Tunable STAT Activation by Synthetic Pathway Activators (spas) Increases Engineered T-cell Potency and Persistence

Abstract Clinically effective adoptive T cell therapy for the treatment of solid tumors will require robust T cell expansion, persistence, and potency. The Janus-kinase signal transducer and activator of transcription (JAK-STAT) pathway governs T cell activation and differentiation, and can thereby serve as a critical regulator of these properties. To take advantage of the benefits of STAT signaling in programming an antitumor T-cell response, we used synthetic biology to create a library of proteins, termed Synthetic Pathway Activators (SPAs) which constitutively drive STAT signaling without the need for external cytokine input. SPAs can be designed to engage activity of multiple STAT family transcription factors at variable levels through rational design. We have developed several classes of SPAs, including but not limited to Class I SPAs, which primarily increase pSTAT3 activity, and Class II SPAs, which increase pSTAT5 activity. When constitutively expressed in ArsenalBio Integrated Circuit T (ICT) cells, SPAs result in significant enhancements in T-cell potency and expansion. Repetitive stimulation assays, wherein T cells are challenged with tumor cells every 2 days, reveal that Class I SPAs result in 6-log or higher improved tumor cell clearance over a 2-week assay period. Across various mouse xenograft models, SPA-expressing ICTs reach at least 6-fold improved tumor growth inhibition. RNAseq and ATACseq analysis indicate dramatic changes to gene expression profiles in T cells expressing Class I SPAs, with maintenance of T cell stem-like phenotypes, and restricted accessibility of various exhaustion marker genes. Importantly, despite significantly increased levels of expansion, ICTs equipped with SPAs are not immortalized, showing no signs of cytokine-independent outgrowth. In addition, SPA-expressing ICT cells rapidly contract following tumor clearance in-vivo. The SPA platform represents a novel, tunable, and T cell intrinsic approach for engineering cell fates that result in potent anti-tumor properties. Citation Format: Thomas J. Gardner, Beatriz Millare, Anzhi Yao, Ashley Cass, Suchismita Mohanty, Jeremy Chen, Alma Gomez, David DeTomaso, Manching Ku, Lionel Berthoin, Meng Lim, Azalea Ong, Vince Thomas, Nicholas Quant, Brian Hsu, Amy-Jo Casbon, Natalie Bezman, Aaron Cooper, Levi Gray-Rupp, Angela C. Boroughs, W. Nicholas Haining. Tunable STAT activation by synthetic pathway activators (SPAs) increases engineered T-cell potency and persistence. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4073.