Feasibility Of Anti EGFRvⅢ-CAR-T Cells As Drug Carriers In The Treatment Of Glioma

Objective: glioblastoma is the most malignant and invasive intracranial tumor.At present,although surgery combined with radiotherapy and chemotherapy can prolong the survival time of patients,the 5-year survival rate is only 9.8%.The most important reason is that blood-brain barrier and blood tumor barrier limit the entry of chemotherapy drugs with good killing effect into the brain.So how to solve the limitation of blood-brain barrier is the key to the treatment of glioma.Immunotherapy based on chimeric antigen receptor(car T)is a new antitumor therapy,However,due to the heterogeneity of tumor and the immunosuppressive microenvironment,the effect of CAR-T cell in the treatment of glioma is not good;while SN-38 can enter the tumor through vascular leakage,which can overcome the heterogeneity of tumor and the immunosuppressive microenvironment.Due to the existence of intracranial blood-brain barrier and blood-tumor barrier,it lacks effective anti-tumor effect.So can we combine the advantages of the two methods and use the new antitumor strategy to treat malignant glioma with CAR-T cells as drug carriers Methods: SN-38 liposomes were prepared by thin-film ultrasonic method and ethanol injection method.The cytotoxicity of sn-38-l,EGFRvⅢ car-t and sn-38-l-EGFRvⅢ car-t to tumor cells was compared by 51 Cr isotope release assay.Peripheral T cells were isolated by magnetic bead sorting method,and EGFRvⅢ car-t cells were constructed by genetic engineering technology.The construction effect of sn-38-EGFRvⅢ car-t cell vector system and its killing effect on tumor cells in vitro and in vivo were evaluated by immunofluorescence,WB / PCR and cytotoxicity test.Results: the optimal synthesis conditions of sn-38-l liposome were determined by establishing the analytical conditions of SN-38,that is,film-forming temperature 45 ℃,hydration temperature 45 ℃,hydration time 45 ℃,phospholipid concentration 20 mg / ml,phospholipid cholesterol ratio 4:1,PEG concentration 1 mg / ml.After determining the optimal process,we further tested the entrapment efficiency and drug loading of SN-38 liposomes,which were 89% and 3.27 ug,respectively.Through CCK8 experiment,we found that at the same concentration,the killing effect of SN-38 on glioma cell lines U87 and U251 was more than 10 times higher than that of temozolomide,and the killing effect of SN-38 on glioma cell lines and T cells was different at low concentration(< 2ug / ml).Through the transformation of T cells by genetic engineering technology,it was found that about 11.21%-22.71% of T cells expressed car protein.About 30.27-42.35% of the T cells were connected with sn-38-l liposomes.Sn-38-l / EGFRvⅢ-car T cells were co cultured with glioma cells in the ratio of 1:1,1:1,1.5 and 1:2.According to the following groups,the killing effect of sn-38-l / EGFRvⅢ-car t was more obvious than that of other groups.Conclusions: compared with car T cell alone,sn-38-l / EGFRvⅢ-car t can not only exert its own immune killing effect,but also realize the targeted transportation of chemotherapeutic drugs as drug carrier,and realize the combined anti-tumor effect of immunotherapy and drug therapy,which has a better clinical application scenario.