Abstract 2691: Configurable multiplex immunofluorescence panels for emerging ADC targets

Abstract Background: Antibody-drug conjugates (ADCs) are revolutionizing oncology by selectively delivering cytotoxic payloads to cancer cells while sparing healthy tissues. ADC targets are typically cell-surface proteins overexpressed on tumor cells but with limited expression on normal cells, making spatial proteomics assays an ideal complement to this research. However, standard DAB-based immunohistochemistry techniques often lack the specificity and sensitivity needed to confidently quantify the target expression level in tumors with low target abundance, which can result in discordant scoring by pathologists. To support ADC drug development, we present a flexible and scalable approach to the design, development, and validation of mIF panels tailored to ADC targets using the highly sensitive InSituPlex (ISP) technology. Methods: Antibodies targeting ADC-specific markers were initially optimized using DAB chromogenic staining. Following this, antibodies were conjugated to DNA barcodes, and titrations of both antibodies and fluorophores were performed to achieve optimal signal intensity and staining patterns as evaluated by a pathologist. Singleplex DAB and ISP assays were conducted on serial FFPE sections across multiple tumor types. Validation experiments were designed and performed to test the platform's precision and configurability for ADC-related targets. AI-driven STARVUE software was used for image analysis and assessment of reproducibility of key metrics including cell density and signal intensity. Results: This approach integrates adaptability to diverse ADC targets, optimized antibody titration, fluorophore selection, and panel construction, achieving high specificity and sensitivity for multiple biomarkers in a single tissue section. The inclusion of STARVUE image analysis enhances the evaluation of spatial relationships between ADC targets and downstream effectors, offering a deeper understanding of drug efficacy and resistance mechanisms. The platform demonstrated high concordance across configurations, with single-target coefficients of variation (CVs) consistently below 20%. Spatial and intensity metrics further confirmed the robustness of the assay in multiple tumor types. Conclusions: This validated and scalable mIF platform for ADC targets represents a significant advancement in spatial proteomics, providing a robust solution for researchers and clinicians to accelerate ADC development, optimize therapeutic strategies, and improve clinical outcomes. Citation Format: Yvette Cajigas, Cassandra Kysilovsky, Lauren Duro, Kevin Hwang, Lorenz Rognoni, Angela Vasaturo. Configurable multiplex immunofluorescence panels for emerging ADC targets [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 2691.