Pan-alphabeta-T-cell reactive anti-TCR VHH bispecifics demonstrate potent cytotoxicity through novel constant region targeting

Introduction: All approved T-cell engagers target CD3ε, but alternative engagement sites on the TCR complex may yield differentiated functional properties. We developed VHH-based T-cell-engaging (TCE) arms that bind a previously unexploited quaternary epitope formed at the TCRalpha/beta constant-region interface. These binders exhibit pan-alphabeta-T-cell reactivity with inherent human-cynomolgus cross-reactivity. Methods: Anti-TCR VHHs were identified using yeast-based selections against recombinant human and cynomolgus TCRalpha/beta heterodimers, employing both synthetic libraries and llama immunization. Affinity optimization of VHHs maintained cross-species recognition. Epitope mapping was performed using TCR constant-region mutant libraries generated by error-prone PCR. Structures of VHH-TCR complexes were solved. Bispecifics incorporating anti-tumor associated antigen (TAA) domains were assessed in T-cell-dependent cytotoxicity (TDCC) assays across tumor models and benchmarked against clinical CD3 controls. Results: Multiple VHH lineages that recognize a conserved quaternary epitope at the TCRalpha/beta constant-region interface were isolated, providing TRBC1/C2 coverage and human-cynomolgus cross-reactivity. Structural analyses established the binding mode. Despite moderate monovalent affinities, anti-TCR bispecifics demonstrated potent TDCC activity comparable to or exceeding benchmark CD3 bispecifics and clinically active controls. Functional activity was confirmed in both human and cynomolgus assay systems, including preliminary cytokine readouts. Conclusions: VHH-based bispecifics that target a quaternary epitope within the TCR constant region constitute an emerging class of T-cell-engagers with potent cytotoxic activity and broad alphabeta-T-cell coverage. The combination of TRBC1/C2 inclusivity, structural definition, and human-cynomolgus cross-reactivity positions TCR constant-region targeting as a promising alternative for next-generation T-cell-redirecting therapeutics.