Quantitative live-imaging platform to probe the spatio-temporal internalization and lysosomal degradation of therapeutic antibodies and ADCs in cells

The therapeutic efficacy of antibody-drug conjugates (ADCs) is governed not only by target binding but also by the efficiency of receptor-mediated internalization, intracellular trafficking, and lysosomal degradation. However, most conventional assays provide static or endpoint measurements, limiting their ability to resolve these dynamic, multistep processes. Here, we report a quantitative, dual-probe live-cell imaging platform that simultaneously tracks antibody internalization and lysosomal degradation with spatiotemporal resolution. By combining an internalization tracer with a cathepsin-activated fluorogenic sensor, this assay enables direct, real-time measurement of lysosomal routing following cellular uptake. Using trastuzumab and clinically approved HER2-targeting ADCs as model systems, we demonstrate that internalization and lysosomal degradation are temporally uncoupled and occur with distinct kinetics. Moreover, ADCs sharing the same antibody backbone exhibited markedly different processing profiles, highlighting the influence of linker chemistry and payload properties on their intracellular fate. These results underscore the importance of evaluating ADCs as integrated molecular systems rather than as modular components. This platform provides a scalable and mechanistically informative tool for ADC development, enabling candidate ranking, linker screening, and rational payload - linker optimization based on functional intracellular behavior rather than uptake alone. As such, it offers a framework for data-driven design of next-generation ADCs with improved therapeutic index.