Differential Expression of ABC Transporter Genes in Brain Vessels vs. Peripheral Tissues and Vessels from Human, Mouse and Rat.

BACKGROUND: ATP-binding cassette (ABC) transporters comprise a superfamily of genes encoding membrane proteins with nucleotide-binding domains (NBD). These transporters, including drug efflux across the blood-brain barrier (BBB), carry a variety of substrates through plasma membranes against substrate gradients, fueled by hydrolyzing ATP. The expression patterns/enrichment of ABC transporter genes in brain microvessels compared to peripheral vessels and tissues are largely uncharacterized. METHODS: In this study, the expression patterns of ABC transporter genes in brain microvessels, peripheral tissues (lung, liver and spleen) and lung vessels were investigated using RNA-seq and WesTM analyses in three species: human, mouse and rat. RESULTS: The study demonstrated that ABC drug efflux transporter genes (including ABCB1, ABCG2, ABCC4 and ABCC5) were highly expressed in isolated brain microvessels in all three species studied; the expression of ABCB1, ABCG2, ABCC1, ABCC4 and ABCC5 was generally higher in rodent brain microvessels compared to those of humans. In contrast, ABCC2 and ABCC3 expression was low in brain microvessels, but high in rodent liver and lung vessels. Overall, most ABC transporters (with the exception of drug efflux transporters) were enriched in peripheral tissues compared to brain microvessels in humans, while in rodent species, additional ABC transporters were found to be enriched in brain microvessels. CONCLUSIONS: This study furthers the understanding of species similarities and differences in the expression patterns of ABC transporter genes; this is important for translational studies in drug development. In particular, CNS drug delivery and toxicity may vary among species depending on their unique profiles of ABC transporter expression in brain microvessels and BBB.

Epitope mapping of a blood-brain barrier crossing antibody targeting the cysteine-rich region of IGF1R using hydrogen-exchange mass spectrometry enabled by electrochemical reduction.

Pathologies of the central nervous system impact a significant portion of our population, and the delivery of therapeutics for effective treatment is challenging. The insulin-like growth factor-1 receptor (IGF1R) has emerged as a target for receptor-mediated transcytosis, a process by which antibodies are shuttled across the blood-brain barrier (BBB). Here, we describe the biophysical characterization of VHH-IR4, a BBB-crossing single-domain antibody (sdAb). Binding was confirmed by isothermal titration calorimetry and an epitope was highlighted by surface plasmon resonance that does not overlap with the IGF-1 binding site or other known BBB-crossing sdAbs. The epitope was mapped with a combination of linear peptide scanning and hydrogen-deuterium exchange mass spectrometry (HDX-MS). IGF1R is large and heavily disulphide bonded, and comprehensive HDX analysis was achieved only through the use of online electrochemical reduction coupled with a multiprotease approach, which identified an epitope for VHH-IR4 within the cysteine-rich region (CRR) of IGF1R spanning residues W244-G265. This is the first report of an sdAb binding the CRR. We show that VHH-IR4 inhibits ligand induced auto-phosphorylation of IGF1R and that this effect is mediated by downstream conformational effects. Our results will guide the selection of antibodies with improved trafficking and optimized IGF1R binding characteristics.

Brain Delivery of IGF1R5, a Single-Domain Antibody Targeting Insulin-like Growth Factor-1 Receptor.

The ability of drugs and therapeutic antibodies to reach central nervous system (CNS) targets is greatly diminished by the blood-brain barrier (BBB). Receptor-mediated transcytosis (RMT), which is responsible for the transport of natural protein ligands across the BBB, was identified as a way to increase drug delivery to the brain. In this study, we characterized IGF1R5, which is a single-domain antibody (sdAb) that binds to insulin-like growth factor-1 receptor (IGF1R) at the BBB, as a ligand that triggers RMT and could deliver cargo molecules that otherwise do not cross the BBB. Surface plasmon resonance binding analyses demonstrated the species cross-reactivity of IGF1R5 toward IGF1R from multiple species. To overcome the short serum half-life of sdAbs, we fused IGF1R5 to the human (hFc) or mouse Fc domain (mFc). IGF1R5 in both N- and C-terminal mFc fusion showed enhanced transmigration across a rat BBB model (SV-ARBEC) in vitro. Increased levels of hFc-IGF1R5 in the cerebrospinal fluid and vessel-depleted brain parenchyma fractions further confirmed the ability of IGF1R5 to cross the BBB in vivo. We next tested whether this carrier was able to ferry a pharmacologically active payload across the BBB by measuring the hypothermic and analgesic properties of neurotensin and galanin, respectively. The fusion of IGF1R5-hFc to neurotensin induced a dose-dependent reduction in the core temperature. The reversal of hyperalgesia by galanin that was chemically linked to IGF1R5-mFc was demonstrated using the Hargreaves model of inflammatory pain. Taken together, our results provided a proof of concept that appropriate antibodies, such as IGF1R5 against IGF1R, are suitable as RMT carriers for the delivery of therapeutic cargos for CNS applications.

Neuroprotection against supra-lethal 'stroke in a dish' insults by an anti-excitotoxic receptor antagonist cocktail.

The goal of this study was to identify cocktails of drugs able to protect cultured rodent cortical neurons against increasing durations of oxygen-glucose deprivation (OGD). As expected, a cocktail composed of an NMDA and AMPA receptor antagonists and a voltage gated Ca2+ channel blocker (MK-801, CNQX and nifedipine, respectively) provided complete neuroprotection against mild OGD. Increasingly longer durations of OGD necessitated increasing the doses of MK-801 and CNQX, until these cocktails ultimately failed to provide neuroprotection against supra-lethal OGD, even at maximal drug concentrations. Surprisingly, supplementation of any of these cocktails with blockers of TRPM7 channels for increasing OGD durations was not neuroprotective, unless these blockers possessed the ability to inhibit NMDA receptors. Supplementation of the maximally effective cocktail with other NMDA receptor antagonists augmented neuroprotection, suggesting insufficient NMDAR blockade by MK-801. Substitution of MK-801 in cocktails with high concentrations of a glycine site NMDA receptor antagonist caused the greatest improvements in neuroprotection, with the more potent SM-31900 superior to L689,560. Substitution of CQNX in cocktails with AMPA receptor antagonists at high concentrations also improved neuroprotection, particularly with the combination of SYM2206 and NBQX. The most neuroprotective cocktail was thus composed of SM-31900, SYM2206, NBQX, nifedipine and the antioxidant trolox. Thus, the cumulative properties of antagonist potency and concentration in a cocktail dictate neuroprotective efficacy. The central target of supra-lethal OGD is excitotoxicity, which must be blocked to the greatest extent possible to minimize ion influx.

Targeting insulin-like growth factor-1 receptor (IGF1R) for brain delivery of biologics.

The blood-brain barrier (BBB) prevents the majority of drugs from crossing into the brain and reaching neurons. To overcome this challenge, safe and non-invasive technologies targeting receptor-mediated pathways have been developed. In this study, three single-domain antibodies (sdAbs; IGF1R3, IGF1R4, and IGF1R5) targeting the extracellular domain of the human insulin-like growth factor-1 receptor (IGF1R), generated by llama immunization, showed enhanced transmigration across the rat BBB model (SV-ARBEC) in vitro. The rate of brain uptake of these sdAbs fused to mouse Fc (sdAb-mFc) in vivo was estimated using the fluorescent in situ brain perfusion (ISBP) technique followed by optical brain imaging and distribution volume evaluation. Compared to the brains perfused with the negative control A20.1-mFc, the brains perfused with anti-IGF1R sdAbs showed a significant increase of the total fluorescence intensity (~2-fold, p < .01) and the distribution volume (~4-fold, p < .01). The concentration curve for IGF1R4-mFc demonstrated a linear accumulation plateauing at approximately 400 µg (~1 µM), suggesting a saturable mechanism of transport. Capillary depletion and mass spectrometry analyses of brain parenchyma post-ISBP confirmed the IGF1R4-mFc brain uptake with ~25% of the total amount being accumulated in the parenchymal fraction in contrast to undetectable levels of A20.1-mFc after a 5-min perfusion protocol. Systemic administration of IGF1R4-mFc fused with the non-BBB crossing analgesic peptide galanin (2 and 5 mg/kg) induced a dose-dependent suppression of thermal hyperalgesia in the Hargreaves pain model. In conclusion, novel anti-IGF1R sdAbs showed receptor-mediated brain uptake with pharmacologically effective parenchymal delivery of non-permeable neuroactive peptides.

Defining the epitope of a blood-brain barrier crossing single domain antibody specific for the type 1 insulin-like growth factor receptor.

