CAR-T-EGFRvⅢ Combined With PD-1 Blocks The Dual-targeted Anti-glioma Effect

Part One: The clinical significance of EGFR,PD-1 and PD-L1 in glioma【Objective】EGFR,PD-1 and PD-L1 are hot molecules in tumor research,which are closely related to the development,and treatment resistance of tumor.We aim to explore the clinical significance of EGFR,PD-1 and PD-L1 in gliomas.【Methods】The association of EGFR mutation and expression,PD-1,PD-L1 expression with glioma grade,IDH mutation and prognosis by bioinformatics analysis.Further the relationship between EGFR signal and PD-1/PD-L1 signal and prognosis of glioma was analyzed.【Results】EGFR mutation frequency was the highest in WHO grade IV gliomas(22.97%).The survival time of patients with EGFR mutant glioma was significantly shorter than that of patients with EGFR wild glioma.The expression of EGFR was positively correlated with the grade of glioma.Compared with IDH mutant gliomas,EGFR expression was significantly higher in IDH wild gliomas.The expression of PD-1 and PD-L1 was positively correlated with the grade of glioma.Compared with IDH mutant gliomas,the expression of both genes in IDH wild gliomas was significantly higher.The patients with higher expression of PD-1 or PD-L1 has the worse the prognosis.Combined analysis showed that the prognosis of glioma patients with EGFR mutation and high expression of PD-1 or PD-L1 was the worst;the prognosis of glioma patients with high expression of EGFR and high expression of PD-1 or PD-L1 was the worst.【Conclusion】Based on the glioma genomics database,we found that EGFR,PD-1 and PD-L1 play an important role in the malignant progression and clinical prognosis of glioma,which provides a new target and strategy for the diagnosis and treatment of glioma.Part Two: EGFRvIII-CAR-T Cells with PD-1 knockdown have improved anti-glioma activity【Objective】EGFR,PD-1 and PD-L1 are important targets of glioma,but the treatment of these targets has not achieved satisfactory results.Car-T technology is developing rapidly,and it has shown amazing curative effect in the treatment of blood cancer.It has also been rapidly applied to the treatment of solid tumors.This project aims to use CAR-T technology to target EGFRvIII and block PD-1 / PD-L1 signal,then to analyze the effect of its combined treatment of glioma and provide theoretical and experimental basis for the development of its application in the treatment of glioma.【Methods】First,PD-L1WT-EGFRvIII+U373 cells were constructed by lentiviral vector of EGFRvIII,and then PD-L1KO/EGFRvIII+ U373 cells were constructed by CRISPR /cas9 technology.PD-1WT/EGFRvIII-CAR-T cells and PD-1KD/EGFRvIII-CAR-Tcells were constructed by CAR-T technology.We demonstrated the biological effect of dual targeting EGFRvIII/PD-1 signal on glioma in vivo and in vitro by molecular,cell biology and nude mouse xenograft tumor model.【Results】PD-1WT/EGFRvIII-CAR-T cells and PD-1KD/EGFRvIII-CAR-T cells were generated.Cytokine production and lytic activity of these two CAR-T cells against to PD-L1WT/EGFRvIII+ U373 cells or PD-L1KO/EGFRvIII+ U373 cells were evaluated.The results showed that PD-1KD/EGFRvIII-CAR-T cells and PD-1WT/EGFRvIII-CAR-T cells showed same levels of interferon-γ(IFN-γ)and interleukin-2(IL-2)production as well as cytolytic activity against PD-L1KO/EGFRvIII+ U373 cells;however,PD-1KD/EGFRvIII-CAR-T cells exhibited higher levels of IFN-γ and IL-2 production as well as cytolytic activity against PD-L1WT/EGFRvIII+ U373 cells than that of PD-1WT/EGFRvIII-CAR-T cells.PD-1KD/EGFRvIII-CAR-T cells also exhibited higher anti-glioma activity and longer survival in mice in vivo than that of PD-1WT/EGFRvIII-CAR-T cells.【Conclusion】PD-1 knockdown enhanced the lytic activity of EGFRvIII-CAR-T cells against PD-L1WT/EGFRvIII+ U373 cells,and blocking EGFRvIII-CAR-T cells against PD-L1WT/EGFRvIII+ U373 cells may be an effective method for PD-L1+EGFRvIII+GBM therapy.Our work provides a new idea for the therapeutic strategy of CAR T cells for glioma.