The precise mechanisms governing the central distribution of macromolecules from the cerebrospinal fluid (CSF) to the brain and spinal cord remain poorly understood, despite their importance for physiological processes such as antibody trafficking for central immune surveillance, as well as several ongoing intrathecal clinical trials. In the present study, we clarify how IgG and smaller single-domain antibodies (sdAb) distribute throughout the whole brain in a size-dependent manner after intrathecal infusion in rats using ex vivo fluorescence and in vivo three-dimensional magnetic resonance imaging. Antibody distribution was characterized by diffusion at the brain surface and widespread distribution to deep brain regions along the perivascular spaces of all vessel types, with sdAb accessing a four- to seven-fold greater brain area than IgG. Perivascular transport involved blood vessels of all caliber and putative smooth muscle and astroglial basement membrane compartments. Perivascular access to smooth muscle basement membrane compartments also exhibited size-dependence. Electron microscopy was used to show stomata on leptomeningeal coverings of blood vessels in the subarachnoid space as potential access points allowing substances in the CSF to enter the perivascular space. Osmolyte co-infusion significantly enhanced perivascular access of the larger antibody from the CSF, with intrathecal 0.75 m mannitol increasing the number of perivascular profiles per slice area accessed by IgG by ∼50%. The results of the present study reveal potential distribution mechanisms for endogenous IgG, which is one of the most abundant proteins in the CSF, as well as provide new insights with respect to understanding and improving the drug delivery of macromolecules to the central nervous system via the intrathecal route.
The blood-brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic "arm" is combined with a BBB-transcytosing arm can significantly enhance their brain delivery. The BBB-permeable single-domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single-domain antibody phage display library. In this study, FC5 was engineered as a mono- and bivalent fusion with the human Fc domain to optimize it as a modular brain delivery platform. In vitro studies demonstrated that the bivalent fusion of FC5 with Fc increased the rate of transcytosis (Papp) across brain endothelial monolayer by 25% compared with monovalent fusion. Up to a 30-fold enhanced apparent brain exposure (derived from serum and cerebrospinal fluid pharmacokinetic profiles) of FC5- compared with control domain antibody-Fc fusions after systemic dosing in rats was observed. Systemic pharmacological potency was evaluated in the Hargreaves model of inflammatory pain using the BBB-impermeable neuropeptides dalargin and neuropeptide Y chemically conjugated with FC5-Fc fusion proteins. Improved serum pharmacokinetics of Fc-fused FC5 contributed to a 60-fold increase in pharmacological potency compared with the single-domain version of FC5; bivalent and monovalent FC5 fusions with Fc exhibited similar systemic pharmacological potency. The study demonstrates that modular incorporation of FC5 as the BBB-carrier arm in bispecific antibodies or antibody-drug conjugates offers an avenue to develop pharmacologically active biotherapeutics for CNS indications.
Receptor mediated transcytosis harnessing the cellular uptake and transport of natural ligands across the blood-brain barrier (BBB) has been identified as a means for antibody delivery to the CNS. In this study, we characterized bispecific antibodies in which a BBB-crossing antibody fragment FC5 was used as a BBB carrier. Cargo antibodies were either a high-affinity, selective antibody antagonist of the metabotropic glutamate receptor-1 (BBB-mGluR1), a widely abundant CNS target, or an IgG that does not bind the CNS target (BBB-NiP). Both BBB-NiP and BBB-mGluR1 demonstrated a similar 20-fold enhanced rate of transcytosis across an in vitro BBB model compared with mGluR1 IgG fused to a control antibody fragment. All 3 bispecific antibodies exhibited identical pharmacokinetics in vivo Comparative assessment of BBB-NiP and BBB-mGluR1 revealed that, whereas their serum pharmacokinetics and BBB penetration were identical, their central disposition (brain levels) and elimination (cerebrospinal fluid levels) were widely different, due to central target-mediated removal of the mGluR1-engaging antibody. Central mGluR1 target engagement after systemic administration was demonstrated by a dose-dependent inhibition of mGluR-1-mediated thermal hyperalgesia and by colocalization of the antibody with thalamic neurons involved in mGluR1-mediated pain processing. We demonstrate the feasibility of targeting central G-protein-coupled receptors using a BBB-crossing bispecific antibody approach and emerging principles that govern brain distribution and disposition of these antibodies. These data will be important for designing safe and selective CNS antibody therapeutics.-Webster, C. I., Caram-Salas, N., Haqqani, A. S., Thom, G., Brown, L., Rennie, K., Yogi, A., Costain, W., Brunette, E., Stanimirovic, D. B. Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.
FC5 and FC44 are single-domain antibodies (VHHs), selected by functional panning of phage-display llama VHH library for their ability to internalize human brain endothelial cells (BEC) and to transmigrate the in vitro BBB model. Quantification of brain delivery of FC5 and FC44 in vivo was challenging using classical methods because of their short plasma half-life and their loss of functionality with radioactive labeling. A highly sensitive (detection limit <2 ng/mL) and specific SRM-ILIS method to detect and quantify unlabeled VHHs in multiplexed assays was developed and applied to comparatively evaluate brain delivery of FC5 and FC44, and two control VHHs, EG2 and A20.1. FC5 and FC44 compared to control VHHs demonstrated significantly (p < 0.01) enhanced transport (50-100-fold) across rat in vitro BBB model as well as in vivo brain targeting assessed by optical imaging. The multiplexed SRM-ILIS analyses of plasma and CSF levels of codosed VHHs demonstrated that while all 4 VHHs have similar blood pharmacokinetics, only FC5 and FC44 show elevated CSF levels, suggesting that they are potential novel carriers for delivery of drugs and macromolecules across the BBB.
Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood-brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. Constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of antibodies or protein ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter SLC3A2/CD98hc and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, brain parenchyma and peripheral organs of the mouse and the human using RNA-seq approach. IGF1R, INSR and LRP8 were highly enriched in mouse brain microvessels compared to peripheral tissues. In human brain microvessels only INSR was enriched compared to either the brain or the lung. The expression levels of SLC2A1, LRP1, IGF1R, LRP8 and TFRC were significantly higher in the mouse compared to human brain microvessels. The protein expression of these receptors analyzed by Western blot and immunofluorescent staining of the brain microvessels correlated with their transcript abundance. This study provides a molecular transcriptomics map of key RMT receptors in mouse and human brain microvessels and peripheral tissues, important to translational studies of biodistribution, efficacy and safety of antibodies developed against these receptors.
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