Abstract 7467: Real-time detection of ADC internalization kinetics using a pH-sensitive fluorescent dye

Abstract BACKGROUND Monitoring antibody-drug conjugate (ADC) internalization demands highly sensitive and specific methods to differentiate surface-bound from internalized molecules. To address this need, we developed an internalization detection reagent based on pH-sensitive fluorescent dyes that enable real-time visualization of endocytosis events in live cells. This assay allows quantitative monitoring of dynamic parameters such as initiation rate, uptake duration, and total internalization, providing critical insights for understanding ADC mechanisms of action, optimizing dosing regimens, and refining administration strategies. METHOD AND MATERIALS The primary antibody (2 μg/mL) and Internalization Detection reagent (1μg/mL) were prepared in cell culture medium, mixed, and incubated at room temperature for 10 min to form the labeling complex. For suspension cells, 50 μL of cell suspension (1×106 cells/mL) was seeded into each well of a 96-well plate and combined with 50 μL of the labeling complex. For adherent cells, 5,000-10,000 cells were plated per well and allow to attach. The medium was then replaced with 50 μL of fresh medium prior to the addition of 50 μL of the labeling complex. Cells were incubated with the labeling complex for 0.5-24 h, followed by analysis via flow cytometry or high-content imaging at designated time points. RESULTS SK-BR-3 (HER2+) cells were incubated with various Anti-HER2 mAbs and our internalization detection reagent. Throughout the incubation period, there was a time-dependent increase in fluorescence across all tested primary antibodies, with signal intensity gradually increasing over time. In contrast, no positive signal was detected in the negative control cell line MDA-MB-468(HER2-). SK-BR-3(HER2+) cells were also incubated with anti-HER2 mAbs and their corresponding ADC drugs in presence of the internalization detection reagent. The intensity of the internalization detection signal showed a dose-dependent upward trend with increasing reagent concentration. The internalization detection signal for anti-HER2 antibody in SK-BR-3 cells increased gradually over the initial 7 hours, then the signal reached a plateau and maintained stable for up to 18 hours. Upon removal of the reagent and antibody mixture at the 7-hour mark, the internalization signal in the 2 nM anti-HER2 antibody group showed a gradual decline over the next 24 hours. CONCLUSION The developed internalization detection reagent enables real-time, quantitative monitoring of antibody and ADC internalization kinetics with high sensitivity, reproducibility, and specificity. The reagent demonstrates consistent performance across different targets such as HER2 and CD20, offering a robust and versatile platform for studying endocytosis mechanisms, comparing candidate antibodies, and accelerating ADC development and optimization. Citation Format: An Ouyang, Linlin Ma, Di Shi, Siwen Wang, Tianfu Zhang, Zhicheng Dong, YuehChun Hsieh. Real-time detection of ADC internalization kinetics using a pH-sensitive fluorescent dye abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7467.