The influence of co-stimulatory domains on the metabolic regulation of chimeric antigen receptor (CAR) T cell function

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T-cells are a vital component of the immune system and promoting/engineering T-cell responses is now at the forefront of cancer immuno-therapies. The development of chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical responses in treating certain subsets of B cell leukemia or lymphoma. CAR's are composed of an extracellular antigen-binding region against the tumour target linked to intracellular signalling domains to signal for T- cell activation and effector function. Upon recognising antigen, clustering and immobilisation of CAR molecules occurs which induces phosphorylation of the CD3-zeta (CD3z) chain akin to signalling via the natural T cell receptor. This triggers T-cell proliferation, cytokine release, metabolic changes and cytotoxicity; the T-cell effector response. CAR T- cell therapy has been highly effective in leukemic and lymphatic cancers, however, so far, has shown disappointing results for the treatment of solid tumours. The ability of a T-cell to display full effector functions (e.g. proliferation, cytotoxicity) crucially depends on optimal cellular energy utilisation and the availability of nutrients in the environment. Tumour cells in solid tumours can counteract this immune response by manipulating the availability of nutrients in the tumour microenvironment (TME), leading to treatment failure. T-cell co-stimulation can be delivered through both CD28 and 4-1BB. CAR T cells currently in the clinic carry either costimulatory domain, termed 2nd generation CAR, and 3rd generation CAR have also been developed in which both co-stimulatory domains were placed in line as part of the CAR. Disappointingly 3rd generation CAR did not show improved performance compared to 2nd generation CAR in the clinic (1). Now, our lab has generated a parallel-CAR (pCAR) construct to optimise costimulatory signalling delivering both CD28 and 4-1BB, one as part of the CAR and one as a proximal but independent chimeric costimulatory molecule (Muliaditan, Flaherty, Maher, Schurich, manuscript under review). These pCAR T-cells have shown enhanced tumour killing capacity in vitro and in vivo compared with CAR T-cells currently in the clinic. In vitro, pCAR T-cells have shown to be more resistant to T-cell exhaustion and senescence, sustain proliferation and cytokine release. We hypothesise that the increased functionality of pCAR T-cells is due to improved T-cell metabolism and mitochondrial function. Our preliminary findings have supported this hypothesis, showing that following stimulation, pCAR T-cells have significantly higher expression of nutrient transporters, and have a higher proportion of functional mitochondria when compared to conventional CAR counterparts. The increased ability of the pCAR T-cells to take up essential nutrients like glucose and iron suggests that they can sustain a high rate metabolism, contributing to their function and proliferation. Furthermore, the enhanced functionality of their mitochondria suggests increased longevity and resistance to exhaustion, a process we have previously shown vital in chronic viral infection (2). We believe these factors will contribute to a more effective therapeutic CAR T-cell product to withstand the nutrient lacking tumour microenvironment.