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

Current 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 endothelia...

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Bibliographic Details
Published in:Journal of cerebral blood flow and metabolism 2018-04, Vol.38 (4), p.727-740
Main Authors: Haqqani, Arsalan S, Delaney, Christie E, Brunette, Eric, Baumann, Ewa, Farrington, Graham K, Sisk, William, Eldredge, John, Ding, Wen, Tremblay, Tammy-Lynn, Stanimirovic, Danica B
Format: Article
Language:English
Subjects:
Animals
Antibodies - cerebrospinal fluid
Antibodies - metabolism
Antigens, CD
Blood-Brain Barrier - metabolism
Blood-Brain Barrier - physiology
Endosomes - physiology
Endothelial Cells - metabolism
Lysosomes - metabolism
Mass Spectrometry
Original
Rats
Rats, Sprague-Dawley
Receptors, Transferrin
Transcytosis - physiology
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Summary:Current 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.
ISSN:0271-678X
1559-7016
DOI:10.1177/0271678X17740031