Covert T Cells: Engineering T Cells to Interrogate Intracellular Antigens

Adoptive T-cell therapy is an emerging cancer treatment paradigm in which tumor-reactive T cells are expanded ex vivo prior to infusion into a patient. While recent FDA approvals highlight the remarkable curative potential of CD19-targeted T cells for patients with advanced B-cell malignancies, several obstacles currently prevent the clinical application of adoptive T-cell therapy for the vast majority of cancers. In particular, T-cell lytic selectivity is normally defined via receptor-mediated recognition of surface-bound antigens, which prompts the targeted delivery of cytotoxic protein payloads including granzyme B (GrB). Unfortunately, surface-antigen expression is rarely restricted to tumor cells, and misidentification of healthy tissues frequently results in on-target, off-tumor toxicities. In response, several studies have explored multi-input systems that require the simultaneous recognition of multiple surface antigens for tumor identification. However, such strategies remain constrained by antigen availability on the cell surface, thereby excluding a potentially vast repertoire of intracellular tumor markers. Here, we present a strategy to reprogram T cells to interrogate target cells for intracellular disease signatures prior to triggering lytic function. Specifically, we engineered synthetic GrB switches, termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT), which are delivered from T cells into target cells but require intracellular-antigen encounter within the target cell to initiate target-cell apoptosis. One example of an intracellular disease signature is the overexpression of Sentrin-specific protease 1 (SENP1) in prostate, pancreatic, and oncocytic thyroid tumors. As proof of concept, we developed a small ubiquitin-like modifier (SUMO)-GrB fusion that is specifically cleaved to activate GrB in SENP1-overexpressing cells. We further demonstrate that COVERT molecules are efficiently packaged into T-cell lytic granules and specifically degranulated upon receptor-mediated surface-antigen recognition. We anticipate that increasingly robust mammalian genome-editing technologies will enable the COVERT system to complement existing surface-receptor technologies and refine the therapeutic precision of adoptive T-cell therapy.