Abstract
Many tools for studying the pharmacokinetics of biologics lack single-cell resolution to quantify the heterogeneous tissue distribution and subsequent therapeutic degradation in vivo. This protocol describes a dual-labeling technique using two near-infrared dyes with widely differing residualization rates to efficiently quantify in vivo therapeutic protein distribution and degradation rates at the single cell level (number of proteins/cell) via ex vivo flow cytometry and histology. Examples are shown for four biologics with varying rates of receptor internalization and degradation and a secondary dye pair for use in systems with lower receptor expression. Organ biodistribution, tissue-level confocal microscopy, and cellular-level flow cytometry were used to image the multi-scale distribution of these agents in tumor xenograft mouse models. The single-cell measurements reveal highly heterogeneous delivery, and degradation results show the delay between peak tumor uptake and maximum protein degradation. This approach has broad applicability in tracking the tissue and cellular distribution of protein therapeutics for drug development and dose determination.
Highlights
Therapeutic proteins remain one of the fastest growing areas of pharmaceutical development in the treatment of many diseases including cancer and autoimmune disorders [1,2,3]
In the case of antibody–drug conjugates (ADCs), efficacy can be enhanced by understanding the internalization/degradation and payload release at the subcellular scale, the average number and variability of payload molecules required to achieve cell death in vivo at the cellular scale [8], the number of cells in the tumor receiving a therapeutic dose at the tissue scale, and the healthy tissue exposure and resulting toxicity at the whole organ level [9]
DDAO/BoDIPY-FL, approximates the intact protein, since it is cleared upon degradation, while IRDye/AF647 approximates the cumulative uptake in the cell, since it is ‘trapped’ within the cell
Summary
Therapeutic proteins remain one of the fastest growing areas of pharmaceutical development in the treatment of many diseases including cancer and autoimmune disorders [1,2,3]. This method provides robust results for tracking organ biodistribution and degradation at the whole animal and organ level (e.g., [14]) Motivated by this approach, we measured the residualization properties of NIR fluorophores, identifying both residualizing and non-residualizing dyes [15]. The current protocol is similar in concept to the radiolabeling approach but uses NIR fluorescence to increase spatial and temporal resolution This allows measurement of degradation and distribution across multiple length scales using the high spatial resolution of fluorescence and ability to quantify kinetic rates, such as degradation at the cellular level in vivo while reducing safety concerns, time/half-life constraints, and expense of radioactivity. Multiple length scales can be analyzed in vivo for the same animal, providing insight into heterogeneity and inter-animal variability
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