An Enzyme-Responsive Imiquimod Prodrug for Precision Immune Activation within the Tumor Microenvironment

Small-molecule immune modulators offer a promising alternative to biologics, such as antibodies, for cancer immunotherapy. A key example is the TLR7 agonist imiquimod (<b>IMQ</b>), which has already been approved for the treatment of various dermatological malignancies. Nevertheless, the clinical use of <b>IMQ</b> is limited to tumors amenable to topical application, as its systemic administration poses a high risk of severe inflammatory toxicity due to the widespread expression of TLRs. Therefore, to extend the use of TLR7 agonists to the treatment of other solid tumor types, we developed a β-glucuronidase-responsive albumin-binding prodrug designed for the selective delivery of <b>IMQ</b> within the tumor microenvironment. This prodrug masks <b>IMQ</b>'s immunogenicity, allowing for its administration in immunocompetent mice without eliciting the systemic side effects associated with TLR7 agonists. However, the β-glucuronidase-catalyzed prodrug activation enables the selective, tumor site-specific release of <b>IMQ</b>, thereby restoring its biological activities. This controlled delivery promotes M1 macrophage polarization, T cell activation, and an increase in IgG levels exclusively within malignant tissues without affecting the healthy organs that are sensitive to TLR7 agonists. This study demonstrates that targeting tumor microenvironment specificities represents a promising approach for developing selective cancer immunotherapies based on small-molecule immune modulators.