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Preparation method of CAR-T cell with CD133 specificity, based on non-viral vector and capable of automatically secreting PD1 scFv
Pending Publication Date: 2021-11-23
夏建川
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[0005] The purpose of the present invention is to provide a method for preparing CD133-specific and self-secreting PD1 scFv CAR-T cells based on non-viral vectors, which can solve the problem that the current use of viruses to prepare CAR-T cells and plasmid-modified T cells often carry anti- Insufficient safety issues such as sex genes, through the combination of Sleeping Beauty transposon and microcircle carrier technology, the stable integration of CAR gene into T cells is achieved, and the potential risk of plasmid resistance genes in human applications is also avoided
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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...
[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 CellBiotin 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 cancercell 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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Abstract
The invention discloses a preparation method of CAR-T (Chimeric Antigen Receptor-T) cells with CD133 specificity, based on a non-viral vector and capable of automatically secreting PD1scFv. The preparation method disclosed by the invention comprises the following steps: constructing a sleeping beauty CAR minicircle parent plasmid and a sleeping beauty transposaseminicircle parent plasmid; preparing the sleeping beauty CAR minicircleplasmid and the sleeping beauty transposase minicircle plasmid; carrying out electrotransfection to prepare CAR-T cells; expanding and enriching the CAR-T cells; and harvesting and characterizing the CAR-T cells. The CAR-T cells capable of secreting PD1scFv and targeting CD133 are obtained through a non-virus method, the CAR-T cells not only can effectively combine the advantages of the CAR-T cells and immune checkpoint inhibitors, but also can further remove resistance genes on plasmids by adopting the non-virus sleep beauty transposon and minicircle combined method, so that the safety of the CAR-T cells in clinical application can be ensured to the greatest extent.
Description
technical field [0001] The invention belongs to the field of biomedicine, and relates to a method for preparing non-viral vector CD133-specific and autocrine PD1 single-chain antibody scFv CAR-T cells, in particular to a technology based on Sleeping Beauty transposon and microcircle vectors. A non-viral CAR-T cell approach. Background technique [0002] Primary liver cancer is one of the malignant tumors with the highest morbidity and mortality. Traditional surgery, radiotherapy and chemotherapy are still difficult to effectively treat tumors. There is an urgent need to develop new treatment methods. Immunotherapy has become a hot spot in current tumor treatment research and clinical application . Among them, CAR-T cell therapy, which uses genetic engineering to modify T cells in vitro and then reinfuse them into patients, has shown great advantages in the treatment of hematological malignancies, but its therapeutic effect in solid tumors is not good. Therefore, it is nece...
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