Abstract A136: Comparison of the bystander effects and killing capabilities of the HER2-targeting antibody drug conjugates T-DM1 and T-DXd

Abstract Antibody-drug conjugates (ADCs) are a class of targeted therapies designed to selectively deliver cytotoxic agents (payloads) to cancer cells. ADCs achieve this specificity by binding to surface antigens that are highly expressed on tumor cells, such as human epidermal growth factor receptor 2 (HER2). Upon binding to the target antigen and subsequent internalization, the cytotoxic payload is released inside the cell, leading to cell death. In addition to their antigen-specific action, ADCs can also affect neighboring cells that do not express the target antigen - a phenomenon known as the bystander effect. This occurs when the payload is released extracellularly before internalization, or when it diffuses out of the target cell after intracellular release. The extent of the bystander effect is influenced by the linker chemistry and physicochemical properties of the payload. This study investigates the bystander effect of two FDA-approved HER2-targeting ADCs: trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd). Both ADCs use the same anti-HER2 monoclonal antibody, trastuzumab, but differ in linker type and payload. T-DM1 employs a non-cleavable thioether linker (SMCC) conjugated to the microtubule inhibitor DM1, whereas T-DXd uses a cleavable tetrapeptide-based linker that is enzymatically degraded in lysosomes to release DXd, a membrane-permeable topoisomerase I inhibitor. HER2 expression was quantified across a panel of cancer cell lines using flow cytometry with Quantibrite PE beads. The SK-BR-3 breast cancer cell line was identified as highly HER2-positive, expressing approximately 53,000 receptors per cell. In contrast, the U-87MG ATCC glioblastoma cell line showed no detectable HER2 expression. Cell viability assays were performed to determine the cytotoxic effects of T-DM1 and T-DXd. In monoculture, both ADCs induced potent cell death in HER2-positive SK-BR-3 cells, with no effect on HER2-negative U-87MG cells. However, in co-culture experiments, T-DXd induced significant cytotoxicity in HER2-negative U-87 MG cells, indicative of a strong bystander effect. T-DM1, in contrast, did not show this effect. These findings highlight key differences in the bystander killing capabilities of ADCs based on their linker and payload design, and may therefore contribute to the future development of more effective ADC therapies. Citation Format: Eef F.P. Smits, Janneke J.T.M. Melis, Daphne J.F. Kluitmans, Jeffrey J. Kooijman, Mandy W.E. Drossaert - Smeets, Guido J.R. Zaman. Comparison of the bystander effects and killing capabilities of the HER2-targeting antibody drug conjugates T-DM1 and T-DXd [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A136.