Construction and In Vitro Potency Investigation of Hypoxia-Inducible CAR-T Targeting PSCA
Zhu Xiuxiu1, Wang Ling2, Shen Junjie1, Chen Xuejiao2, Qian Cheng1*
Considering anoxic tumor microenvironment, we designed a hypoxia-inducible chimeric antigen receptor-modified T cells (CAR-T) targeting prostate stem cell antigen (PSCA), and tested the cytotoxicity of these cells against tumor cells in vitro. 5H1P and the sequences encoding the light and heavy chain variable regions of a monoclonal antibody targeting PSCA were synthesized and integrated into lentiviral vectors using restriction enzymes. Then lentivirus were infected to human T lymphocytes isolated from peripheral blood mononuclear cells to preparation CAR-T cells. Taking use of CoCl2, we emulated the hypoxia model in vitro. The positivity rate of PSCA-CAR expression in the T lymphocytes was detected by flow cytometry with or without CoCl2 treatment. There was 42% weak positive subpopulation in uninduced group and increased to 84.4% after CoCl2 induction, in which the fluorescence intensity increased significantly. Cytotoxic assay was performed to evaluate the specific cytotoxicity of the genetically modified T cells against the PSCA-positive HeLa cells, and ELISA was used to detect cytokine secretion by the cells. Results showed that induction group had better cytotoxicity than noninduced control group (P<0.05), and secreted significantly higher levels of cytokines (P<0.01). In conclusion, we successfully constructed the hypoxia-inducible CAR-T targeting PSCA with enhanced potency in anoxic environment. It will provide a new solution for CAR-T treatment in solid tumors.
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