Chimeric Antigen Receptor (CAR)-T cells have great efficacy against CD19
+
leukemia but little success for solid tumors. This study explored the effectiveness of third generation anti-HER2 CAR-T cells alone or in combination with anti-PD1 antibody on breast tumor cells expressing HER2
in vitro
and in immune competent mouse model. The PDL1-positive mouse mammary tumor cell line 4T1 engineered to express luciferase and human HER2 was used as the target cell line (4T1-Luc-HER2). Anti-HER2 CAR-T cells were generated by transducing mouse spleen T cells with recombinant lentiviruses. ELISA analysis showed that IL-2 and IFN-γ secretion was increased in CAR-T cells co-cultured with the target cells, and the secretion of these two cytokines was increased further with the addition of anti-PD1 antibody. Lactate dehydrogenase assay revealed that CAR-T cells displayed a potent cytotoxicity against the target cells, and the addition of anti-PD1 antibody further enhanced the cytotoxicity. At the effector: target ratio of 16:1, cytotoxicity was 39.8% with CAR-T cells alone, and increased to 49.5% with the addition of anti-PD1 antibody. In immune competent syngeneic mouse model, CAR-T cells were found to be present in tumor stroma, inhibited tumor growth and increased tumor apoptosis significantly. Addition of anti-PD1 antibody further enhanced these anti-tumor activities. Twenty-one days after treatment, tumor weight was reduced by 50.0% and 73.3% in CAR-T group and CAR-T plus anti-PD1 group compared with blank T group. Our results indicate that anti-PD1 antibody can greatly increase the efficacy of anti-HER2 CAR-T against HER2-positive solid tumors.
Without effective treatment, glioblastoma is one of the deadliest cancers worldwide. The aim of the present study was to explore whether combinational immunotherapy is effective for treating malignant glioblastoma in vitro. The therapeutic efficacy of third generation anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR)-T cells alone and in combination with PD1 blockade was investigated for the treatment of malignant glioblastoma cells in vitro. Anti-HER2 CART cells were prepared by transducing activated primary human T cells with lentiviruses which expressed third generation anti-HER2 CAR. The CAR-positive cell ratio was detected using flow cytometry. The expression level of CAR was detected by western blot analysis. The binding of anti-HER2 CART cells to HER2 + U251 glioblastoma cells was examined under a fluorescence microscope. The cytokine secretion of CART cells induced by target cells was analyzed via ELISA. The cytotoxicity of anti-HER2 CART cells alone or in combination with anti-programmed death-1 (PD1) antibody against HER2 + /PDL1 + U251 cells was examined using an LDH assay. The CAR-positive cell ratio and expression level of CAR in prepared CART cells were both high enough. Anti-HER2 CART cells could specifically bind to U251 cells. The IL-2 and IFN-γ secretion of CART cells increased after being co-cultured with U251 cells, and further increased in the presence of anti-PD1 antibody. Anti-HER2 CART cells displayed a potent cytotoxicity against U251 cells. In addition, the presence of anti-PD1 antibody further enhanced the efficacy of anti-HER2 CART cells against U251 cells. The present results indicated that blocking PD1 immuno-suppression can increase the activation of CART cells after they are activated by a targeting antigen. Third generation anti-HER2 CART cells along with PD1 blockade have a great therapeutic potential for combatting malignant glioblastoma.
We optimized the preparation of recombinant lentivirus that can express third-generation anti-Her2 CAR in T cells, which should lay the foundation for improving the efficacy of CAR-T cells with respect to killing target cells.
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