BACH2 dosage establishes the hierarchy of stemness and fine-tunes antitumor immunity in CAR T cells

Self-renewing stem-like T cells promote the efficacy of cancer immunotherapy and are a heterogeneous population with sub-lineages demonstrating different degrees of stemness. At the apex of this hierarchy is long-term (LT) stem-like T cells with the highest capacity of persistence, repopulation and response to immune checkpoint inhibitors (ICI). However, the pathway that establishes the hierarchy of stemness in chimeric antigen receptor (CAR) T cells and its role in antitumor efficacy of CAR T cells are unclear. Here, we demonstrate that LT stem-like differentiation and antitumor immunity of CAR T cells are dose-dependently regulated by BACH2. Pre-infusion LT stem-like CAR T cells showed epigenetic activation of BACH2 and superior antitumor response in mice and humans. After clearing tumor in vivo, LT stem-like T cells emerged as a CAR subset that transcriptionally and epigenetically upregulated BACH2 and downregulated TOX. Loss-of-function experiments revealed an essential role of BACH2 in the antitumor response of CAR T cells and the transcriptional program of LT stem-like CAR T cells. In exhaustion-prone GD2 CAR T cells with high tonic signaling, we showed that quantitative control of BACH2 protein level by a small molecule drug fine-tuned the degree of stemness and exhaustion in CAR T cells. Chemically inducing temporal BACH2 activation during manufacture of GD2 CAR T cells imprinted greater antitumor immunity against solid tumor after infusion. Together, we show that BACH2 dosage establishes the hierarchy of stem-like CAR T cells and can be temporally and tunably controlled in CAR T cells to optimize differentiation and antitumor immunity. Overall design: We performed single-cell ATAC+RNA sequencing on in vitro clutured CAR T cells and CAR T cells isolated from tumor bearing mice on day 20 after infusion. We performed scRNAseq on WT and BACH2 inducible knock out CAR T cells isolated from tumor bearing mice on day 7 after infusion. We performed ChIP-seq