Development of enhanced enhertu-resistant HER2-positive breast cancer models for ADC resistance research

Antibody-drug conjugates (ADCs) combine the precision of a monoclonal antibody with a potent cytotoxic payload to selectively target cancer cells and represent a promising strategy for targeted cancer therapy. Enhertu (trastuzumab deruxtecan, DS-8201), an ADC targeting HER2-positive cancers, has achieved remarkable clinical efficacy and set a new benchmark for anti-HER2 ADCs, yet acquired resistance remains a major challenge. To elucidate resistance mechanisms and support development of next-generation ADCs, we established a stepwise preclinical platform for Enhertu resistance using the HER2-positive breast cancer cell line JIMT-1. JIMT-1 tumor bearing mice were first subjected to repeated in vivo Enhertu dosing, and subsequent residual tumors were harvested to derive a resistant cell line. This resistant cell line exhibited stable resistance to Enhertu both in vitro and in vivo . In vitro , resistant cells showed a right-shifted IC50 to the DXd payload, while flow cytometry and immunohistochemistry demonstrated the line maintained HER2 expression. RNA sequencing and whole-exome sequencing revealed upregulation of ABCG2 and other transporters, indicating a payload-centric resistance mechanism driven by altered payload sensitivity and enhanced drug efflux rather than target downregulation. To generate a more stringent model that better reflects long-term, high-intensity clinical exposure, the resistant cell line was re-implanted and serially passaged in vivo under high-dose Enhertu treatment, yielding an “enhanced” high-dose resistant tumor model. These enhanced tumors remained HER2-positive by IHC and displayed transcriptional changes similar to the resistant cell line, including pronounced ABCG2 upregulation and broader alterations in genes related to drug transport and cell-cycle regulation. Together, these Enhertu-resistant models offer a practical system to investigate resistance mechanisms that may not yet be evident in clinical samples and to identify candidate biomarkers and therapeutic targets. These cell lines also enable preclinical testing of biomarker-guided combination therapies and next-generation ADCs designed to overcome Enhertu resistance in HER2-positive malignancies.