T-cell Receptor (TCR) Targeting with Multivalent T-cell Engagers

T-cell engagers (TCEs) for cancer immunotherapy have traditionally relied on high affinity single chain fragment variable (scFv) domains to target CD3, specifically the ε chain, for the activation of T-cells. Despite their clinical success, there have been reports of TCEs driving systemic toxicity, non-specific T-cell activation, on-target off-tumor effects, and severe inflammation due to cytokine release. To address these limitations, we designed multivalent TCEs using Chemically Self-Assembled Nanorings (CSANs) that target the α/β constant region of the T-cell receptor (TCR) in the TCR/CD3 complex using a moderate affinity αTCR nanobody (αTCR _VHH ). Nanobodies offer superior physical and chemical properties over scFvs-including higher solubility, stability and lower production cost-making them increasingly popular as structural units of TCEs. We compared the efficacy and safety profile of this moderate affinity, nanobody-based TCR binder against high affinity αCD3 _scFv based CSANs across EGFR and PSMA expressing solid tumor models. While the αCD3 _scFv CSANs offered potent cytotoxicity, they also induced antigen independent T-cell activation bypassing the requirement of tumor crosslinking for cytotoxicity. In contrast the αTCR _VHH CSANs required strict antigen engagement to trigger cytotoxicity, significantly reducing non-specific T-cell activation and thus enhancing the safety profile. Although the initiation of cytotoxicity was kinetically slower than the αCD3 _scFv counterpart, αTCR _VHH CSANs achieved comparable end point cytotoxicity across multiple antigen densities, as well as in 3D tumor spheroids. Through this study we demonstrate the applicability of nanobodies as T-cell targeting domains, enhanced specificity and safety of moderate affinity T-cells binders and the diversification of T-cell targeting epitopes without compromising the efficacy of TCEs.