Cis- and trans-binding chimeric costimulatory receptors enhance T-cell fitness and tumor control

T-cell-based therapies have shown remarkable success in combatting hematologic malignancies; however, their efficacy in solid tumors is hindered by the immunosuppressive microenvironment and restricted antigen availability. The use of chimeric costimulatory receptors (CCRs) has emerged as a strategy to improve T-cell function. However, most designs target antigens distinct from the primary antigen receptor, complicating their application across heterogeneous tumors. Here, we characterized the molecular requirements for a platform enabling costimulation in engineered T cells on the basis of dual targeting of a single antigen via a TCR and a CCR. We applied this strategy to the stress ligand BTN3A, which is broadly expressed in solid tumors and is a part of the antigen complex recognized by the γ9δ2TCR. Through structural modeling, alanine scanning, and antibody screening, we determined that 103-4-1BB, a BTN3A-specific CCR, bound to an epitope on BTN3A that was distinct from the γ9δ2TCR epitope. This epitope separation is critical for enabling synergistic coengagement of a single antigen, and the resulting increase in T-cell activation requires both γ9δ2TCR signaling and the trans-acting functionality of the anti-BTN3A-CCR. Moreover, the extracellular domain of 103-4-1BB stabilized T-cell–tumor cell interactions and increased γ9δ2TCR sensitivity, whereas its intracellular 4-1BB signaling domain drove robust proliferation, improved T-cell fitness, and mediated potent tumor control in vivo. Notably, cis-binding of the CCR to BTN3A on engineered T cells promoted survival in the absence of tumor cells, while transbinding to tumor-expressed BTN3A was required for infiltration, tumor clearance, and memory formation. These findings establish a modular framework for designing cis/trans-active CCRs that enhance T-cell function through single-antigen dual engagement, enabling broadly applicable strategies to improve solid tumor immunotherapy. (Figure presented.)