Abstract 1601: Advancing cancer immunotherapy: Unveiling the potential of PD-L1-targeted IFN-α2a dutaflip molecule through patient-derived tumor explants

Abstract This study aims to elucidate the mode of action (MoA) of the programmed cell death ligand 1 (PD-L1)-targeted Interferon alpha (IFN-α2a) Dutaflip molecule (RO7694744) in human patient-derived tumor explants (PTEs). PTEs are highly translationally relevant models due to their ability to closely mimic the native tumor microenvironment (TME) and maintain the heterogeneity of patient-specific tumor biology. The goal is to evaluate the specificity and efficacy of immune responses in preclinical development. RO7694744 is a trivalent bispecific antibody-cytokine fusion protein designed to specifically activate IFN-α on PD-L1 positive cells. This targeted activation enhances anti-tumor immune responses while minimizing systemic toxicity and non-specific activity. Fresh human PTEs from various cancer types (non-small cell lung cancer (NSCLC) n=16 and bladder cancer (BLCA) n=5) were cultivated ex vivo in a dynamic flow 3D cultivation system (MIVO), and treated with RO7694744 for 20 hours. Untreated samples and those treated with recombinant human IFN-α2a (rhIFNAα2a) served as negative and positive controls, respectively. After cultivation, cytokine secretion was analyzed, and single nucleus RNA sequencing (snRNA-seq) was performed to evaluate immune responses and transcriptional changes at the single-cell level. Comprehensive baseline characterization, including clinically relevant histopathological analysis, bulk RNA sequencing, and flow cytometry analyses, was conducted to correlate drug response with tumor-intrinsic features. RO7694744 treatment induced a consistent cytokine response (IP-10, ISG-15, IFNg) across NSCLC and BLCA samples, comparable to rhIFNAα2a but to a slightly lower extent. Pearson correlation revealed a positive relationship between PD-L1 positive cells and elevated cytokine response levels in samples treated with RO7694744, a specificity not observed with rhIFNAα2a. SnRNA-seq data analysis identified 44 cellular subpopulations, with a preferential activation of PD-L1 positive tumor cells and immune cells by the RO7694744. This activation was characterized by an up-regulation of an inflammatory gene signature in PD-L1 positive cells, not seen in untreated conditions. In summary, RO7694744 demonstrates a specific immunomodulatory effect on PD-L1 positive tumor and immune cells, inducing a targeted inflammatory response. The use of fresh human PTEs in this study resulted in translationally-relevant findings, underscoring the PTE model as an advanced next-generation tumor model for the precise evaluation of cancer immunotherapies. These findings enhance the understanding of RO7694744's MOA, confirming its PD-L1-dependent tumor targeting and activity within the TME. This supports a patient selection strategy and identifies clinically-relevant pharmacodynamic biomarkers. Citation Format: Steffen Dettling,1 Martina Antoniolli,2 Kerstin Pätzold,2 Manuela Gies,2 Jakob Rosenhauer,2 Jens Liedke,2 Laura Krümpelmann,2 Marion Lichtenauer,2 David Lamparter,1 Tai-Hsien Ou Yang,3 Petra Schwalie,1 Gabriel Schnetzler,1 Andreas Roller,1 Moritz Rapp,2 Meher Majety,2 Pablo Umaña,4 Marina Bacac4. Advancing cancer immunotherapy: Unveiling the potential of PD-L1-targeted IFN-α2a dutaflip molecule through patient-derived tumor explants [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 1601.