Protease-activated prodrugs leverage the increased activity of proteases in the tumor microenvironment and the tight regulation in healthy tissues to provide selective activation of cytotoxins in the tumor while minimizing toxicity to normal tissues. One of the largest classes of protease-activated prodrugs are composed of therapeutic agents conjugated to macromolecular carriers via peptide motifs that are substrates for cathepsin B, and antibody-drug conjugates are one of the most successful designs within this class. However, many of these peptide motifs are also cleaved by extracellular enzymes such as elastase and carboxylesterase 1C. Additionally, some peptide sequences have little selectivity for other lysosomal cathepsins, which have also been found to have extracellular activity in normal physiological processes. A lack of selectivity or oversensitivity to other extracellular enzymes can lead to off-target release of the cytotoxic payload and subsequent toxicities. In this report, we describe an approach for modulating cathepsin-mediated release of the cytotoxic payload through steric shielding provided by the synergistic effects of appropriately designed hydrophilic linkers and the conjugated carrier. We prepared a fluorogenic model payload with a Val-Cit cleavable trigger and attached the trigger-payload to a variety of PEG-based linker architectures with different numbers of PEG arms (y), different numbers of ethylene oxide units in each arm (n), and different distances between the cleavable trigger and PEG branch point (D'). These linker-payloads were then used to prepare DAR2 conjugates with the cleavable triggers at three different distances (D) from the antibody, and cathepsin-mediated payload release was monitored with in vitro assays. The results show that structural variables of the linker architectures can be manipulated to effectively shield enzymatically labile trigger-payloads from enzymes with readily accessible binding sites, and may offer an additional strategy for balancing off-target and tumor-targeted payload release.

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