Nanobody-Targeted Conditional Antimicrobial Therapeutics.

Conditional therapeutics that rely on disease microenvironment-specific triggers for activation are a promising strategy to improve therapeutic cargos. Among the investigated triggers, protease activity is used most often because of its dysregulation in several diseases. How to optimally fine-tune protease activation for different therapeutic cargos remains a challenge. Here, we designed nanobody-targeted conditional antimicrobial therapeutics to deliver a model therapeutic peptide and protein to the site of bacterial infection. We explored several parameters that influence proteolytic activation. We report the use of targeting nanobodies to enhance the activation of therapeutics that are otherwise activated inefficiently despite extensive optimization of the cleavable linker. Specifically, the pairing of Ly6G/C or ADAM10-targeting nanobodies with ADAM10-cleavable linkers improved activation via proximity-enabled reactivity. This study demonstrates a distinct role of active targeting in conditional therapeutic activation. More broadly, this optimization framework provides a guideline for the development of conditional therapeutics to treat various diseases in which protease activity is dysregulated.