Abstract 4449: Functional organoid screening uncovers target dependency and bystander killing in TROP2 ADCs

Abstract Introduction: TROP2-targeted antibody-drug conjugates (ADCs) represent a promising therapeutic class for multiple tumor types with elevated TROP2 expression. Membrane-permeable payloads can induce bystander killing, making functional, physiologically relevant testing crucial. We developed an advanced organoid-based ADC evaluation workflow combining large-scale patient-derived (xenograft) organoid (PD(X)O) screening, CRISPR-engineered isogenic models, and 3D high-content imaging (HCI) to assess potency, target dependency, and bystander effects. Methods: Two TROP2 ADCs - an experimental exatecan-based ADC linked via a hydrophilic, cleavable linker, and datopotamab-deruxtecan (Dato-Dxd; DS-1062) - were evaluated in the organoid screening platform, where models were selected irrespective of TROP2 expression. The exatecan-based ADC was tested in 109 PD(X)Os (101 tumor, 8 normal) across ten cancer types using nine-point dose-response. Dato-Dxd was screened using six-point dose-response assays in 142 PD(X)Os (132 tumor; 10 normal) spanning nine tumor types. Drug incubation ran over five days, and efficacy was determined by ATP-based cell viability assay. Free payloads exatecan and Dxd (both naked and linker-conjugated) served as controls. CRISPR/Cas9-generated TROP2 knockout (KO) organoid pairs were labeled with fluorescent/luminescent reporters for target HCI-based dependency and bystander effect assays. Results: The exatecan-based ADC demonstrated high overall efficacy (68.6% showed >50% inhibition at the highest dose of 1 µM), with pancreatic models even showing 100% sensitivity, and 32.4% of organoids achieving IC50 < 0.1 µM. Payloads alone were strongly cytotoxic in most models (93.3% of models sensitive with IC50 < 0.03 µM) with similar potency for exatecan and Dxd, whereas linker-conjugated Dxd was significantly less cytotoxic (average IC50 > 0.1µM). Dato-Dxd responses varied across the PD(X)O panel (54% showed >50% inhibition at 0.1µM, 37% IC50 < 0.1µM). For both TROP2 ADCs, a correlation between efficacy and TROP2 mRNA abundance was observed. In isogenic KO organoid models, loss of TROP2 abrogated Dato-Dxd responses, confirming target dependency. Mixed-culture assays quantified Dato-Dxd bystander killing, validating membrane-permeable payload action in neighboring cells. Conclusion: The combined organoid screening and HCI workflow provides a robust, translationally relevant platform for ADC evaluation, integrating efficacy profiling, mechanistic validation, and functional bystander effect modeling. CRISPR-generated isogenic organoids enable precise dissection of target dependency and resistance, supporting the preclinical optimization of TROP2-targeted ADCs. Citation Format: Jialei Sun, Marten Hornsveld, Lenno Krenning, Dorrith Verstegen, Peng Han, Zhongman Sun, Caitlyn Hulsebosch, Dione Blok, Peter van Schaik, Mali He, Huike Ju, Yi Sun, Mariusz Madej, Hester Bange, Jun Zhou, Peng Wang, Ludovic Bourre, Marrit Putker. Functional organoid screening uncovers target dependency and bystander killing in TROP2 ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4449.