Abstract
We have adapted an in vitro model of the human blood-brain barrier, the immortalized human cerebral microvascular endothelial cells (hCMEC/D3), to quantitatively measure protein transcytosis. After validating the receptor-mediated transport using transferrin, the system was used to measure transcytosis rates of antibodies directed against potential brain shuttle receptors. While an antibody to the insulin-like growth factor 1 receptor (IGF1R) was exclusively recycled to the apical compartment, the fate of antibodies to the transferrin receptor (TfR) was determined by their relative affinities at extracellular and endosomal pH. An antibody with reduced affinity at pH5.5 showed significant transcytosis, while pH-independent antibodies of comparable affinities at pH 7.4 remained associated with intracellular vesicular compartments and were finally targeted for degradation.
Highlights
Despite decades of intensive research in the labs of academic institutions and the pharmaceutical industry, the blood-brain barrier has remained a significant hurdle for treatment of CNS diseases with growing unmet medical need [1]
The most important physiological entry route for proteins into the brain is through receptor-mediated transcytosis (RMT), the exploitation of which has already been proposed for the transport of biologics into the brain [2]
Immunofluorescence using Confocal Microscopy To investigate the localization of the 128.1 antibody and the natural ligand holotransferrin, monolayers of hCMEC/D3 cells grown to confluence on collagen-coated cover slips were incubated with 5 mg/ml FITC tagged holotransferrin (Invitrogen) or 1 mg/ml 128.1, MEM-189 or the IGF-1R antibodies for 10 min following which the medium was removed and replaced with fresh medium
Summary
Despite decades of intensive research in the labs of academic institutions and the pharmaceutical industry, the blood-brain barrier has remained a significant hurdle for treatment of CNS diseases with growing unmet medical need [1]. The pathways and sorting mechanisms of transcytosis in blood-brain barrier endothelial cells are poorly understood, preventing the targeted generation of ‘‘brain shuttle’’ molecules. Another way to further our understanding on the molecular properties predisposing a protein, or an antibody, to efficient BBB passage, would be a robust in vitro transcytosis assay, enabling the screening of many antibodies and correlating their transcytosis capacity with other molecular properties like receptor specificity or affinity. Many in vitro transcytosis assays have been described in the literature (for review see [3]), published data are often not in agreement with the calculated transcytosis capacity of brain endothelial cells, and may rather represent paracellular flux than transcytosis. We found a correlation between the transcytosis capacity of the antitransferrin receptor antibodies and pH-dependence of the receptor-antibody interaction, identifying a potential new mechanism for the enhancement of transcytosis
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