Abstract 3486: Click-to-release for controlled immune cell activation: tumor-targeted unmasking of an IL-12 prodrug <i>in vivo</i>

Abstract Immunotherapy utilizing immune stimulating cytokines such as IL-12 holds great promise for the treatment of cancer. However, clinical use of IL-12 is hampered by severe toxicity following systemic administration resulting in a narrow therapeutic window. Tagworks is pioneering the use of in vivo click chemistry (Click-to-Release platform) to enable effective on-tumor activation of prodrugs, with the aim to increase the therapeutic index and application scope of potent anticancer drugs. We here present a novel treatment strategy in which IL-12 is inactivated by lysine conjugation to trans-cyclooctene(TCO)-linked PEG masks that can be released through the Click-to-Release reaction between the TCO linker and a tetrazine molecule (the trigger), restoring the native cytokine. Selective activation in the tumor microenvironment is achieved by pre-targeting the trigger to the tumor by i.v. injection of a tetrazine-protein conjugate, triggering local activation of the masked IL-12 when it is given in a subsequent i.v. injection. Evaluation of several Click-to-Release designs in vitro resulted in the selection of an IL-12-TCO-PEG construct with 9 conjugated lysines, exhibiting superior masking (&amp;gt;100 fold attenuated) combined with instantaneous and complete re-activation upon reaction with tetrazine. Next, we developed a tumor-targeted tetrazine based on a diabody against TAG72, a pan-carcinoma marker in the tumor microenvironment, modified with 4 tetrazine moieties. The diabody-tetrazine showed good tumor uptake in mice bearing colorectal (LS174T) xenografts combined with rapid blood clearance (&amp;lt; 0.5 %ID/g 24h p.i.). Also IL-12-TCO-PEG showed nearly complete clearance in 24 hours (&amp;lt; 0.5 %ID/g 24h p.i.). Administration of diabody-tetrazine to tumor-bearing mice followed by IL-12-TCO-PEG 24 hours later afforded selective IL12 unmasking in tumors and not in blood, demonstrating the feasibility of locally activating systemically administered immune stimulating cytokines with the versatile Click-to-Release platform. Citation Format: Martijn H. den Brok, Kim E. de Roode, Luc M. Zijlmans, Laurens H. Kleijn, Marleen H. van Stevendaal, Ron M. Versteegen, Lieke W. Wouters, Raffaella Rossin, Marc S. Robillard. Click-to-release for controlled immune cell activation: tumor-targeted unmasking of an IL-12 prodrug in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3486.