Activation-optimized anti-CD3 valency control enables combinatorial discovery of multi-specific nanoantibodies for solid tumor immunotherapy.

Multi-specific antibodies that engage CD3 to redirect T cells have provided substantial clinical benefit in the treatment of hematologic malignancies, but their application to solid tumors remains constrained by a narrow therapeutic window and fixed molecular geometries that limit systematic optimization. Here we used a modular nano-adaptor framework to adjust anti-CD3 (αCD3) surface valency while maintaining antibody affinity, total antibody loading, and tumor-targeting capacity. By titrating the αCD3 stoichiometric fraction, we identified an activation-optimized conformation that preserves tumor-directed cytotoxicity while reducing non-specific immune activation and T cell exhaustion. This platform enabled rapid screening of 91 tetraspecific nanoantibody configurations that integrate immune checkpoint blockade, co-stimulatory signaling, and innate immune modulation. The lead candidates enhanced immune-tumor engagement, promoted intra-tumoral immune infiltration, and suppressed tumor growth in peripheral blood mononuclear cell (PBMC)-humanized mouse models with acceptable tolerability. These findings demonstrate that anti-CD3 valency control combined with modular combinatorial assembly is a potentially valuable and scalable strategy for the development of multi-specific immunotherapies against solid tumors.