Abstract 6395: Synthetic pathway activators enhance CAR-T functional persistence and prevent T cell exhaustion

Abstract The emergence of T cell exhaustion in CAR-T cells used to treat solid tumors remains a barrier to clinical efficacy. T cell engineering approaches are increasingly used to enhance CAR-T cell function, but to date few approaches directly modulate the development of T cell exhaustion. We have used large scale screening of synthetic transmembrane signaling molecules to identify a class of SPA (Synthetic Pathway Activators) that result in constitutive STAT1/STAT3 activity with marked gains of CAR-T cell anti-tumor potency, expansion, and persistence both in-vitro and in-vivo. In vitro, CAR-T cells expressing SPA displayed higher proliferation in response to both TCR and tumor antigen stimulation, and secreted more Granzyme B and IL-10 cytokines, but did not show cytokine-independent proliferation. SPA-expressing CAR-T cells also retained superior function relative to control CAR-T cells in a repetitive stimulation assay that recapitulates T cell exhaustion. In vivo, SPA-expressing CAR-T cells showed greatly enhanced potency in both 786-O and A498 xenograft models of Renal Cell Carcinoma, and demonstrated increased T cell expansion followed by rapid contraction after tumor clearance. Mechanistic studies utilizing temporal RNAseq data and pseudotime analysis of SPA expressing cells throughout the 14-day RSA assay revealed a divergent trajectory of T cell differentiation in comparison to control CAR-T cells, with greater maintenance of memory marker expression. Importantly, SPA-expressing CAR-T failed to induce TOX - a canonical transcriptional regulator of exhausted T cells - compared to control CAR-T cells. ATAC-Seq analysis further confirmed that the TOX locus remains inactive in SPA cells following chronic antigen stimulation, while unmodified CAR-T cells develop characteristic open chromatin regions surrounding the TOX locus. Thus the SPA module is a T cell intrinsic enhancement that boosts CAR-T potency and persistence by limiting the development of exhaustion. Moreover, it suggests that persistent STAT3/STAT1 activation may provide a general approach to modulate the emergence of T cell exhaustion. Citation Format: Lan Chen, Thomas Gardner, Beatriz Millare, Anzhi Yao, Josephine Giles, Ashley Cass, Suchismita Mohanty, Jeremy Chen, Alma Gomez, David DeTomaso, Aaron Cooper, Levi Gray-Rupp, John Wherry, Nicholas Haining. Synthetic pathway activators enhance CAR-T functional persistence and prevent T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6395.