Improving dual targeting selectivity in T-cell engagers via synapse-gated and affinity-tuned trispecific antibody design.

T-cell engagers (TCEs) represent a powerful drug modality for redirecting a patient's own T cells to recognize and eradicate cancer cells. Although TCEs have been effective in treating hematological cancers, their broad application for solid tumors has been more challenging due to the absence of tumor-specific antigens. This often leads to on-target, off-tumor toxicities and a low therapeutic index (TI). Strategies for dual-antigen targeting of double-positive cancer cells over single-positive normal tissue may improve the TI of TCEs. In this study, we report the development and characterization of a conditional dual tumor-associated antigen (TAA)-targeting trispecific antibody (TriMab) TCE composed of a non-active anchoring arm (. anti-TAA1), deficient in mediating an active immunological synapse, and an affinity-tuned active arm (. anti-TAA2), paired with an anti-CD3 domain to drive AND-gated targeting and elimination of dual-TAA tumors while sparing single-TAA healthy cells. Using an anti-receptor tyrosine kinase-like orphan receptor 1 (ROR1) mAb as a proof-of-concept anchoring arm and an array of affinity-modulated variants of the anti-epidermal growth factor receptor (EGFR) GA201 mAb as active arms, we show conditional engagement and elimination of double-positive human NCI-H358 non-small cell lung cancer cells over single-positive, non-target NCI-H358.ROR1.KO cells by affinity-modulated TriMab TCEs. , the TriMab TCE exhibits selective targeting and eradication of ROR1/EGFR double-positive tumors in a mouse xenograft model. We further demonstrate the generality of the anchoring arm in TriMab using anti-HER2 mAbs targeting different binding epitopes and discuss the interplay of factors regulating immunological synapse formation. Lastly, we demonstrate that the TriMab modality exhibits a favorable developability profile and mAb-like pharmacokinetic properties in human neonatal Fc receptor transgenic mice. Overall, this work presents a generalizable approach to utilizing the TriMab modality by leveraging avidity effects and molecular geometry to achieve conditional AND-gated dual TAA-targeting with a significantly improved TI.