<i>De Novo</i> Design of Peptide Masks Enables Rapid Generation of Conditionally-Active Miniprotein Binders

The widespread expression of therapeutic targets in both diseased and healthy tissues poses a major challenge for protein-based therapeutics, often leading to dose-limiting side effects. One promising strategy to enhance selectivity is reversible inactivation via affinity masks tethered through cleavable linkers responsive to disease-specific cues. Here, we introduce a workflow for the <i>de novo</i> design of peptide masks that reversibly inactivate miniprotein binders. By extending the C-terminus of the binder with a protease-cleavable linker and a masking helix, we generated minimal constructs that sterically block the receptor-binding interface. We applied this strategy to four therapeutically relevant targets, EGFR domains I and III, FGFR2, and IL7Rα, demonstrating broad applicability. Nearly half of the 20 designs achieved >100-fold affinity reduction, with the most effective mask decreasing EGFR binding by over 3 orders of magnitude. Upon cleavage by tumor-associated proteases, binding was restored in 19 out of 20 cases, confirming reversibility. We further show that micromolar or weaker affinity between the binder and the isolated mask is sufficient for robust inactivation and rapid activation. Additionally, by chemically conjugating a photocleavable linker, we created a light-responsive version of the masked binder, enabling external control with comparable efficiency to protease-sensitive designs. This work establishes a generalizable, rapid, and efficient platform for designing cleavable peptide masks from scratch, paving the way for conditionally active protein therapeutics responsive to endogenous or exogenous stimuli.