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

Abstract

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 in vivo 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 in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (K d from 5 to174 nM) 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 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co‐localized with late‐endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co‐localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency.Open Practices Open Science: This manuscript was awarded with the Open Materials Badge.For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.

Antibody screening using a human iPSC‐based blood‐brain barrier model identifies antibodies that accumulate in the CNS

“…In particular, scFv 46.1 had a unique uptake pattern, namely there were few cytoplasmic 46.1-containing structures and most antibody could be found in puncta at the cell-cell junctions, seemingly distinct from recycling or degradative compartments typical of targeted BBB receptors like the TfR. 43,44 Moreover, confocal microscopy indicated the rapid, vectorial transport of 46.1 from the apical membrane to the basolateral junctions. While the identification of the receptor targeted by 46.1 and the elucidation of the detailed intracellular transport mechanism are the focus of future work, we have not observed any literature describing such an antibody transport profile at the BBB.…”

Brain Shuttle Neprilysin reduces central Amyloid-β levels

“…The short plasma half-life of the sFab OX26-NEP construct is either due to target-mediated drug deposition (TMDD) through TfR on various peripheral cells or a clearance through the glycol-profile on NEP. It was previously shown that a reduction in affinity of a bivalent OX26 was sufficient to prolong the plasma half-life and improve the brain exposure [20]. The longer plasma half-life is likely explaining at least in part the improved brain expose as it provides TfR binders over an extended time period circulating in the peripheral compartment.…”

Smart Strategies for Therapeutic Agent Delivery into Brain across the Blood–Brain Barrier Using Receptor-Mediated Transcytosis

“…Nonetheless, RmAb158-scFv8D3 was preferred due to the efficiency of Aβ reduction. 23) OX26 is the rat TfR bivalent antibody. Its variants, OX26 5 , OX26 76 , OX26 108 , and OX26 174 possess dissociation constant (K d ) values of 5, 76, 108, and 174 nM, against the same single epitope on TfR, respectively.…”