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Archive > Volume 30 Issue 4 > 2023,30(4):296-301. DOI:10.3872/j.issn.1007-385X.2023.04.003 Prev Next
Basic Research
Targeted killing effect of bispecific CAR-T cells on EGFRvⅢⅢ+/CD133+ glioma stem cells
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Abstract:
Objective: To prepare bsCAR-T cells and observe its targeted killing effect on epidermal growth factor variant Ⅲ(EGFRvⅢ+,simplified as vⅢ+) and CD133+ glioma stem cells. Methods: Based on the previously generated vⅢ/CD133 minibody and second-generation CAR, bispecific CAR (bsCAR) was constructed. Lentiviral bsCAR were prepared for the transfection of human peripheral blood T cells. Flow cytometry (FCM) and Western blot test were used to detect the transfection efficiency and expression of bsCAR. After bsCAR-T cells were co-cultured with vⅢ+/CD133+ U87 glioma stem cells, their killing effects were detected by LDH release test and cytokine IFN-γ secretion. vⅢ+/CD133+ U87 stem cell transplantation tumor model of nude mouse was established to test the inhibition of bsCAR-T cells on transplanted tumors. Results: vⅢscFv and CD133scFv were joined seamlessly by over-lap PCR with CAR expression cassette (S-vⅢ/CD133scFv-Hinge-TM-CD137-CD3z). The above bsCAR construct was then cloned into EcoRⅠand BamHⅠsites of pCDH-CMV-MCS-EF1-copGFP (pbsCAR). Lentiviral bsCAR were prepared by co-transfection of three plasmid (pVSV-G, pCMV-dR8.9 and pbsCAR) into HEK293T cells and later transfected human peripheral blood T cells. The expression of bsCAR detected by flow cytometry was 71.1%.Western blot analysis showed correct expression of bsCAR. The co-culture assay of bsCAR-T cells and vⅢ+/CD133+ U87 stem cells showed that bsCAR-T cells had specific killing effect on glioma stem cells, which was proportional to the effector-target ratio. IFN-γ secretion was (2 350.6±92) pg?mL-1, which was significantly higher than that of the control group (P<0.01). Nude mice transplantation tumor model demonstrated the transplantation tumor inhibition effect of bsCAR-T cells in vivo (P<0.01). Conclusion: bsCAR-T cells can kill specifically vⅢ+/CD133+ glioma stem cells, which provides experimental basis for cell immunotherapy of solid tumors.
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