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

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.