Engineering Cathepsin S Selective Chemical Probes and Antibody−Drug Conjugates through Substrate Profiling with Unnatural Amino Acids

Cysteine cathepsins, particularly cathepsin S, regulate proteolytic signaling in cancer progression and immune modulation, yet selective tools for individual cathepsins remain limited. Here, we report the design of cathepsin S-selective probes and cathepsin S-cleavable antibody-drug conjugates (ADCs) using substrate profiling with unnatural amino acids. Hybrid Combinatorial Substrate Library (HyCoSuL) screening identified selective tetrapeptide motifs that were used to develop optimized fluorogenic substrates, irreversible inhibitors, and fluorescent activity-based probes with high selectivity for cathepsin S in biochemical and cellular assays. These peptide motifs were then incorporated as cleavable linkers in MMAE-based ADCs targeting HER-2 or TROP-2, enabling cathepsin S-dependent cytotoxicity across breast cancer models with distinct target-expression profiles. Finally, anti-cathepsin S antibodies combined with CyTOF analysis revealed the spatial distribution of cathepsin S and its coexpression with HER-2 and TROP-2 in breast cancer patient samples, suggesting that cathepsin S profiling may help inform future patient stratification strategies for cathepsin S-activated ADC therapy.