Payload Diversification Overcomes Resistance and Guides Sequential Antibody–Drug Conjugate Therapy in Breast Cancer

PURPOSE: Antibody-drug conjugates (ADC) have transformed the treatment of breast cancer. FDA-approved ADC such as trastuzumab deruxtecan (T-DXd), sacituzumab govitecan (SG), and datopotamab deruxtecan (Dato-DXd) have demonstrated clinical benefit; however, drug resistance will almost inevitably emerge in the metastatic setting, and there is no established strategy for selecting subsequent ADC after disease progression following the first exposure to the ADC. This study aimed to define mechanisms of acquired resistance and evaluate rational sequencing approaches. EXPERIMENTAL DESIGN: We generated breast cancer cell lines resistant to T-DXd (TDXd-R) and SG (SG-R). We assessed antigen expression, internalization, and resistance pathways, including efflux transporter activity. To identify therapeutic strategies, we tested the efficacy of ADC carrying non-topoisomerase I (Topo1) payloads in in vitro and in vivo models. RESULTS: Resistance in both TDXd-R and SG-R models was primarily mediated by payload-specific factors, notably upregulation of efflux transporters, rather than loss of antigen expression. Switching to ADC with mechanistically different payloads, including microtubule inhibitors, restored antitumor activity in vitro and in vivo. CONCLUSIONS: Cross-resistance to sequential ADC is primarily driven by shared payload mechanisms rather than loss of the target. Our data also suggest that the clinical benefit of sequential Topo1-based ADC may be reduced following disease progression on a Topo1-based ADC, likely due to these shared resistance pathways. Importantly, switching to ADC with non-cross-resistant payload classes may offer a more effective approach to guide ADC sequencing in patients with metastatic breast cancer, supporting payload diversification as a clinically actionable strategy.