In Vivo Labeling of Brain Capillary Endothelial Cells after Intravenous Injection of Monoclonal Antibodies Targeting the Transferrin Receptor

Abstract

The development of vectors for drug delivery to the central nervous system remains a major pharmaceutical challenge. Here, we have characterized the brain distribution of two monoclonal antibodies (MAbs) targeting the mouse transferrin receptor (TfR) (clones Ri7 and 8D3) compared with control IgGs after intravenous injection into mice. MAbs were fluorolabeled with either Alexa Fluor (AF) dyes 647 or 750. Intravenous injection of Ri7 or 8D3 MAb coupled with AF750 led to higher fluorescence emission in brain homogenates compared with control IgGs, indicating retention in the brain. Fluorescence microscopy analysis revealed that AF647-Ri7 signal was confined to brain cerebrovasculature, colocalizing with an antibody against collagen IV, a marker of basal lamina. Confocal microscopy analysis confirmed the delivery of injected Ri7 MAb into brain endothelial cells using the pericyte marker anti-␣-smooth muscle actin, the endothelial marker CD31, and the collagen IV antibody. No evidence of colocalization was detected with neurons or astrocytes identified using antibodies specific for neuronal nuclei or glial fibrillary acidic protein, respectively. Our data show that anti-TfR vectors injected intravenously readily accumulate into brain capillary endothelial cells, thus displaying strong drugtargeting potential.

Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics

“…Other targets that have been used for BBB-targeting of siRNA include the insulin receptor [104], and the LDL receptor [105]. Transferrin receptor targeted delivery has shown some success for protein and drug uptake into the brain [102,106] but in other cases, trapping in the brain microvasculature has been reported [107,108]. Recently, a more detailed insight into the mechanisms has revealed that the affinity (or avidity) of the peptide or protein ligand to the transferrin receptor is crucial in deciding the fate [103,106,109].…”

Brain Bioavailability of Human Intravenous Immunoglobulin and its Transport through the Murine Blood–Brain Barrier

“…Indeed, IVIg is a heterogeneous product representative of the hIgG repertoire of thousands of healthy donors 5 with highly variable amino-acid composition. 33 Thus, a fraction of these IgG molecules may cross the BBB because of characteristics such as their isoelectric point, 34 degree of hydrophobicity, amino-acid composition, or antigen specificity, 31 whereas the majority might be excluded from the brain. Thus, our data underscore the possibility that deciphering the characteristics of IVIg molecules that allows their migration into the CNS as well as the transporters involved could lead to the identification of brain-penetrant IgG and to the optimization of IVIg use in prevalent CNS disorders.…”

“…Recent studies indicate that IgG binding to transferrin or insulin receptors can access brain cells by mimicking their normal substrates. 31 This strategy was used to engineer chimeric antibodies to promote blood-to-brain transport of therapeutic (A and B) Nontransgenic (NonTg) and 3xTg-AD mice received three injections of IVIg (subchronic treatment) or equivalent volumes of vehicle (Ctrl) and were perfused with 50 mL phosphate-buffered saline (PBS) buffer followed with 50 mL 4% paraformaldehyde. Representative (A) immunohistochemistry and (B) immunofluorescent labeling of IVIg on 35-mm-thick brain sections of 4-to 9-month-old 3xTg-AD and NonTg mice (4-8 animals per group).…”

Time to Open the Blood–Brain Barrier Gate for Biologics?

“…Bivalent binding can induce dimerization or higher order oligomerization of the receptor at the cell surface and/or inside the cell and is likely to change the natural arrangement and density of the receptor. Interestingly, recent studies showed that bivalent binding of an antibody to its cognate receptor leads to trapping of the antibody inside the brain endothelial cells of the BBB [11,12]. Consequently, the fraction of the antibody that actually gets through the BBB and into the brain parenchyma is very small.…”