Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood-brain barrier in vitro is dependent on its binding affinity.

The blood-brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their invivo evaluation demonstrated that either low affinity or monovalent receptor binding re-directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter-study comparisons. In this study, the efficiency of transcytosis invitro and intracellular trafficking in endosomal compartments were evaluated in an invitro BBB model for affinity variants (K-d from 5 to174nM) of the rat TfR-binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics-selected reaction monitoring and fluorescent imaging with markers of early- and late endosomes. The OX26 variants with affinities of 76 and 108nM showed improved trancytosis (P-app values) across the invitro BBB model compared with a 5nM OX26. Although 40% of the 5nM OX26 and 35% of TfR co-localized with late-endosome/lysosome compartment, 76 and 108nM affinity variants showed lower amounts in lysosomes and a predominant co-localization with early endosome markers. The study li nks bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency. Open PracticesOpen Science: This manuscript was awarded with the Open Materials Badge. For more information see: