Abstract 3416: Advancing cancer immunotherapy: PD-L1-targeted IFNa2a fusion protein RO7694744

Abstract Interferon alpha (IFNa) is recognized for its potential in cancer immunotherapy by activating immune responses against tumor cells. However, untargeted IFNa's application is limited by systemic toxicity and non-specific activity, affecting both tumor and healthy tissues and resulting in undesirable side effects. To overcome these challenges, we developed the novel trivalent bispecific antibody-cytokine fusion protein RO7694744 designed to provide a specific activation of IFNa on programmed cell death ligand 1 (PD-L1) positive cells, such as tumor cells and immune cells. One antibody arm binds to human PD-L1 with high affinity, whereas the other arm can bind mutually exclusive either to human PD-L1 (with lower affinity) or to IFNa2a, which is fused to the antibody via a peptide linker. Both PD-L1 binders are non-blocking. This antibody design, called DutaFlip, protects the IFNa2a moiety from interacting with IFNa receptors (IFNAR1/2) until it is activated by binding to PD-L1 on the target cells. Our research explored the PD-L1 selective activity of RO7694744 in multiple preclinical models. Using reporter cell assays, human whole blood cell assays, and various tumor cell lines with different PD-L1 expression levels, we demonstrated that IFNa activity depends on PD-L1 expression. Cells with elevated PD-L1 expression showed significantly higher activation compared to those with low PD-L1 levels. In vivo studies further validated these findings. In MDA-MB-231 tumor-bearing mice, RO7694744 treatment resulted in significant tumor growth inhibition (TGI), accompanied by intratumoral immune cell activation, and demonstrated a synergistic effect when combined with checkpoint inhibitor (CPI) treatment. To ensure translational relevance, we confirmed these in vivo observations in patient-derived tumor explants that mimic the human tumor environment. The explant data supported our previous findings, demonstrating PD-L1 specificity and dependency of RO7694744. Our preclinical findings illustrate that RO7694744 selectively targets and activates PD-L1 expressing tumor and immune cells. Clinical experience is needed to confirm whether this targeted strategy can enhance therapeutic efficacy while minimizing adverse effects, thus addressing the key challenge of untargeted IFNa in cancer immunotherapy. Citation Format: Moritz Rapp, Harald Duerr, Gabriel Schnetzler, Davide Bertoldo, Jack Bates, Laura Kruempelmann, Alexandra Baumgartner, Marion Lichtenauer, Sandra Bunte, Kerstin Paetzold, Martina Antoniolli, Malika Ait-Goughoulte, Yvonnne Kienast, Johannes Sam, Steffen Dettling, Martin Lechmann, Robert Mader, Daniela Willend, Barbara Leutgeb, Anja Irmisch, Claudia Mueller, Metodi Donev, Andreas Roller, Nino Gochitashvili, Kerstin Meier, Yan Li, Meher Majety, Marina Bacac, Christian Klein, Pablo Umana. Advancing cancer immunotherapy: PD-L1-targeted IFNa2a fusion protein RO7694744 [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 3416.