High-grade serous ovarian cancer (HGSC) remains a lethal malignancy with limited targeted therapies. Antibody-drug conjugates (ADCs) targeting trophoblast cell-surface antigen 2 (TROP2) and folate receptor alpha (FRα) are a promising systemic option in recurrent ovarian cancer. Patient-derived long-term organoids (LTOs) provide a physiologically relevant platform to study targeted cytotoxicity, resistance mechanisms, and immune interactions.

We established five HGSC LTOs from surgical specimens and performed single-cell RNA sequencing of LTOs and corresponding tumors. Two TROP2-targeting ADCs, datopotamab deruxtecan (Dato-DXd) and sacituzumab tirumotecan (Sac-TMT), were tested in dose–response titration experiments, including a non-targeting isotype control ADC to assess target-specific effects. Organoids were embedded in basement membrane extract (BME) prior to ADC exposure, followed by media replacement after approximately 48 hours to mimic transient clinical dosing. Viability was monitored over two weeks using PrestoBlue®, with planned retreatment to characterize resistant populations. A FRα-targeting ADC, mirvetuximab soravtansine (MIRV), is planned for future testing. Ongoing experiments include single-cell RNA sequencing to study resistance mechanisms and identify candidate pathways for functional validation, as well as immune cell co-culture to investigate ADC effects in the presence of T cells. Additional organoids will be tested to validate findings.

Single-cell RNA sequencing of five organoids and corresponding tumors revealed variable expression of ADC targets (TROP2 and FRα). We observed dose-dependent reductions in viability for TROP2-targeting ADCs. Sac-TMT showed greater overall cytotoxicity than Dato-DXd, although higher concentrations were required at early time points to achieve comparable effects, possibly reflecting delayed payload release or limited early penetration into the organoid matrix. These differences may also reflect ADC design features, including drug-to-antibody ratio and payload release mechanisms, with Sac-TMT undergoing both extracellular and intracellular cleavage, compared with predominantly intracellular release for Dato-DXd. Toxicity observed with the Sac-TMT isotype control supports a contribution of extracellular payload release.

Patient-derived HGSC LTOs provide a preclinical platform to evaluate ADC efficacy, capture differential responses and potential resistance. Preliminary data suggest that therapeutic activity is influenced by cleavage mechanisms and payload kinetics rather than intrinsic payload potency. Ongoing studies aim to further define the underlying mechanisms and support personalized therapeutic strategies.

This study was co-funded by the European Research Council (ERC), Research Council of Finland, and Cancer Foundation Finland.