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Background HER2 (human epidermal growth factor receptor 2) remains a critical therapeutic target in oncology, with alterations present in approximately 30% of breast cancers and 20% of non-small cell lung cancers. These alterations—ranging from amplification and mutation to overexpression—are associated with aggressive disease and poor clinical outcomes. FDA-approved therapies include monoclonal antibodies, tyrosine kinase inhibitors (TKIs), and antibody-drug conjugates (ADCs). Recent attention has focused on ADCs for their targeted cytotoxic delivery and reduced systemic toxicity. However, key ADC components—linker, payload, and antibody—are rarely evaluated independently in clinically relevant preclinical models, limiting optimization and translational predictability.Methods Patient-derived xenograft organoids (PDXOs) from Champions Oncology’s CTG3D platform were screened for HER2 positivity via IHC, with selected models exhibiting complete, intense membrane staining. Organoids were seeded at 5,000 cells per well in 96-well plates and allowed to form uniform 3D structures (>150 µm) within 3 days. Plates included standard controls (10 µM staurosporine, 0.1% DMSO) to establish Z′-factor metrics. Organoids were treated with HER2-targeting ADCs (e.g., trastuzumab emtansine), payload-only controls, and isotype antibodies. After 6 days of incubation, CellTiter-Glo® was used to assess cell viability and calculate IC50 values.Results The assay demonstrated high reproducibility, with Z′-factors above 0.5 and robust signal-to-noise ratios. HER2+ PDXOs displayed classic spheroidal morphology and consistent proliferation. Differential cytotoxicity was observed among ADCs and free payloads, revealing that linker chemistry significantly influenced drug activity in the organoid context. The ex vivo platform reliably captured drug responses in a high-throughput setting, highlighting the functional relevance of evaluating individual ADC components in a native-like tumor microenvironment.Conclusions Champions Oncology’s HER2-positive CTG3D PDXO platform provides a robust, translationally relevant model for preclinical ADC evaluation. By preserving tumor heterogeneity and 3D architecture, this system enables detailed interrogation of ADC pharmacology, including linker and payload effects. The platform offers a powerful tool for screening and optimizing targeted therapies in HER2-driven cancers and supports rational therapeutic design prior to clinical development.