Development of a Homogeneous Trastuzumab-Triptolide Conjugate for Targeted Therapy of HER2-Overexpressing Ovarian Cancer

Abstract Ovarian cancer remains a lethal malignancy due to chemoresistance and toxicity, which limits the dose of chemotherapy that can be used, necessitating the development of more targeted therapies such as antibody-drug conjugates (ADCs). However, conventional ADCs suffer from heterogeneity. This study aimed to develop a stable, homogeneous ADC by utilizing a bifunctional dibromomaleimide (DBM) linker to cross-link antibody cysteine residues via disulfide-bridging. A DBM linker was synthesized from 3,4-dibromofuran-2,5-dione and bound to the cytotoxic agent triptolide. This triptolide payload was then conjugated to trastuzumab via site-specific disulfide rebridging to yield a homogeneous trastuzumab-triptolide conjugate to target human epithelial growth factor receptor 2 (HER2) on ovarian cancer cells. The study evaluated the ADC’s efficacy against SKOV-3 (high HER2 expression) and OVCAR-8 (low HER2 expression) cell lines. The results showed that the ADC was slightly more potent in SKOV-3 cells, yielding lower IC50 values compared to OVCAR-8. Mechanistic studies of the ADC via flow cytometry revealed that the ADC induced significant apoptosis and cell cycle arrest, characterized by a concentration-dependent increase in Caspase-3/7 expression and distinct alterations in cell population distribution. Furthermore, ADC treatment led to a concentration-dependent decrease in HER2 levels in SKOV-3 cells, confirming successful targeting. The study demonstrates that converting conventional maleimides into bifunctional DBM linkers allows the production of a homogeneous ADC via disulfide-bridging. This approach offers a promising strategy for developing potent anticancer therapeutics with improved selectivity for HER2-overexpressing ovarian cancers.