Dynamic control of transcriptional regulator BACH2 overcomes T cell dysfunction driven by tonic CAR signaling

Chimeric antigen receptors (CARs) integrate multiple lymphocyte-derived functional domains to enable selective antigen binding and robust T cell activation in a single synthetic protein. Through various biochemical mechanisms, nearly all CARs activate intracellular signaling in the absence of antigen, referred to as “tonic signaling”. Previous work has shown that tonic signaling of CARs containing the CD28 costimulatory domain drives T cell exhaustion; in contrast, we have shown that tonic signaling of 41BB-containing CARs enhances T cell function. Using a model of tonically signaling CARs targeting the B cell antigen CD22, we undertook studies to determine the molecular determinants of the divergent impact of tonic CAR signaling on T cell fitness. We identified that tonic 41BB signaling induces activation of BACH2, a transcriptional regulator that directs stem and memory programs to maintain T cell homeostasis. Transgenic expression of BACH2 prevented the development of exhaustion and promoted memory-like programs in tonically signaling CD28-containing CAR T cells. This enhanced acute cytotoxic function but impaired durable anti-tumor function. We linked transgenic BACH2 to a degradation domain, enabling precise control of BACH2 expression and found that low-level expression of BACH2 enabled robust and persistent tumor control in multiple cancer models. At the conclusion of CAR T cell manufacturing, T cells were purified to enrich CAR+ cells using a truncated CD34 selection marker and BACH2+ cells using a truncated EGFR selection marker. From these purified populations, we isolated and sequenced RNA. Studies were performed in technical triplicate using cells manufactured from three independent donors.