Modulation of chimeric antigen receptor T cell (CAR-T) signalling to enhance anti-cancer T cell function

Chimeric antigen receptor (CAR) T cell technology has shown great potential to combat cancer. However, several bottlenecks, e.g. cytokine release syndrome (CRS), efficacy in solid tumours, tumour escape etc., have hindered its further development. Fine-tuning CAR signaling may hold the promise to overcome these challenges. Yet, the mechanism of CAR signal transduction is poorly understood. Thus, there has been relatively little attempt to enhance CAR-T functions and innovate CAR-T technology by modulation of CAR signaling. We have found that signaling by CD28-CAR-T but not CD137-CAR-T is LCK-independent. In the case of CD28-CAR-T, signaling relies on another SRC family kinase – FYN – and the traditionally crucial kinase LCK is dispensable. The study proposes to further understand the mechanism and significance of LCK-independent CAR signal initiation in CAR-T technology. Primary human T cells and T cell lines are used to characterize CAR-T cell signalling in the absence of LCK. The findings are important steps towards unveiling the mechanism of CAR signal transduction and to understand how co-stimulatory domains can interplay with downstream signaling. LCK-deficient CAR-T cells can be engineered by knocking out LCK in primary T cells. These LCK-deficient CAR-T cells showed more specific recognition and had a more persistent phenotype than normal CAR-T. The proposed study hypothesizes that LCK-deficient CAR-T cells will have a better performance in vivo and less severe CRS. The in vivo evaluation will be performed with clinical samples both in a xenograft mouse model and an immunocompetent mouse model, where the impact of LCK-deficient CAR-T on tumor immunity will be investigated. In addition, the study will explore the generation of novel CAR-T technologies based on LCK-independent signaling, allogeneic CAR-T and AND-gate CAR-T. The exploration will demonstrate the potential to innovate CAR-T technologies via signaling modulation.