Anti-Apoptotic c-FLIP Reduces the Anti-Tumour Activity of Chimeric Antigen Receptor T Cells

Simple Summary Chimeric antigen receptor (CAR) T cell treatment is a promising adoptive cell therapy that utilises CAR-expressing primary T cells to target specific tumour antigens. Since its first approval in 2017, the FDA has approved six CAR T cell therapies for blood cancer treatment. Despite their success in treating B cell malignancies, solid-tumour-directed CAR T cells face multifactorial challenges, often resulting in a lack of anti-tumour response in tumour elimination. The aim of this study was to evaluate the potency of expressing anti-apoptotic gene FLICE-like inhibitory protein p43 (c-FLIPp43) in Her2-CAR T cells to resist activation-induced cell death (AICD). This study confirmed the anti-apoptotic activity of c-FLIPp43. However, the expression of c-FLIPp43 reduced the CAR activity in a Her-2 breast cancer xenograft model relative to the control Her2-CAR T cells. This work provides insight into the role of c-FLIP in T cells and its impact on anti-tumour immunity in CAR T cells. CAR T cell treatment of solid tumours is limited by poor persistence partly due to CD95 ligand (CD95L)-induced apoptosis. Both T cells and cells within the tumour microenvironment (TME) may express CD95L, triggering apoptosis in CD95-receptor-positive CAR T cells. Tonic signalling of CAR T cells may also increase CD95-dependent AICD. Because the intracellular protein c-FLIP protects T cells from AICD, we expressed c-FLIPp43 within a Her-2 targeted CAR cassette and evaluated the potential of c-FLIPp43 through in vitro functional assays and in vivo tumour-bearing xenograft model. cFLIP expression protected against CD95L-induced cell death in the Jurkat T cell lines. However, in primary human CAR T cells containing CAR-CD28 domains, c-FLIPp43 overexpression had minimal additional impact on resistance to CD95L-induded cell death. In vitro cytotoxicity against a breast cancer tumour cell line was not altered by c-FLIPp43 expression, but the expression of c-FLIPp43 in Her2-CAR T cells lowered interferon-gamma secretion, without markedly affecting IL-2 levels, and c-FLIPp43-Her2-CAR T cells showed reduced anti-tumour activity in immunodeficient mice with breast cancer. The findings of this study provide a new understanding of the effects of controlling extrinsic apoptosis pathway suppression in CAR T cells, suggesting that c-FLIPp43 expression reduces anti-tumour immunity through the modulation of effector T cell pathways.