation, add the liquid to the LS separation column, After sorting by magnetic beads, the cells that come down from the column are T cells; take 1×10 7 Put T cells into a 6-well plate, add 2ml X-vivo, IL-21000IU / ml, and add CD3 / CD28 to stimulate overnight; on the same day, use DPBS to coat CD133 antig...
Embodiment 3
[0041] Example 3: Killing test of CAR-T cells
[0042] The liver cancer cell lines SK-Hep1 (SK) and Hep-3B (3B) were selected, and the run-flow analysis showed that there was almost no expression of CD133 on SK, and high expression of CD133 on 3B (>90%). Mock T and MC133&PD1 scFv CAR- After co-incubating T with these two kinds of tumor cells according to the effect-target ratio of 1:1, 5:1, and 10:1 for 4 hours, the apoptosis and death of tumor cells were detected by the apoptosis kit (PI / Annexin-V), and the results It has been shown that the prepared CAR-T cells have stronger killing ability than ordinary T cells (see image 3 ).
[0043] For further verification, the killing ability of MC133 CAR-T alone and MC133&PD1 scFv CAR-T was compared. After co-incubating these two kinds of cells with 3B cells at an effect-to-target ratio of 1:4 for 3 days, it was detected by flow cytometry: MC133&PD1scFv CAR-T cells can almost completely kill tumor cells, while tumor cells still rem...
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