Autophagy disruption primes CAR-T cell metabolism for sustained rejection of ovarian tumors

Carleton, G. A.; Levesque, S.; Zacharias, L. G.; San Patricio, J.; Sutcliffe, T.; Watson, P. H.; DeBerardinis, R. J.; Doyon, Y.; Lum, J. J.

doi:10.1101/2025.10.09.681473

bioRxiv · 10.1101/2025.10.09.681473

Autophagy disruption primes CAR-T cell metabolism for sustained rejection of ovarian tumors

Carleton, G. A.·Levesque, S.·Zacharias, L. G.·San Patricio, J.·Sutcliffe, T.·Watson, P. H.·DeBerardinis, R. J.·Doyon, Y.·Lum, J. J.

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Abstract

T-cell based immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy face substantial hurdles when confronting solid tumors such as ovarian cancer, where metabolic constraints in the tumor microenvironment limit T cell infiltration and function. In particular, T cells exposed to nutrient deprivation and hypoxia upregulate autophagy, a lysosomal degradation pathway that negatively regulates effector responses. Here, we used CRISPR-Cas9 to target a folate receptor alpha (FR) CAR expression cassette into the locus of the essential autophagy gene ATG5, thereby generating autophagy-deficient CAR-T cells in a single editing step. Targeted metabolite profiling revealed that deletion of ATG5 induced widespread metabolic reprogramming characterized by increased glucose and amino acid uptake. Functionally, ATG5-knockout CAR-T cells maintained high cytolytic activity when assayed in patient-derived ascites in vitro, and exhibited superior and long-lasting tumor control against ovarian tumors in vivo. Taken together, our results suggest that deletion of ATG5 metabolically primes CAR-T cells for enhanced cytotoxicity in immune-suppressive conditions, thereby improving the therapeutic potential of FR CAR-T cells for ovarian cancer immunotherapy.

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Carleton, G. A., Levesque, S., Zacharias, L. G., San Patricio, J., Sutcliffe, T., Watson, P. H., DeBerardinis, R. J., Doyon, Y., Lum, J. J.. 2025-10-10. Autophagy disruption primes CAR-T cell metabolism for sustained rejection of ovarian tumors. https://doi.org/10.1101/2025.10.09.681473

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