Abstract 2128: Uncovering the multiple therapeutic mechanisms of trastuzumab deruxtecan (T-DXd): effective extracellular payload release and immunomodulatory stimulation

Trastuzumab deruxtecan (T-DXd), also known as fam-trastuzumab-deruxtecan-nxki, is a novel HER2-targeting antibody-drug conjugate (ADC) that has shown remarkable clinical efficacy in breast cancer (BC) and other cancers with varying levels of HER2 expression. However, the mechanisms underlying its activity in HER2-low and HER2-ultralow BC, and the role of immune activation, remain poorly understood. Here, we demonstrate that T-DXd’s efficacy in HER2-low/ultralow BC is predominantly driven by the extracellular tumor-specific protease Cathepsin L (CTSL), but not Ccathepsin B (CTSB). Our in vitro and in vivo studies reveal that extracellular CTSL cleaves T-DXd’s linker, releasing the DXd payload (validated by mass spectrometry) and inducing cytotoxicity within HER2-low/ultralow tumor microenvironments. This mechanism mediates potent anti-tumor effects across multiple HER2-negative cancers. Importantly, analysis of BC biopsies and tissue microarrays from T-DXd-treated patients revealed high CTSL expression in tumor and stromal compartments, independent of HER2 levels, suggesting broad therapeutic potential. In addition to extracellular cleavage, we also found that T-DXd elicits immune activation as an anti-tumor mechanism in HER2-positive BC. The DXd payload induces immunogenic cell death, activating nearby myeloid immune cells via TLR4 and STING pathways, while its antibody backbone engages Fcγ-receptors to stimulate Antibody-Dependent Cellular Phagocytosis (ADCP). This process enhances tumor antigen uptake and drives the expansion of antigen-specific CD8+ T cells. Furthermore, DXd upregulates CD47 on tumor cells, making them more susceptible to immune clearance. Combining CD47 blockade with T-DXd significantly enhanced anti-tumor efficacy in an immune suppressive HER2-transgenic BC mouse model, promoting CD8+ T cell memory and preventing tumor recurrence post-treatment. These studies suggest that strategies targeting the CD47-SIRPa axis may enhance the ability of ADCs to elicit tumor-specific immunity, thus extending the efficacy of these agents and generating long term memory responses against non-immunogenic cancers. Citation Format: Li-Chung Tsao, John S. Wang, Xingru Ma, Sirajbir Sodhi, Joey V. Ragusa, Bushangqing Liu, Jason McBane, Tao Wang, Junping Wei, Cong-Xiao Liu, Xiao-Yi Yang, Gang-jun Lei, Ivan Spasojevic, Ping Fan, Timothy N. Trotter, Michael A. Morse, Herbert K. Lyerly, Zachary C. Hartman. Uncovering the multiple therapeutic mechanisms of trastuzumab deruxtecan (T-DXd): effective extracellular payload release and immunomodulatory stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2128.