More than 50% of patients treated with chimeric antigen receptor (CAR)–T cells for B cell malignancies develop progressive disease after CAR therapy, and these agents have not demonstrated consistent activity against solid tumors. CAR-T cell efficacy is often limited by T cell exhaustion, wherein global transcriptional and epigenetic alterations drive overexpression of immune inhibitory proteins and diminish function. Current therapeutic approaches for targeting T cell exhaustion, including immune checkpoint inhibitors, do not remodel the exhaustion-associated epigenome, which has led some to conclude that T cell exhaustion is an epigenetically fixed state with limited potential for reversal.

We had previously demonstrated that human T cell exhaustion can be induced and maintained by tonic CAR signaling, and we therefore hypothesized that the inhibition of CAR signaling, or rest, could prevent and potentially reverse exhaustion in CAR-T cell populations. Rest was induced using a drug-regulatable degron system, whereby CAR expression and tonic CAR signaling were controlled by the presence (ON) or absence (OFF) of a small molecule. In an alternative approach, CAR-T cells were rested using dasatinib, a clinically available tyrosine kinase inhibitor which potently and reversibly inhibits essential proximal CAR signaling kinases. Rest was induced either just before the manifestation of the exhaustion or after cells had acquired full phenotypic, functional, transcriptomic, and epigenetic hallmarks of exhaustion. Multiomics analyses and functional studies in vitro and in xenograft models were conducted to examine the effects of rest on tonically signaling CAR-T cells and nontonically signaling CAR-T cells exposed to high tumor burdens.

Tonically signaling CAR-T cells expanded ex vivo in the ON state manifested phenotypic, transcriptional, and epigenetic hallmarks of exhaustion, whereas CAR-T cells expanded exclusively in the OFF state or in the presence of dasatinib exhibited diminished tonic CAR signaling, which resulted in a memory-like phenotype and superior antitumor activity both in vitro and following adoptive transfer into xenograft-bearing mice. The induction of rest in tonically signaling, pre-exhausted CAR-T cells redirected their cell fate away from exhaustion and toward a memory-like state. In CAR-T cells that had already acquired hallmark features of exhaustion, the induction of rest for as few as 4 days reversed the exhaustion phenotype and induced transcriptional reprogramming and global epigenetic remodeling to resemble healthy, nonexhausted controls. Further, exhausted CAR-T cells subjected to rest, including those previously exposed to 6 weeks of tonic CAR signaling, demonstrated restored antitumor functionality. The degree of functional reinvigoration was correlated with the duration of rest and was associated with decreased expression of the exhaustion-associated transcription factor TOX and increased expression of memory-associated transcription factors LEF1 and TCF1. These findings were not attributable to the outgrowth of a small subset of exhaustion-resistant cells because they were not associated with marked changes in proliferation, apoptosis, or clonal restriction of the T cell receptor (TCR) repertoire. Rather, functional reinvigoration was dependent on the activity of the histone methyltransferase EZH2, consistent with epigenetic remodeling in response to rest. Using dasatinib-insensitive liquid and solid-tumor xenograft models, CAR-T cells subjected to intermittent rest through the oscillation of CAR expression or pulsed dasatinib in vivo exhibited superior tumor control …