Abstract PD3-11: Overcoming ADC Resistance: Payload Diversification as a Strategy for Sequential Therapy

Abstract BACKGROUND: The current sequential use of antibody-drug conjugates (ADCs) for metastatic breast cancer in clinics lacks preclinical evidence. There are no data to justify sequencing topoisomerase I (Topo1) payloads, such as sacituzumab govitecan (SG), trastuzumab deruxtecan (T-DXd), and datopotamab deruxtecan (Dato-DXd). As use of these ADCs expands in metastatic disease, resistance driven by shared payload mechanisms raises concerns about cross-resistance and cumulative toxicity. We hypothesized that switching to antibody-drug conjugates (ADCs) with distinct cytotoxic payloads could overcome Topo1 inhibitor resistance and restore therapeutic efficacy. To test this hypothesis, we utilized breast cancer cell line models with acquired resistance to Topo1 inhibitor-based ADCs and evaluated sequential treatment regimens incorporating ADCs with alternative payloads. MATERIALS AND METHODS: We utilized two T-DXd-resistant (T-DXd-R) HER2+ breast cancer cell lines (SUM190-TDXd-R, HCC1954-TDXd-R) and two SG-resistant (SG-R) triple-negative breast cancer (TNBC) cell lines (HCC1806-SG-R, MDA-MB-468-SG-R), along with their respective parental control cells. HER2 and Trop2 expression following chronic ADC exposure was evaluated by Western blotting and flow cytometry. ADC internalization was evaluated using endocytosis assays. To assess ADC and payload sensitivity, we performed sulforhodamine B (SRB) proliferation assays using a panel that included ADCs conjugated to Topo1-inhibitor (T-DXd, SG, Dato-DXd), Topo1-inhibiting payloads (DXd, SN38), microtubule-disrupting payloads (MMAF Monomethyl auristatin F, MMAE Monomethyl auristatin E), and ADCs conjugated to tubulin inhibitors (T-DM1, trastuzumab-MMAF, sacituzumab-MMAF). In vivo efficacy studies were conducted using xenograft models derived from SUM190-TDXdR and HCC1806-SGR cells. RESULTS: HER2 expression was significantly reduced in T-DXd-R HER2+ breast cancer cells (p 0.002), while Trop2 expression remained stable in both T-DXd-R and SG-R triple-negative breast cancer models, compared to parental cells. ADC internalization correlated directly with surface antigen levels (p 0.01). Both resistant models showed reduced sensitivity not only to their respective ADCs but also to other Topo1 inhibitor-based ADCs and free Topo1-inhibiting payloads (p 0.01 for all comparisons with parental controls). In contrast, microtubule-disrupting agents such as MMAE and MMAF, as well as ADCs containing MMAF, demonstrated strong antitumor effects. In the T-DXd-R HER2+ breast cancer model, tumor growth inhibition was negligible with SG (not significant), but significant with T-DXd (50.4%, p 0.0001), trastuzumab-MMAF (83.9%, p 0.0001), and sacituzumab-MMAF (76.7%, p 0.0001) compared to control. Similarly, in SG-R TNBC xenografts, SG had no significant effect, whereas sacituzumab-MMAF achieved 86.9% tumor growth inhibition (p 0.0001) compared to 31.7% with Dato-DXd (not significant). CONCLUSIONS: This first preclinical report demonstrates that resistance to Topo1 inhibitor-based ADCs in breast cancer is primarily driven by payload-specific insensitivity rather than complete loss of target antigen expression. In contrast, switching to ADCs with distinct payloads, such as microtubule inhibitors (MMAF or MMAE), restored antitumor efficacy in tumors that had lost responsiveness to Topo1 inhibitor-based ADCs. These findings underscore the payload class as a critical determinant of ADC anti-cancer activity. We need to advance the diversification of ADC payloads by integrating novel cytotoxic agents and immune-modulatory compounds, thereby expanding therapeutic options and addressing heterogeneous resistance mechanisms. Citation Format: D. Rampa, N. Ogata, N. Sridhar, F. Takeo, C. Wannaphut, J. Maynard, K. Tsuchikama, G. Sledge, N. Ueno, J. Lee. Overcoming ADC Resistance: Payload Diversification as a Strategy for Sequential Therapy abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PD3-11.