Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier

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DOIResolve DOI: http://doi.org/10.1177/0271678X17740031
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TypeArticle
Journal titleJournal of Cerebral Blood Flow & Metabolism
ISSN0271-678X
1559-7016
Pages0271678X1774003
Subjectintracellular trafficking; blood–brain barrier; mass spectrometry; selected reaction monitoring
AbstractCurrent methods for examining antibody trafficking are either non-quantitative such as immunocytochemistry or require antibody labeling with tracers. We have developed a multiplexed quantitative method for antibody ‘tracking’ in endosomal compartments of brain endothelial cells. Rat brain endothelial cells were co-incubated with blood-brain barrier (BBB)-crossing FC5, monovalent FC5Fc or bivalent FC5Fc fusion antibodies and control antibodies. Endosomes were separated using sucrose-density gradient ultracentrifugation and analyzed using multiplexed mass spectrometry to simultaneously quantify endosomal markers, receptor-mediated transcytosis (RMT) receptors and the co-incubated antibodies in each fraction. The quantitation showed that markers of early endosomes were enriched in high-density fractions (HDF), whereas markers of late endosomes and lysosomes were enriched in low-density fractions (LDF). RMT receptors, including transferrin receptor, showed a profile similar to that of early endosome markers. The in vitro BBB transcytosis rates of antibodies were directly proportional to their partition into early endosome fractions of brain endothelial cells. Addition of the Fc domain resulted in facilitated antibody ‘redistribution’ from LDF into HDF and additionally into multivesicular bodies (MVB). Sorting of various FC5 antibody formats away from late endosomes and lysosomes and into early endosomes and a subset of MVB results in increased antibody transcytosis at the abluminal side of the BBB.
Publication date
PublisherSAGE
LanguageEnglish
AffiliationHuman Health Therapeutics; National Research Council Canada
Peer reviewedYes
NPARC number23002491
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Record identifier33302487-9987-42d0-bb7f-3b59445d5f72
Record created2017-11-16
Record modified2017-11-16
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