Thread by @EvanWeberPhD

I am thrilled to share our new paper out in @ScienceMagazine! We show that transient disruption of CAR signaling, or "rest", can reinvigorate exhausted CAR-T cells and boost anti-tumor functionality #CART #immunotherapy #IO

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doi.org/10.1126/scienc…

This project began with a simple question: if persistent CAR signaling alone can promote exhaustion, what happens if you turn the CAR off? To test this, we first teamed up with Tom Wandless to create a drug-regulatable CAR system (DD-CAR) that enabled tunable CAR expression
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Using a DD-CAR targeting GD2, which exhibits antigen-independent tonic CAR signaling, we observed that "hiding" the CAR during ex vivo expansion dramatically improved CAR-T cell activity, underscoring the importance of preventing tonic CAR signaling to preserve function
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We then asked whether CAR-T cells could be redirected away from exhaustion by transiently resting them. Using a DD version of the tonic signaling CAR exhaustion model published in (Lynn et al, Nature, 2019), we observed that rested cells skewed towards a more memory-like fate
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But what about CAR-T cells on which exhaustion was already imprinted (day 11 cells)? It turns out even these cells benefited from rest, demonstrating reversal of phenotypic hallmarks of exhaustion and global transcriptional reprogramming (Rested D11-15)
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Do rested cells actually respond better to tumor once CAR expression is re-normalized. Indeed, they do! Rested cells exhibited restored killing and cytokine secretion, polyfunctionality, and responsiveness to lower antigen density compared to Always ON exhausted cells
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This functional reinvigoration suggested to us that we might be altering the exhaustion-associated epigenome, which @EJohnWherry @nickhaining @SchietingerLab @UtzschneiderD and many others have shown is critical for locking exhausted T cells in a dysfunctional state.
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Surprisingly, CAR-T cell rest induced whole-scale epigenetic remodeling (measured by ATAC-seq), whereby the chromatin accessibility landscape of rested cells closely resembled that of healthy, non-exhausted cells.
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Notably, epigenetic remodeling was required for functional reinvigoration, as inhibiting one component of this remodeling (the histone methyltransferase EZH2) disrupted the rescue of IL-2 secretion and killing in rested cells, but minimally affected healthy Always OFF cells.
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Rest could also be induced using the drug dasatinib, which we previously identified as a potent and reversible inhibitor of CAR signaling. The longer we rested with dasatinib, the greater the effects we saw on phenotype (including reversal of TOX expression) and function.
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"BUT, does rest work in vivo," you ask? Remarkably, intermittent CAR-T rest using pulsed dasatinib augmented tumor control in das-insensitive liquid and solid xenograft models. Tumor-infiltrating CAR-T cells in das-treated mice were more memory-like and more functional.
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In summary, CAR-T cell rest is a maneuver that could enhance CAR-T cell therapeutics and warrants clinical testing. Our work also indicates that exhausted human CAR-T cells retain plasticity for functional reinvigoration and epigenetic remodeling.
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This incredibly labor-intensive study could not have been done without the amazing team science @StanfordMed. A big THANK YOU to all of the co-authors, especially my mentor @cm35c, Kevin Parker, @molecular_elena, and Rachel Lynn, and collaborators @HowardYChang @Satpathology 13/

Other co-authors include @GoodZinaida @hima_anbunathan @Dori_Na_Krzemie @LouaiLabanieh @juliabelk @BenceDaniel2 @gromcology. Thank you all!
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Also @SandorDanielne! (sorry, couldn’t find your handle earlier)