Enzyme‐Activated Orthogonal Proteolysis Chimeras for Tumor Microenvironment‐Responsive Immunomodulation

Abstract Precise modulation of dynamic and complex tumor microenvironment (TME) to disrupt tumorigenesis and reshape intratumoral immune infiltration has emerged as promising approaches for enhanced cancer therapy. Among recent innovations, proteolysis‐targeting chimeras (PROTACs) represent a burgeoning chemical knockdown technology capable of degrading oncogenic protein homeostasis and inducing dynamic alternations within carcinoma settings, offering potential for antitumor manipulation. However, achieving selectivity in PROTACs that respond to disease environmental stimulation and precisely perturb on‐target proteins remains challenging. The multi‐step synthesis and limited permeability, attributed to high‐molecular‐weight and heterobifunctional structures, further hinder their in vivo efficacy. Herein, we present a unique TME‐responsive enzyme‐activated clickable PROTACs, which features a short peptide‐tagged pomalidomide derivative to undergo tumor‐specific cleavage by cathepsin protease to induce orthogonal crosslinking of the exposed cysteine with 2‐cyanobenzothiazole‐labeled epigenetic protein‐ligand JQ1, facilitating in situ degrader formation within tumor regions only. Systematic protein profiling and proteomic analysis revealed that such TME‐specific clickable‐PROTACs not only selectively eliminate epigenetic proteins without tedious pre‐synthesis to bridge disparate small‐molecule bi‐warhead fragments, but also demonstrated superior tumor penetration compared to conventional high‐molecular‐weight PROTACs. Importantly, these clickable‐PROTACs efficiently downregulated immune checkpoint programmed death‐ligand 1 (PD−L1) both in vitro and in vivo, remodeling TME for enhanced therapeutics, especially in anti‐tumoral immunomodulation.