Abstract A059: Uncovering Bystander Killing Mechanisms of Trastuzumab Deruxtecan (T-DXd): Effective Extracellular Payload Release via Cathepsin L in HER2-low Breast Cancer.

Trastuzumab deruxtecan (T-DXd), or fam-trastuzumab-deruxtecan-nxki, is a novel antibody-drug-conjugate (ADC) targeting HER2 that has demonstrated profound clinical efficacy across HER2-positive breast cancer (BC), including in HER2-low and HER2 “ultra-low” BC. However, the precise mechanism underlying its efficacy in HER2-low BC is still poorly understood. To determine the mechanism of DXd release and cytotoxicity, we compared in vivo T-DXd treatment of human BC xenografts with varying HER2 expression profiles, coupled with in vitro bystander killing analysis of co-cultured HER2-negative cells, and liquid chromatography–mass spectrometry of DXd payload levels in the tumor microenvironment. Our findings revealed that the efficacy of T-DXd in HER2-low and HER2-negative BC is mediated by extracellular proteases, specifically cathepsin L (CTSL), but not cathepsin B, within the tumor microenvironment (TME). Unlike traditional ADCs such as trastuzumab emtansine (T-DM1), which contain a non-cleavable linker and require efficient intracellular endocytosis for payload release and cytotoxicity, HER2-low and HER2 “ultra-low” BC demonstrated limited ADC uptake. Despite this, these cancer types exhibited strong sensitivity to T-DXd in vivo, but not to T-DM1. Critically, using CTSL overexpression and knockout strategies on HER2 “ultra-low” MDA-MB-231 cells, we found that extracellular CTSL released by tumor cells mediates the proteolytic cleavage of T-DXd’s linker. This enables broader DXd payload release and cytotoxicity, bypassing traditional ADC-resistance mechanisms, including HER2 downregulation and resistance to ADC internalization. Our studies reveal a crucial payload release mechanism of T-DXd within the TME, mediated by extracellular CTSL cleavage of T-DXd’s tetrapeptide linker. This mechanism likely contributes to the broad efficacy of T-DXd in HER2-low and even HER2 “ultra-low” BC over T-DM1. Collectively, our findings suggest that CTSL and other extracellular proteases in the TME may serve as important biomarkers for predicting T-DXd efficacy.Citation Format: Li-Chung Tsao, John S. Wang, Joey Ragusa, Bushangqing Liu, Jason McBane, Ivan Spasojevic, Ping Fan, Timothy N. Trotter, Herbert Kim Lyerly, Zachary C Hartman. Uncovering Bystander Killing Mechanisms of Trastuzumab Deruxtecan (T-DXd): Effective Extracellular Payload Release via Cathepsin L in HER2-low Breast Cancer. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A059.