Glutathione-Triggered Unleashing of Protease K Prodrug Suppressed Primary Tumors and Its Metastasis with Stimulated Anti-tumor Immunity.

Protein therapeutics have garnered significant enthusiasm for oncological applications, owing to their exquisite specificity and intrinsic bioactivity. Yet, proteins remain constrained by limited transcellular permeability and proteolytic susceptibility during delivery. To circumvent these impediments, we engineered a redox-responsive protein prodrug, RP-ss-PK, via covalent conjugation of cytotoxic proteinase K (PK) to a multifunctional polymer, RGD-PEG-ss-NPC. This linear hetero-difunctional PEG scaffold contains a cyclic RGD peptide at the α-terminus for integrin-mediated tumor targeting and a 4-nitrophenyl ethyl carbonate (NPC) group at the ω-terminus for site-specific conjugation to amine-rich lysine residues on PK. An internal disulfide linker (-ss-) renders the prodrug cleavable in the presence of intracellular glutathione (GSH). Upon systemic administration, RP-ss-PK selectively accumulates within neoplastic lesions via RGD-integrin (αvβ3/αvβ5) recognition. Endocytosis exposes the disulfide tether to the cytosolic glutathione milieu, triggering reductive cleavage and subsequent liberation of native, fully active PK. The unleashed protease initiates indiscriminate proteolysis that precipitates rapid tumor cell death, accompanied by plasma-membrane permeabilization. This membrane disruption facilitates the extracellular release of damage-associated molecular patterns (DAMPs), thereby igniting anti-tumor immunity and remodeling the tumor microenvironment. Collectively, this prodrug paradigm not only amplifies the tumor-selective delivery of protein therapeutics but also exemplifies a facile, broadly applicable strategy for intracellular activation and functional restoration of protein cargos. Our findings establish RP-ss-PK as a versatile platform poised for the targeted protein treatment of diverse pathologies.