De Novo Design of Peptide Masks Enables Rapid Generation of Conditionally-Active Miniprotein Binders

ABSTRACT The widespread expression of many therapeutic targets in both diseased and healthy tissues presents a significant challenge for protein therapeutics, often resulting in dose-limiting side effects. To focus the action of biotherapeutics at the disease site, they can be reversibly inactivated by tethering an affinity mask through a linker cleavable by a disease-specific cue. However, current methods to generate affinity masks require extensive screening of masking sequence libraries. Here, we report a workflow to design the first de novo peptide masks for the reversible inactivation of miniprotein binders and we show its application on an EGFR antagonist miniprotein. By extending the C-terminus of the miniprotein to cover the binding interface, a masking sequence is generated that decreases binding to EGFR over 1000 times. Binding of the parental miniprotein can be fully rescued by cleavage of the linker between the binder and the mask with tumor-specific proteases. Additionally, by site-specifically conjugating the peptide mask through a chemical linker, we show we can render the protein responsive to alternative stimuli such as light. Our approach opens the doors to dramatically accelerating the development of affinity-based masks for other proteins with therapeutic potential to render them conditionally active to any stimulus.