Optimized CD147-CART Cells Against Glioblastoma

Glioblastoma Multiforme（GBM）is the most common and aggressive primary malignancy in central nervous system,with a median survival of only 14-16 months.Standard treatments,including surgery,radiation and drug therapy（temozolomide chemotherapy）,have not significantly improved GBM patients’survival,and patients generally have a dismal prognosis.Chimeric antigen receptor T cell（CAR-T）therapy,as an emerging cell therapy method,has achieved remarkable clinical responses in hematological malignancies.Despite the recent preclinical and clinical studies have proved the feasibility of CAR-T therapy in solid tumor,due to the heterogeneity of tumor antigen,“off-target”effect and immunosuppressive tumor microenvironment,the treatment effect of CAR-T in solid tumor is unsatisfactory.It is urgent to improve the efficiency of CAR-T cells in treating solid tumor.CD147 is a type I transmembrane glycoprotein and highly expressed in GBM and could serve as a potential target against GBM.Therefore,this study intends to generate CD147-CART cells and investigate their efficiency against GBM,and explore the new methods for improving their anti-glioblastoma function.This study is composed of the following three parts.PartⅠStudy on anti-glioblastoma ability of CD147-CART cellsUsing CGGA datasets,we confirmed significant upregulation of CD147 m RNA in GBM and increased expression of CD147 is negatively associated with overall survival of patients.At the same time,immunohistochemical staining results of glioma tissue also confirmed the high expression of CD147 in GBM.In addition,the results of flow cytometry,western blot and immunofluorescent staining showed that the three glioma cell lines we selected all expressed high levels of CD147,and CD147 was positively correlated with the invasion,migration and proliferation of glioma cells,proved by transwell,wound healing and CCK8assays relatively.Activated primary human T cells were transduced with CD147-CAR lentivirus to generate CD147-CART cells.The expression of chimeric antigen receptor was detected by flow cytometry and RT-PCR.The results of cytotoxicity assay showed that CD147-CART cells could specifically lyse CD147 positive glioma cells in a dose-dependent and time-dependent manner.Meanwhile,after coculturing with target cells,CD147-CART cells could release multiple cytokines.The results of xenograft tumor mouse model exhibited CD147-CART cells could effectively suppress tumor growth in vivo.PartⅡConstruction and function assessment of optimized CD147-CART cellsSince CD147 on the surface of T cells can induce fratricide among CD147-CART cells,we decided to intervene the expression of CD147 on the surface of CART cells by gene editing to effectively solve this problem.Therefore,we first evaluated the effect of down-regulated CD147 expression on T cell function.We used lentivirus infection to stably interfere with CD147 on T cells.Flow cytometry and RT-PCR showed that the expression of CD147 was significantly down-regulated.The results of proliferation assay and apoptosis assay demonstrated that interference of CD147 could inhibit the proliferation of T cells in some degree.However,the cytotoxicity and cytokines release of T cells were enhanced after downregulation of CD147.Moreover,the restimulation ability of T cells was improved after downregulation of CD147.Meanwhile,compared to UTD cells,there is no significant change in the ratio of CD4⁺/CD8⁺in sh CD147-T cells.Intriguingly,the proportion of central memory T cells was significantly upregulated and the exhausted status of T cells was inhibited effectively after downregulation of CD147.These results showed that it was feasible to construct CART cells which interfered with CD147.CD147^KD-CART cells were successfully constructed by dual-lentivirus infection.The expression efficiency of chimeric antigen receptor was no different from that of CD147-CART cells,but the expression of CD147 was significantly downregulated,and the fratricide among CART cells was effectively repressed.Cytotoxicity assay showed that CD147^KD-CART cells had an enhanced ability to lyse CD147 positive glioma cells.Intracellular molecular staining results also confirmed that CD147^KD-CART cells could release higher levels of killing related cytokines.Moreover,there was a significant increase in the proportion of central memory T cells in CD147^KD-CART cells and downregulation of CD147 could efficiently suppressed the exhaustion of CD147-CART cells.Meanwhile,CD147^KD-CART cells demonstrated better persistence and anti-tumor capacity in vivo.Part Ⅲ Study on the role of CD147 in anti-tumor ability of CAR-T cellsFinally,we successfully constructed CD147^KD-EGFRvⅢ-CART cells by dual-lentivirus infection,and investigated their anti-tumor ability to explore the role of CD147 in anti-tumor ability of CAR-T cells.Flow cytometry showed the expression of CD147 on CART cells was significantly downregulated.Cytotoxicity assay revealed that CD147^KD-EGFRvⅢ-CART cells had an enhanced ability to lyse EGFRvⅢpositive glioma cells.ELISA results also confirmed that CD147^KD-EGFRvⅢ-CART cells could release higher levels of killing related cytokines.Moreover,the proportion of central memory T cells in CD147^KD-EGFRvⅢ-CART cells was significantly increased and the exhaustion of CART cells after cocultured with tumor cells was efficiently suppressed.Meanwhile,CD147^KD-EGFRvⅢ-CART cells demonstrated better persistence and anti-tumor capacity in vivo.In conclusion,above results indicate that CD147-CART cells display remarkable anti-tumor ability against CD147 positive GBM.Downregulation of CD147 on T cells could effectively inhibit fratricide among CD147-CART cells and enhance the survival and anti-tumor capacity of CD147-CART cells in vitro and in vivo.Moreover,down-regulation of CD147 also enhance the antitumor activity of EGFRvⅢ-CART cells,suggesting that interfering with the expression of CD147 could be a general and effective method to improve the therapeutic effect of CAR-T cells.Our study provides a theoretical and experimental basis for promoting the clinical transformation of CD147-CART cells in the treatment of GBM,and a new effective strategy for enhancing the therapeutic effect of CAR-T cells in solid tumors.