Abstract LB-264: Autophagy modulates intracellular dynamics of [fam-] trastuzumab deruxtecan (DS-8201a), a novel HER2 antibody-drug conjugate (HER2-ADC)

Background of the study: [fam-] trastuzumab deruxtecan (DS-8201a) is a HER2-targeting antibody-drug conjugate (ADC) with a humanized anti-HER2 monoclonal antibody attached to a topoisomerase I inhibitor payload by a peptide-based cleavable linker. [fam-] trastuzumab deruxtecan has been shown to be highly effective in preclinical and clinical studies. In addition to payload potency, drug-to-antibody ratio and linker stability, the effectiveness of the ADC is expected to be defined by intracellular molecular dynamics of the ADC/HER2 complex such as internalization activity, translocation to lysosomes and lysosomal enzyme-mediated linker cleavage. However, the machinery of ADC dynamics has not been studied quantitatively. Methods: First, in vitro quantitative assays to study [fam-] trastuzumab deruxtecan intracellular dynamics were established. Second, differentially expressed gene (DEG) analysis among lysosome trafficking-high/low HER2 expressing cell lines was performed to understand intracellular dynamics. Third, functional relevance of the gene extracted from DEG analysis was assessed by siRNA-mediated in vitro knockdown study. Finally, to comprehend contribution of lysosome activity to intracellular dynamics of [fam-] trastuzumab deruxtecan, in vitro lysosome inhibition studies were performed using bafilomycin A1. Then, impact of autophagy activation on the dynamics was studied with mTOR inhibitor everolimus. Results: [Fam-] trastuzumab deruxtecan bound to the cell surface of HER2 expressing cell lines in expression level-dependent manner. Lysosome trafficking study revealed that two cell lines, JIMT1 and MDA-MB-453, showed high trafficking activity for the modest HER2 expression. DEG analysis result exhibited that lysosome-related genes were significantly upregulated in the two cell lines, which suggested involvement of lysosome activity in ADC dynamics. Lysosome trafficking of [fam-] trastuzumab deruxtecan was hampered by knockdown of such genes. Lysosome is supposed to be the endpoint of ADC processing and its activity itself might affect the ADC activity. Inhibition of lysosome activity by bafilomycin A1 resulted in weakening ADC dynamics in the cell lines. In addition, contribution of lysosome activation via autophagy stimulation using everolimus resulted in activation of lysosome trafficking of the ADC in MDA-MB-453. Conclusion: In vitro quantitative analyses of intracellular [fam-] trastuzumab deruxtecan dynamics revealed that lysosome related activity played important roles in the ADC dynamics. As lysosome activity is known to be modulated by autophagy mechanism, influence of autophagy activation to ADC dynamics was elucidated in this study. The results obtained suggest a scientific rationale for combo treatment of ADC and autophagy activator as well as the advantage of ADC on autophagy-active cancers. Citation Format: Manabu Abe, Motoko Nagata, Kumiko Koyama, Satoru Yasuda, Tsuyoshi Hirata, Yusuke Kuwahara, Koichiro Inaki, Naomi Kasanuki, Megumi Minami, Katsunobu Hagihara, Kenichi Wakita, Eric Slosberg, Masato Murakami. Autophagy modulates intracellular dynamics of [fam-] trastuzumab deruxtecan (DS-8201a), a novel HER2 antibody-drug conjugate (HER2-ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-264.