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Reprogramming T Cells to Interrogate Intracellular Disease Signatures


Adoptive T-cell therapy—an emerging paradigm in which tumor-targeting T cells serve as living therapeutic modalities administered to cancer patients—has demonstrated remarkable curative potential in patients with relapsing B-cell malignancies, but clinical translation has not been extended to the vast majority of diseases. In particular, the lack of tumor-exclusive surface-bound antigens presents a fundamental challenge to conventional targeting approaches involving surface receptor engineering, due to an inherent risk for ‘on-target, off-tumor’ toxicities. To expand the pool of candidate disease signatures for adoptive T-cell therapy, we devised a strategy to reprogram T cells to interrogate target cells for intracellular antigen expression prior to enacting cytolytic programs. Specifically, we genetically replaced the endogenous and constitutively cytotoxic granzyme B (GrB) payload with a GrB-based switch, termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT), which remains inactive until encounter with an intracellular disease signature. In this manner, the COVERT system complements surface receptor technologies to enable AND-gate Boolean logic computation for spatiotemporally segregated input signals, first at the target-cell surface, and then from within the target cell. Here, we report the design of a small ubiquitin-like modifier protein (SUMO)-GrB fusion that is specifically activated by the intracellular tumor-associated protease, SENP1, to selectively initiate apoptosis. We systematically optimized the T-cell genome-editing workflow to facilitate the efficient manufacture of primary human CD8+ T cells that express SUMO-GrB and a chimeric antigen receptor (CAR) instead of wild-type GrB and the endogenous T-cell receptor, respectively. We also provide the first demonstration of selective T-cell lytic function in response to intracellular antigen expression. Finally, we explore the design of allosterically regulated COVERT switches by nanobody domain insertion, with the aim of developing a modular architecture that supports the rapid optimization of switch behavior for any intracellular disease biomarker. As medical strategies continue to shift toward precision medicine, the COVERT platform represents a potentially transformative technology that can enhance the safety of adoptive T-cell therapy to enable implementation as a front-line treatment option for a broader range of diseases.

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