Tuning CAR-T cell response with <i>TNFAIP3</i> variants.

Abstract

e14527 Background: The clinical success of Chimeric Antigen Receptor (CAR)-T cell therapy is frequently limited by two major, interconnected challenges: T cell exhaustion and subsequent tumour-mediated immune escape associated with chronic antigen stimulation, which represent strategic opportunities to improve therapeutic outcomes. Global deletion of TNFAIP3 is embryonically lethal in animal models and haploinsufficiency is poorly tolerated in humans, while selective loss of TNFAIP3 in immune cells confers protection to infections rather than detriment due to a reduction in the immune threshold response boosting immune stimulation. Further to this, we have previously shown that coding gene variants of TNFAIP3 can modify protein enzymic function and hence tune the level of TNFAIP3’s anti-inflammatory effect – contrasting the classical view that TNFAIP3 acts a binary control point. We propose the strategic engineering of CAR-T cells with specific TNFAIP3 variants will enhance CAR-T cell efficacy, overcome exhaustion, and improve therapeutic persistence. Methods: Here we tested the impact of two reduction of function TNFAIP3 variants on CAR-T response. Variant-1 was identified from our ongoing functional genomic screen of super responders to checkpoint inhibition therapy; Variant-2 was reported in a family with hereditary inflammatory disease and validated by us as a reduction of function variant in functional studies. We genetically modified human T cell lines and primary T cells to express variant-1 or variant-2 with an anti-CD19 directed CAR and investigated their impact on CAR-T cell activation and function. For this study, we designed a CAR construct based on 3 rd generation parameters. Results: Modifying CAR-T cells with TNFAIP3 variant-1 and variant-2 led to robust upregulation of NF-κB signalling and increased stimulus dependent levels of NF-κB target genes. Modified cells showed significantly higher levels of activation and migration marker expression with enhanced secretion of key effector cytokines compared to control CAR-T cells. Quantitatively, the tested TNFAIP3 variants enhanced cytotoxic ability above baseline in static assays systems. Current experiments are focused on investigating real-time target cell death kinetics upon engagement with tumour cells (high content imaging). Conclusions: Our data supports the hypothesis that TNFAIP3 variants can successfully tune CAR-T cell response by lowering the CAR-T cell activation threshold for more favourable anti-tumour outcomes.