BACH2 regulates T cell lineage states to overcome dysfunction driven by tonic CAR signaling Free

Like T cell receptors (TCRs), chimeric antigen receptors (CARs) trigger potent intracellular signaling upon engagement with antigen, a process which initiates cell state transitions that direct T cell functionality. Unlike TCRs, CARs have an intrinsic predisposition to initiate intracellular signaling in the absence of target antigen, referred to as “tonic” signaling. Tonic CAR signaling of CARs containing the CD28 costimulatory domain has previously been shown to be a potent driver of T cell exhaustion. We previously showed that, in contrast to CD28, CARs containing the 41BB costimulatory domain acquire enhanced functionality from tonic signaling. These data indicate that the impact of tonic CAR signaling is directly dependent on the CAR costimulatory domain. To understand the molecular regulators of the enhanced T cell function induced by tonic 41BB signaling, we generated CARs targeting the B cell antigen CD22 that contained either CD28 or 41BB costimulatory domains that did (22/BB-S, 22/28-S) or did not (22/BB-L, 22/28-L) signal tonically. We confirmed that tonic signaling of CD28 impaired T cell function upon coculture with the CD22⁺ human ALL cell line Nalm6, while tonic 41BB-based CAR signaling enhanced function. RNA sequencing demonstrated 22/BB-S expressed higher memory-associated genes (ie. CCR7, TCF7) and a gene program regulated by BACH2. To investigate if BACH2 is responsible for the enhanced function of 22/BB-S we generated 22/28-S cells that overexpressed BACH2 (22/28-S+BACH2^OE). This transgenic over-expression completely rescued acute anti-tumor function, with cytotoxicity equivalent to 22/BB-S. Disruption of endogenous BACH2 expression in 22/BB-S cells reduced impaired tumor killing, rendering function similar to 22/28-S cells. 22/28-S+BACH2^OE were significantly enriched for central memory cells (T_CM) with a concomitant reduction in effector memory cells (T_EM). Mass cytometry revealed that 22/28-S+BACH2^OE cells were highly phenotypically similar to 22/BB-S cells at the conclusion of manufacturing and after stimulation with target cells. Transcriptome analysis demonstrated that 22/28-S+BACH2^OE expressed more memory-related genes and flow cytometry revealed that 22/28-S+BACH2^OE cells expressed high levels of TCF1, critical for directing T_CM formation, and restrained expression of Eomes, necessary for transition to T_EM. Collectively, we found that transgenic expression of BACH2 in 22/28-S cells antagonized tonic CAR-driven exhaustion to promotes early lineage (naïve and central memory) gene programs. To interrogate how BACH2 overexpression impacted long-term function, we subjected CAR T cells to chronic stimulation co-cultures with Nalm6. We found BACH2^OE initially improved function of 22/28-S but restrained their ability to maintain cytotoxic function. We observed that 22/28-S+BACH2^OE were “locked” in a T_CM state over time and failed to transition into T_EM upon antigen engagement. To overcome this limitation of constitutive BACH2 overexpression, we tagged transgenic BACH2 with a degradation domain (DD, BACH2^DD) to fine tune BACH2 expression using the antibiotic trimethroprim (TMP). We confirmed that in the presence of TMP (BACH2^DD ON) BACH2 expression was stabilized, however in the absence of TMP (BACH2^DD OFF) we observed low-level expression of BACH2 indicating leakiness of the DD system. 22/28-S+BACH2^DD ON cells had similar long-term functionality as 22/28-S+BACH2^OE cells but that 22/28-S+BACH2^DD OFF cells significantly enhanced tumor control. Unlike 22/28-S+BACH2^OE and 22/28-S+BACH2^DDON cells, 22/28-S+BACH2^DD OFF cells were able to transit to T_EM with loss of TCF1 and increase in Eomes. In a xenograft model of human ALL, we observed equivalent disease control between 22/28-S+BACH2^DD OFF and 22/BB-S cells. Our findings demonstrate that BACH2 plays a central role in CAR T cell lineage decisions and can be tuned to overcome exhaustion and enhance long-term function. Finally, we analyzed the association between endogenous BACH2 activity and clinical outcomes in pre-infusion CAR T cell products. Regulon analysis of single cell RNAseq data from 12 products confirmed higher BACH2 activity in central memory cells and, notably, identified a clear and significant association between higher endogenous BACH2 activity and enhanced patient survival.