Arsalan S. Haqqani

Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system

Adhesion molecules of the immunoglobulin superfamily are crucial effectors of leukocyte trafficking into the central nervous system. Using a lipid raft-based proteomic approach, we identified ALCAM as an adhesion molecule involved in leukocyte migration across the blood-brain barrier (BBB). ALCAM expressed on BBB endothelium localized together with CD6 on leukocytes and with BBB endothelium transmigratory cups. ALCAM expression on BBB cells was upregulated in active multiple sclerosis and experimental autoimmune encephalomyelitis lesions. Moreover, ALCAM blockade restricted the transmigration of CD4+ lymphocytes and monocytes across BBB endothelium in vitro and in vivo and reduced the severity and delayed the time of onset of experimental autoimmune encephalomyelitis. Our findings indicate an important function for ALCAM in the recruitment of leukocytes into the brain and identify ALCAM as a potential target for the therapeutic dampening of neuroinflammation.

Characterization of vascular protein expression patterns in cerebral ischemia/reperfusion using laser capture microdissection and ICAT-nanoLC-MS/MS.

Cerebral ischemia rapidly initiates structural and functional changes in brain vessels, including blood‐brain barrier disruption, inflammation, and angiogenesis. Molecular events that accompany these changes were investigated in brain microvessels extracted using laser‐capture microdissection (LCM) from Sprague‐Dawley rats subjected to a 20 min transient global cerebral ischemia followed by 1, 6, or 24 h reperfusion. Proteins extracted from ∼300 LCM captured microvessels (20–100 µm) were ICAT‐labeled and analyzed by nanoLC‐MS. In‐house software was used to identify paired ICAT peaks, which were then sequenced by nanoLC‐MS/MS. Pattern analyses using k‐means clustering method classified 57 differentially expressed proteins in 7 distinct dynamic patterns. Protein function was assigned using Panther Classification system. Early reperfusion (1 h) was characterized by down‐regulation of ion pumps, nutrient transporters, and cell structure/motility proteins, and up‐regulation of transcription factors, signal transduction molecules and proteins involved in carbohydrate metabolism. The up‐regulation of inflammatory cytokines and proteins involved in the extracellular matrix remodeling and anti‐oxidative defense was observed in late reperfusion (6–24 h). The up‐regulation of IL‐1β and TGF‐1β in ischemic brain vessels was confirmed by ELISA, quantitative PCR, and/or immunohistochemistry. A biphasic postischemic (1 and 24 h) BBB opening for 3H‐sucrose was evident in the same model. Differentially expressed proteins identified in brain vessels during reperfusion are likely involved in orchestrating functional vascular responses to ischemia, including the observed BBB disruption. Haqqani A. S., Nesic M., Preston E., Baumann E., Kelly J., Stanimirovic D. Characterization of vascular protein expression patterns in cerebral ischemia/reperfusion using laser capture microdissection and ICAT‐nanoLC‐MS/MS. FASEB J. 19, 1809–1821 (2005)

Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

BackgroundIn addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit.MethodsTo study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed.ResultsA total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially interact with both primary astrocytes and cortical neurons, as cell-cell communication vesicles. Finally, brain endothelial cell extracellular microvesicles were shown to contain several receptors previously shown to carry macromolecules across the blood brain barrier, including transferrin receptor, insulin receptor, LRPs, LDL and TMEM30A.ConclusionsThe methods described here permit identification of the molecular signatures for brain endothelial cell-specific extracellular microvesicles under various biological conditions. In addition to being a potential source of useful biomarkers, these vesicles contain potentially novel receptors known for delivering molecules across the blood–brain barrier.

Role of Ninjurin-1 in the migration of myeloid cells to central nervous system inflammatory lesions.

Blood‐derived myeloid antigen‐presenting cells (APCs) account for a significant proportion of the leukocytes found within lesions of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). These APCs along with activated microglia are thought to be pivotal in the initiation of the central nervous system (CNS)‐targeted immune response in MS and EAE. However, the exact molecules that direct the migration of myeloid cells from the periphery across the blood–brain barrier (BBB) remain largely unknown.

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.—Farrington, G. K., Caram‐Salas, N., Haqqani, A. S., Brunette, E., Eldredge, J., Pepinsky, B., Antognetti, G., Baumann, E., Ding, W., Garber, E., Jiang, S., Delaney, C., Boileau, E., Sisk, W. P., Stanimirovic, D. B., A novel platform for engineering blood‐brain barrier‐crossing bispecific biologics. FASEB J. 28, 4764–4778 (2014). www.fasebj.org

Functions of lipid raft membrane microdomains at the blood-brain barrier

The blood–brain barrier (BBB) is a highly specialized structural and functional component of the central nervous system that separates the circulating blood from the brain and spinal cord parenchyma. Brain endothelial cells (BECs) that primarily constitute the BBB are tightly interconnected by multiprotein complexes, the adherens junctions and the tight junctions, thereby creating a highly restrictive cellular barrier. Lipid-enriched membrane microdomain compartmentalization is an inherent property of BECs and allows for the apicobasal polarity of brain endothelium, temporal and spatial coordination of cell signaling events, and actin remodeling. In this manuscript, we review the role of membrane microdomains, in particular lipid rafts, in the BBB under physiological conditions and during leukocyte transmigration/diapedesis. Furthermore, we propose a classification of endothelial membrane microdomains based on their function, or at least on the function ascribed to the molecules included in such heterogeneous rafts: (1) rafts associated with interendothelial junctions and adhesion of BECs to basal lamina (scaffolding rafts); (2) rafts involved in immune cell adhesion and migration across brain endothelium (adhesion rafts); (3) rafts associated with transendothelial transport of nutrients and ions (transporter rafts).

Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantification.

Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label‐free MS‐based approach to identify formerly N‐linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to ∼2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine–protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments.

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 inmGluR1‐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 FASEB J. 30, 1927–1940 (2016). www.fasebj.org

Blood–brain barrier models: in vitro to in vivo translation in preclinical development of CNS-targeting biotherapeutics

Introduction: The majority of therapeutics, small molecule or biologics, developed for the CNS do not penetrate the blood–brain barrier (BBB) sufficiently to induce pharmacologically meaningful effects on CNS targets. To improve the efficiency of CNS drug discovery, several in vitro models of the BBB have been used to aid early selection of molecules with CNS exposure potential. However, correlative studies suggest relatively poor predictability of in vitro BBB models underscoring the need to combine in vitro and in vivo BBB penetration assessment into an integrated preclinical workflow. Areas covered: This review gives a brief general overview of in vitro and in vivo BBB models used in the pre-clinical evaluation of CNS-targeting drugs, with particular focus on the recent progress in developing humanized models. The authors discuss the advantages, limitations, in vitro–in vivo correlation, and integration of these models into CNS drug discovery and development with the aim of improving translation. Expert opinion: Often, a simplistic rationalization of the CNS drug discovery and development process overlooks or even ignores the need for an early and predictive assessment of the BBB permeability. Indeed, past failures of CNS candidates in clinical trials argue strongly that the early deployment of in vitro and in vivo models for assessing BBB permeability, mechanisms of transport and brain exposure of leads, and the co-development of BBB delivery strategies will improve translation and increase the clinical success of CNS pipelines.

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.