Click-to-Release for Controlled Immune Cell Activation: Tumor-Targeted Unmasking of an IL12 Prodrug

<b>Objectives:</b> Immunotherapy utilizing immune-stimulating cytokines such as IL12 holds great promise for the treatment of cancer. However, clinical use of IL12 is hampered due to severe toxicity following systemic administration. We here present a novel treatment strategy in which IL12 is chemically silenced by conjugation to PEG masks that sterically hinder the receptor binding. Subsequently, the masks can be released on demand using a bioorthogonal click reaction, cleaving the linker connecting the masks, thereby restoring the native cytokine. This "click-to-release" approach is based on the highly selective Inverse electron-demand Diels-Alder (IEDDA) pyridazine elimination reaction between a tetrazine (Tz) and a <i>trans</i>-cyclooctene (TCO), optimized for fast reaction kinetics and in vivo compatibility. Selective activation in the tumor microenvironment is achieved by pretargeting one component of this reaction to the tumor, triggering local activation of the masked IL12 once it is given in a secondary i.v. injection. <b>Methods:</b> IL12 masking and unmasking were evaluated in vitro with PAGE and HEK-Blue reporter cells and ex vivo with ELISA. Biodistribution in mice was evaluated with I-125 radiolabeling and biotin-click histochemistry. <b>Results:</b> Several designs were evaluated and optimized in vitro, resulting in an IL12-TCO-PEG construct that exhibited superior masking and subsequent reactivation upon reaction with a tetrazine bound to a TAG-72-targeted diabody. In tumor-bearing mice, we demonstrated that this diabody-tetrazine could efficiently pre-localize tetrazine in the tumor. Administration of IL12-TCO-PEG 24 h later afforded efficient and selective unmasking in tumors, but not in the blood. <b>Conclusions:</b> These results demonstrate proof of principle of the click-cleavable IL12 prodrug approach and showcase the versatility of the click-to-release reaction.