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, pairing of Ly6G/C or ADAM10-targeting nanobodies with ADAM10-cleavable linkers improved activation via proximity-enabled reactivity. More broadly, this optimization framework provides a guideline for the development of conditional therapeutics to treat various diseases where protease activity is dysregulated. ### Competing Interest Statement S.N.B. and C.N. are listed as inventors on patent application related to the content of this work. S.N.B. reports compensation for cofounding, consulting, and/or board membership in Glympse Bio, Satellite Bio, CEND Therapeutics, Catalio Capital, Intergalactic Therapeutics, Port Therapeutics, Vertex Pharmaceuticals, and Moderna, and receives sponsored research funding from Johnson & Johnson, Revitope, and Owlstone. All the other authors declare no competing interests.