Objective To construct genetically modified T cells expressing chimeric antigen receptor(CAR) targeting prostate stem cell antigen(PSCA) and test the cytotoxicity of these cells against tumor cells both in vitro and in a tumor-bearing mouse model. Methods 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. Human T lymphocytes isolated from peripheral blood mononuclear cells were infected with the recombinant lentivirus and the positivity rate of PSCA-CAR expression in the T lymphocytes was detected using flow cytometry. Cytotoxic assay was performed to evaluate the specific cytotoxicity of the genetically modified T cells against the PSCA-positive He La cells and PSCA-negotive T24 cells

and ELISA was used to detect cytokine secretion by the cells. The antitumor effect of PSCA-CAR-T cells was also tested in a NOG mouse model bearing He La cell xenograft. Results DNA sequencing analysis suggested correct synthesis of PSCA-CAR sequences

and enzyme digestion verified successful construction of the lentiviral vector PL-PSCA-G4 H-28 TM-28 BBζ. The positivity rate of CAR expression was 53. 9% in the T lymphocytes after infection with the lentiviral vector. The PSCA-CAR-T cells showed a significantly stronger cytotoxicity against PSCA-positive tumor cells than against PSCA-negative tumor cells(P < 0. 01) both in short-term coincubation at a high effective-target ratio and in long-term co-incubation at a low effective-target ratio. The PSCA-CAR-T cells secreted significantly higher levels of cytokines than the control T cells upon stimulation by PSCA-positive tumor cells(P < 0. 01)

but the secretions were almost undetectable upon stimulation by PSCA-negative tumor cells. In the in vivo experiment

PSCA-CAR-T cells exhibited a significant tumorsuppressive effect in the tumor-bearing mice(P < 0. 01). Conclusion PSCA-CAR-T cells capable of specific killing of PSCA-positive tumor cells are successfully constructed both in vitro and in vivo.