The siRNA-mediated downregulation of PD-1 alone or simultaneously with CTLA-4 shows enhanced in vitro CAR-T-cell functionality for further clinical development towards the potential use in immunotherapy of melanoma.

Abstract

Chimeric antigen receptor (CAR)-T cells have been used successfully for cancer immunotherapy. While substantial tumor regression was observed in leukaemia and lymphoma, CAR therapy of solid tumors needs further improvement. A major obstacle to the efficiency of engineered T cells is posed by triggering of inhibitory receptors, for example programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), leading to an impaired antitumor activity. To boost CAR-T-cell function, we co-electroporated T cells with both, mRNA encoding a CAR specific for chondroitin sulphate proteoglycan 4 (CSPG4) and small-interfering RNAs (siRNAs) to downregulate PD-1 (siPD-1) and CTLA-4 (siCTLA-4). Flow cytometry revealed that activation-induced upregulation of both PD-1 and CTLA-4 was suppressed when compared to CAR-T cells electroporated with negative control siRNA. The siRNA transfection showed no influence on CAR expression of engineered T cells. Functionality assays were performed using PD-L1- and CD80-transfected melanoma cells endogenously expressing CSPG4. CAR-T cells transfected with siPD-1 alone showed improvement in cytokine secretion. Additionally, CAR-T cells transfected with either siPD-1 alone or together with siCTLA-4 exhibited a significantly increased cytotoxicity. No or only little effects were observed when CAR-T cells were co-transfected with siCTLA-4 only. Taken together, it is feasible to optimize CAR-T cells by co-transfection of CAR-encoding mRNA and siRNAs to downregulate inhibitory receptors. Our in vitro data indicate an improvement of the functionality of these CAR-T cells, suggesting that this strategy could represent a novel method to enhance CAR-T-cell immunotherapy of cancer.