Ligand-activated signaling through the type 1 insulin-like growth factor receptor (IGF1R) is implicated in many physiological processes ranging from normal human growth to cancer proliferation and metastasis. IGF1R has also emerged as a target for receptor-mediated transcytosis, a transport phenomenon that can be exploited to shuttle biotherapeutics across the blood-brain barrier (BBB). We employed differential hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) to characterize the interactions of the IGF1R ectodomain with a recently discovered BBB-crossing single-domain antibody (sdAb), VHH-IR5, in comparison with IGF-1 binding. HDX-MS confirmed that IGF-1 induced global conformational shifts in the L1/FnIII-1/-2 domains and α-CT helix of IGF1R. In contrast, the VHH-IR5 sdAb-mediated changes in conformational dynamics were limited to the α-CT helix and its immediate vicinity (L1 domain). High-resolution NMR spectroscopy titration data and linear peptide scanning demonstrated that VHH-IR5 has high-affinity binding interactions with a peptide sequence around the C-terminal region of the α-CT helix. Taken together, these results define a core linear epitope for VHH-IR5 within the α-CT helix, overlapping the IGF-1 binding site, and suggest a potential role for the α-CT helix in sdAb-mediated transcytosis.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human.

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.

Serum albumin-binding VH Hs with variable pH sensitivities enable tailored half-life extension of biologics.

Prolonged serum half-life is required for the efficacy of most protein therapeutics. One strategy for half-life extension is to exploit the long circulating half-life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single-domain antibodies (VH Hs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half-lives of multiple proteins in rats from minutes to days. We serendipitously identified an additional VH H (M75) that is naturally pH-sensitive: at endosomal pH, binding affinity for human serum albumin (HSA) was dramatically weakened and binding to rat serum albumin (RSA) was undetectable. Domain mapping revealed that M75 bound to HSA domain 1 and 2. Moreover, alanine scanning of HSA His residues suggested a critical role for His247, located in HSA domain 2, in M75 binding and its pH dependence. Isothermal titration calorimetry experiments were suggestive of proton-linked binding of M75 to HSA, with differing binding enthalpies observed for full-length HSA and an HSA domain 1-domain 2 fusion protein in which surface-exposed His residues were substituted with Ala. M75 conferred moderate half-life extension in rats, from minutes to hours, likely due to rapid dissociation from RSA during FcRn-mediated endosomal recycling in tandem with albumin conformational changes induced by M75 binding that prevented interaction with FcRn. Humanized VH Hs maintained in vivo half-life extension capabilities. These VH Hs represent a new set of tools for extending protein therapeutic half-life and one (M75) demonstrates a unique pH-sensitive binding interaction that can be exploited to achieve modest in vivo half-life.

Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood-brain barrier in vitro is dependent on its binding affinity.

The blood-brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re-directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter-study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (Kd from 5 to174 nM) of the rat TfR-binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics-selected reaction monitoring and fluorescent imaging with markers of early- and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co-localized with late-endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co-localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.

Enhanced Delivery of Galanin Conjugates to the Brain through Bioengineering of the Anti-Transferrin Receptor Antibody OX26.

The blood-brain barrier (BBB) is a formidable obstacle for brain delivery of therapeutic antibodies. However, antibodies against the transferrin receptor (TfR), enriched in brain endothelial cells, have been developed as delivery carriers of therapeutic cargoes into the brain via a receptor-mediated transcytosis pathway. In vitro and in vivo studies demonstrated that either a low-affinity or monovalent binding of these antibodies to the TfR improves their release on the abluminal side of the BBB and target engagement in brain parenchyma. However, these studies have been performed with mouse-selective TfR antibodies that recognize different TfR epitopes and have varied binding characteristics. In this study, we evaluated serum pharmacokinetics and brain and CSF exposure of the rat TfR-binding antibody OX26 affinity variants, having KDs of 5 nM, 76 nM, 108 nM, and 174 nM, all binding the same epitope in bivalent format. Pharmacodynamic responses were tested in the Hargreaves chronic pain model after conjugation of OX26 affinity variants with the analgesic and antiepileptic peptide, galanin. OX26 variants with affinities of 76 nM and 108 nM showed enhanced brain and cerebrospinal fluid (CSF) exposure and higher potency in the Hargreaves model, compared to a 5 nM affinity variant; lowering affinity to 174 nM resulted in prolonged serum pharmacokinetics, but reduced brain and CSF exposure. The study demonstrates that binding affinity optimization of TfR-binding antibodies could improve their brain and CSF exposure even in the absence of monovalent TfR engagement.

Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport and osmotic enhancement of delivery.

