Therapeutic antibody fragments, such as adv, hold promise for treating central nervous system (CNS) disorders but must overcome the restrictive blood-brain barrier (BBB). One of the best-characterized examples is the 8D3 Fab, a brain-penetrating fragment that binds the transferrin receptor (TfR) and is known to undergo receptor-mediated transcytosis. Here, we employ state-of-the-art four-dimensional (4D) imaging to directly detect and quantify single endocytic and intracellular trafficking events of the 8D3 Fab in murine endothelial cell models. By combining high-resolution imaging with machine learning-driven analysis, we investigate how binding kinetics, pH sensitivity, and trafficking efficiency influence antibody fragment behavior inside endothelial cells, while quantitatively estimating copy numbers per vesicle, resolving recycling, degradation, and release pathways, and assessing the impact of albumin binding on transport efficiency. These insights refine our understanding of TfR-targeted brain-penetrating antibodies, inform the design of next-generation therapeutic formats, and establish methodologies for more accurate evaluation of BBB transport mechanisms.
Margaritaki et al. (Sun,) studied this question.