| Objective: To explore the potential of Her2-CAR-T cells targeting HER2 / neu antigens in the treatment of advanced gastric cancer.At the same time,in order to improve the tumor immunosuppression microenvironment,Her-12 CAR-T cells were used as carriers to carry and continuously secrete IL-12(Her2-PB-IL12-CAR-T)or Her2 / neu antigen to specifically induce the expression of IL-12."Truck" CAR-T cells of cytokines(Her2-NFAT-IL12-CAR-T)."Truck" CAR-T cells carrying IL-12 improve the immune microenvironment by releasing high concentrations of IL-12 locally in the tumor,further increasing the potential of Her2-CAR-T cells to treat advanced gastric cancer.This topic aims to obtain preclinical data on the effectiveness and safety of Her2-CAR-T cells and IL-12 for the treatment of advanced gastric cancer through in vivo and in vitro experimental studies,and provide important experimental evidence for later clinical application research.Methods:(1)Construction of vector and transduction and expansion of CAR-T cells in vitro:Construction of an IL-12 vector with Piggy Bac transposon as a transduction system for sustainable secretion and antigen-induced expression and secretion,and sequencing verification.Sequence verification and amplification of Her2-CAR plasmid with transposon as transduction system(Her2-CAR was donated by Professor Huang Xingxu from Shanghai University of Science and Technology).Three types of CAR-T cells were obtained through electroporation technology and T cell expansion in vitro,namely Her2-CAR-T cells,HER2-PB-IL12-CAR-T cells that continuously express IL-12,and antigen-specific induction expression HER2-NFAT-IL12-CAR-T cells of IL-12,and NT cells that were not transfected in parallel.Flow cytometry was used to detect the transduction efficiency of Her2-CAR and IL-12;the expanded cells were counted every 5-7 days,and the cell growth curve was drawn.The proportion of CD3 +,CD4 +,and CD8 + T cells and the positive rate of CAR-T cells were detected by flow cytometry on days 14,21-28.(2)Screening of target cells and acquisition of over-expressed Luciferase stable cells: The HER2 expression intensity of gastric cancer cell lines NCI-N87,AGS,HGC-27,and SGC-7901 cells was determined by flow cytometry to determine HER2 positive and negative cell lines.A HER2 strong positive cell line was selected to transduce Luciferase plasmid,and a stable overexpressing Luciferase gastric cancer cell line was obtained through continuous selection by geneticin.This cell will be used as a target cell to complete subsequent CAR-T cell function studies in vitro and in vivo.(3)In vitro experiments: Four effector cells and HER2 target cells were tested at different target ratios(10: 1,5: 1,2.5: 1,1.25:1)for 4h and 24 h,and then different CAR-T cells and NT were detected.The killing ability of cells to tumor cells;effector cells and gastric cancer target cells were co-acted for 24 h at 2: 1,and the supernatant was collected.The cytokine(IL-2,IL4,IL6,IL10,IFN-γ,TNF-α)secretion in the supernatant was detected by flow cytometry,and the secretion of IL-12 was detected by ELISA.(3)In vivo experiments: the construction of a nude mouse model of gastric cancer was completed.Select 4-week-old female BALB / c-nu / nu nude mice by subcutaneous injection of5 × 106 / NCI-N87-Luc + and wait for tumors to grow to about 200mm3 with 1 × 106 / only two consecutive CAR-T cell treatments at intervals The time is 7 days.The weight change of the mice was measured every 3-4 days,and the tumor growth was observed and plotted by a vernier caliper and a small animal live imager.The mice were sacrificed after 27 days of immune cell treatment,and tumors were stripped and weighed.Results:(1)The IL-12 expression vector with biological function was successfully constructed based on Piggy Bac system,which can continuously express in T cells and induce antigen-specific expression.(2)The expression of HER2 in gastric cancer cell lines was analyzed by flow cytometry.The expression intensity of Her2 antigen was significantly different in different gastric cancer cells.Among them,NCI-N87 is a Her2 strong expressing cell line.Through transduction and screening,stable overexpression Luciferase NCI-N87-Luc + cells were obtained.(3)Her2-CAR-T cells,Her2-PB-IL12-CAR-T cells,Her2-NFAT-IL12-CAR-T cells and NT cells carrying IL-12 cytokines were successfully prepared.(4)CAR-T cell function experiments in vitro show that,compared with HER2-CAR-T cells,two CAR-T cells that can secrete IL-12 have stronger killing ability to tumor cells.Under the stimulation of Her2 antigen,the induction of IL-12-expressing HE2-NFAT-IL-12 showed no less lethal power than HER2-PB-IL12 cells that continuously secrete IL-12,and the former has stronger safety.Cytokine testing showed that compared with HER2,CART cells that can secrete IL-12 can secrete more cytokines related to cell killing,such as IL-2,TFN-α,IFN-γ,and IL-10.(5)The mouse gastric cancer model was successfully constructed.In vivo experiments show that Her2-CAR-T cells have a certain inhibitory effect on the growth of gastric cancer cells.Her2-PB-IL12-CAR-T cells and Her2-NFAT-IL12-CAR-T cells carrying IL-12 show stronger tumor suppressive capacity in animals than Her2-CAR-T.Three CAR-T cells Shows good safety to mice.Conclusion:1.In vivo experimental studies of Her2-CAR-T cells targeting Her2 antigen have shown good anti-tumor ability and safety to gastric cancer cells..2.In vivo and in vitro studies of HER2-NFAT-IL12-CAR-T and HER2-PB-IL12-CAR-T cells constructed in this study showed that their tumor suppressor effect was superior to HER2-CAR-T cells,because HER2-NFAT-IL12-CAR The IL-12 secreted by-T cells is specifically induced by the antigen,so it is safer than HER2-PB-IL12-CAR-T cells that secrete IL-12 directly. |
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