KEY POINTS: It is unclear precisely how macromolecules (e.g. endogenous proteins and exogenous immunotherapeutics) access brain tissue from the cerebrospinal fluid (CSF). We show that transport at the brain-CSF interface involves a balance between Fickian diffusion in the extracellular spaces at the brain surface and convective transport in perivascular spaces of cerebral blood vessels. Intrathecally-infused antibodies exhibited size-dependent access to the perivascular spaces and tunica media basement membranes of leptomeningeal arteries. Perivascular access and distribution of full-length IgG could be enhanced by intrathecal co-infusion of hyperosmolar mannitol. Pores or stomata present on CSF-facing leptomeningeal cells ensheathing blood vessels in the subarachnoid space may provide unique entry sites into the perivascular spaces from the CSF. These results illuminate new mechanisms likely to govern antibody trafficking at the brain-CSF interface with relevance for immune surveillance in the healthy brain and insights into the distribution of therapeutic antibodies. ABSTRACT: 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.

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 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.

Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.

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.

Failure and rescue of preconditioning-induced neuroprotection in severe stroke-like insults.

Preconditioning is a well established neuroprotective modality. However, the mechanism and relative efficacy of neuroprotection between diverse preconditioners is poorly defined. Cultured neurons were preconditioned by 4-aminopyridine and bicuculline (4-AP/bic), rendering neurons tolerant to normally lethal (sufficient to kill most neurons) oxygen-glucose deprivation (OGD) or a chemical OGD-mimic, ouabain/TBOA, by suppression of extracellular glutamate (glutamateex) elevations. However, subjecting preconditioned neurons to longer-duration supra-lethal insults caused neurotoxic glutamateex elevations, thereby identifying a 'ceiling' to neuroprotection. Neuroprotective 'rescue' of neurons could be obtained by administration of an NMDA receptor antagonist, MK-801, just before glutamateex rose during these supra-lethal insults. Next, we evaluated if these concepts of glutamateex suppression during lethal OGD, and a neuroprotective ceiling requiring MK-801 rescue under supra-lethal OGD, extended to the preconditioning field. In screening a panel of 42 diverse putative preconditioners, neuroprotection against normally lethal OGD was observed in 12 cases, which correlated with glutamateex suppression, both of which could be reversed, either by the inclusion of a glutamate uptake inhibitor (TBOA, to increase glutamateex levels) during OGD or by exposure to supra-lethal OGD. Administrating MK-801 during the latter stages of supra-lethal OGD again rescued neurons, although to varying degrees dependent on the preconditioning agent. Thus, 'stress-testing' against the harshest ischemic-like insults yet tested identifies the most efficacious preconditioners, which dictates how early MK-801 needs to be administered during the insult in order to maintain neuroprotection. Preconditioning delays a neurotoxic rise in glutamateex levels, thereby 'buying time' for acute anti-excitotoxic pharmacologic rescue.

A novel platform for engineering blood-brain barrier-crossing bispecific biologics.

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.

Blood-brain barrier transport of amyloid beta peptides in efflux pump knock-out animals evaluated by in vivo optical imaging.

BACKGROUND: Aß transport (flux) across the blood-brain barrier (BBB) is thought to contribute to the pathogenesis of Alzheimer's disease as well as to elimination of toxic amyloid from the brain by immunotherapy. Several BBB transporters have been implicated in Aß exchange between brain parenchyma and the circulation, including efflux transporters P-glycoprotein/ABCB1 and BCRP/ABCG2. Here we describe an application of in vivo optical imaging methods to study Aß transport across the BBB in wild-type or animals deficient in specific efflux transporters. METHODS/DESIGN: Synthetic human Aß1-40 or scrambled Aß40-1 peptides were labeled with the near-infrared fluorescent tracer, Cy5.5. The free tracer or Cy5.5-labeled peptides were injected intravenously into Abcb1-KO or Abcg2-KO mice or their corresponding wild-type controls. The animals were imaged prospectively at different time points over a period of 8 hours using eXplore Optix small animal imager. At the end of the observation, animals were sacrificed by perfusion, their brains were imaged ex-vivo and sectioned for immunofluorescence analyses. DISCUSSION: After appropriate circulation time, the fluorescence concentration in the head ROI measured in vivo was close to background values in both wild-type and Abcb1-KO or Abcg2-KO mice injected with either free dye or scrambled Aß40-1-Cy5.5. In animals injected with Aß1-40-Cy5.5, the deficiency in either Abcb1 or Abcg2 resulted in significant increases in fluorescence concentration in the head ROIs 2 hours after injection compared to wild-type animals. Fluorescence decay (elimination rate) over 2-8 hours after injection was similar between wild-type (t1/2 = 1.97 h) and Abcg2-KO (t1/2 = 2.34 h) and was slightly faster (t1/2 = 1.38 h) in Abcb1-KO mice. In vivo time-domain imaging method allows prospective, dynamic analyses of brain uptake/elimination of fluorescently-labeled compounds, including Aß. Deficiency of either of the two major efflux pumps, Abcb1 and Abcg2, implicated in Aß trafficking across the BBB, resulted in increased accumulation of peripherally-injected Aß1-40 in the brain.

