ShRNA-mediated silencing of PD-1 augments the efficacy of chimeric antigen receptor T cells on subcutaneous prostate and leukemia xenograft

Abstract

Chimeric antigen receptor T cells (CAR-T) immunotherapy has shown promising clinical results in the treatment of leukemia and lymphoma, but the effectiveness is limited for solid tumors. The PD-1/PD-L1 pathway is a key immunosuppressive mechanism for cancer cells to avoid eradication by CAR-T cells. In this study, the shRNA (short hair RNA) gene-silencing technique was used to construct the third-generation of CAR-T cells with PD-1 silencing which targeted CD19 antigen (CD19/△PD-1 CAR-T) and prostate stem cell antigen (PSCA/△PD-1 CAR-T), thereby blocking the PD-1/PD-L1 pathway in treatment of lymphoma and prostate subcutaneous xenograft and enhancing the anti-tumor effect of CAR-T cells. The cell experiments showed that PD-1 silencing in CAR-T cells effectively blocked the PD-1 / PD-L1 pathway. When the ratio of effector to target cell is 8:1, △PD-1 CAR-T cells exhibited higher killing ability and cytokine releasing ability than normal CAR-T cells did. The subcutaneous tumor models were constructed using human chronic myelogenous leukemia cells expressing CD19 (K562-CD19) and human prostate cancer cells expressing PSCA (PC3-PSCA), and treated with CD19/△PD-1 CAR-T and PSCA/△PD-1 CAR-T cells, respectively. The tumor volumes significantly reduced within one week, indicating the good tumor growth inhibitory effect of △PD-1 CAR-T cells. Mice injected with △PD-1 CAR-T cells showed a significantly prolonged survival time compared to those with normal CAR-T cells. This study proved that shRNA-mediated PD-1 silencing technology is an effective strategy for blocking the PD-1/PD-L1 immunosuppression pathway and enhancing the therapeutic effect of CAR-T cells on subcutaneous xenograft. SUMMARY: The effect of CAR-T in treating solid tumors has not been as successful as that in hematological malignancies. The key immunosuppressive mechanism is the expression of PD-1/PD-L1. We used gene silencing technique mediated by shRNA (short hair RNA) to block the PD-1/PD-L1 pathway in lymphoma and prostate tumors, thus enhancing the anti-tumor effect of CAR-T cells on subcutaneous xenograft.

Keywords: Immunotherapy; PD-1 silenced CAR-T cells; PD-1/PD-L1; ShRNA; Subcutaneous xenograft.