De novo masking domains that gate TNF-family ligand assembly and activity.

Tumor necrosis factor family ligands (TNFLs) are central regulators of immunity and promising agents for cancer therapy, but their clinical use is often limited by dose-limiting systemic toxicity. Conditional activation and genetic fusion with antibodies could improve safety and pharmacokinetics, yet these features are difficult to combine because TNFLs form obligate homotrimers that are structurally mismatched with antibody architectures. Here we use AI-enabled protein design to create de novo protein masks that conditionally control TNFL assembly. The masks are genetically fused to TNFLs through protease-sensitive linkers and prevent trimer formation by competitively binding the TNFL trimerization interface. We demonstrate with TNFα, OX40L, and 4-1BBL that protease-mediated release of the mask promotes trimer assembly, receptor binding and biological activity. These monomeric, switchable TNFLs are readily incorporated into full-length IgG formats, enabling plug-and-play construction of conditionally activatable antibody fusions. These fusions can be designed either to release activated soluble TNFLs upon protease treatment or, by reordering the fusion architecture, to trigger antibody multimerization and signaling by membrane-type TNFLs. When this multimerization strategy is applied to antibody-drug conjugates, conditional trimerization enhances cell killing by ~30-fold, thereby improving the therapeutic window and enabling multiple strategies for selective tumor microenvironment targeting.