Multiplexed evaluation of serum and CSF pharmacokinetics of brain-targeting single-domain antibodies using a NanoLC-SRM-ILIS method.

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.

Preconditioning induces tolerance by suppressing glutamate release in neuron culture ischemia models.

This study determined how preconditioned neurons responded to oxygen-glucose deprivation (OGD) to result in neuroprotection instead of neurotoxicity. Neurons preconditioned using chronically elevated synaptic activity displayed suppressed elevations in extracellular glutamate ([glutamateex ]) and intracellular Ca(2+) (Ca(2+) in ) during OGD. The glutamate uptake inhibitor TBOA induced neurotoxicity, but at a longer OGD duration for preconditioned cultures, suggestive of delayed up-regulation of transporter activity relative to non-preconditioned cultures. This delay was attributed to a critically attenuated release of glutamate, based on tolerance observed against insults mimicking key neurotoxic signaling during OGD (OGD-mimetics). Specifically, in the presence of TBOA, preconditioned neurons displayed potent protection to the OGD-mimetics: ouabain (a Na(+) /K(+) ATPase inhibitor), high 55 mM KCl extracellular buffer (plasma membrane depolarization), veratridine (a Na(+) ionophore), and paraquat (intracellular superoxide producer), which correlated with suppressed [glutamateex ] elevations in the former two insults. Tolerance by preconditioning was reversed by manipulations that increased [glutamateex ], such as by exposure to TBOA or GABAA receptor agonists during OGD, or by exposure to exogenous NMDA or glutamate. Pre-synaptic suppression of neuronal glutamate release by preconditioning, possibly via suppressed exocytic release, represents a key convergence point in neuroprotection during exposure to OGD and OGD-mimetics.

In vitro and in vivo methods for assessing FcRn-mediated reverse transcytosis across the blood-brain barrier.

The neonatal Fc receptor, FcRn, mediates endocytic recycling pathway that prevents degradation of IgG and is expressed in most endothelial cells. The blood-brain barrier (BBB), formed by brain endothelial cells sealed with tight junctions, restricts transport of IgG from the blood to the brain. In contrast, it has been suggested that IgG undergoes efflux from the brain parenchyma via reverse transcytosis across the BBB mediated by FcRn. The fast elimination of therapeutic antibodies from the brain via this route may limit their therapeutic potency. In vitro and in vivo methods described in this chapter were developed to facilitate research into mechanisms and dynamics of brain efflux of compounds, including FcRn-mediated reverse transcytosis across the BBB. The in vitro model uses immortalized adult rat brain endothelial cells which express high levels of FcRn. In vivo models use Prospective optical imaging to measure the clearance rate of intracerebrally injected FcRn-transported molecules tagged with near-infrared fluorescent probes.

In vivo optical imaging of ischemic blood-brain barrier disruption.

The blood-brain barrier (BBB) disruption following cerebral ischemia (stroke) contributes to the development of life-threatening brain edema. Recent studies suggested that the ischemic BBB disruption is not uniform throughout the affected brain region. The aim of this study was to establish in vivo optical imaging methods to assess the size selectivity and spatial distribution of the BBB disruption after a focal cerebral ischemia. The BBB permeability was assessed in mice subjected to a 60-min middle cerebral artery occlusion and 24 h of reperfusion using in vivo time domain near-infrared optical imaging after contrast enhancement with two tracers of different molecular size, Cy5.5 (1 kDa) and Cy5.5 conjugated with bovine serum albumin (BSA) (67 kDa). Volumetric reconstruction of contrast-enhanced brain areas in vivo and ex vivo indicated that the BSA-Cy5.5-enhancement is identical to the volume of infarct determined by TTC staining, whereas the volume of enhancement with Cy5.5 was 40% greater. The volume differential between areas of BBB disruption for small and large-size molecules could be useful for determining the size of peri-infarct tissues (penumbra) that can respond to neuroprotective therapies.