Learnings about Aß from human brain recommend the use of a live-neuron bioassay for the discovery of next generation Alzheimer's disease immunotherapeutics.

Despite ongoing debate, the amyloid ß-protein (Aß) remains the prime therapeutic target for the treatment of Alzheimer's disease (AD). However, rational drug design has been hampered by a lack of knowledge about neuroactive Aß. To help address this deficit, we developed live-cell imaging of iPSC-derived human neurons (iNs) to study the effects of the most disease relevant form of Aß-oligomeric assemblies (oAß) extracted from AD brain. Of ten brains studied, extracts from nine caused neuritotoxicity, and in eight cases this was abrogated by Aß immunodepletion. Here we show that activity in this bioassay agrees relatively well with disruption of hippocampal long-term potentiation, a correlate of learning and memory, and that measurement of neurotoxic oAß can be obscured by more abundant non-toxic forms of Aß. These findings indicate that the development of novel Aß targeting therapeutics may benefit from unbiased activity-based discovery. To test this principle, we directly compared 5 clinical antibodies (aducanumab, bapineuzumab,  BAN2401, gantenerumab, and SAR228810) together with an in-house aggregate-preferring antibody (1C22) and established relative EC50s in protecting human neurons from human Aß. The results yielded objective numerical data on the potency of each antibody in neutralizing human oAß neuritotoxicity. Their relative efficacies in this morphological assay were paralleled by their functional ability to rescue oAß-induced inhibition of hippocampal synaptic plasticity. This novel paradigm provides an unbiased, all-human system for selecting candidate antibodies for advancement to human immunotherapy.

A calcium-sensitive antibody isolates soluble amyloid-ß aggregates and fibrils from Alzheimer's disease brain.

Aqueously soluble oligomers of amyloid-ß peptide may be the principal neurotoxic forms of amyloid-ß in Alzheimer's disease, initiating downstream events that include tau hyperphosphorylation, neuritic/synaptic injury, microgliosis and neuron loss. Synthetic oligomeric amyloid-ß has been studied extensively, but little is known about the biochemistry of natural oligomeric amyloid-ß in human brain, even though it is more potent than simple synthetic peptides and comprises truncated and modified amyloid-ß monomers. We hypothesized that monoclonal antibodies specific to neurotoxic oligomeric amyloid-ß could be used to isolate it for further study. Here we report a unique human monoclonal antibody (B24) raised against synthetic oligomeric amyloid-ß that potently prevents Alzheimer's disease brain oligomeric amyloid-ß-induced impairment of hippocampal long-term potentiation. B24 binds natural and synthetic oligomeric amyloid-ß and a subset of amyloid plaques, but only in the presence of Ca2+. The amyloid-ß N terminus is required for B24 binding. Hydroxyapatite chromatography revealed that natural oligomeric amyloid-ß is highly avid for Ca2+. We took advantage of the reversible Ca2+-dependence of B24 binding to perform non-denaturing immunoaffinity isolation of oligomeric amyloid-ß from Alzheimer's disease brain-soluble extracts. Unexpectedly, the immunopurified material contained amyloid fibrils visualized by electron microscopy and amenable to further structural characterization. B24-purified human oligomeric amyloid-ß inhibited mouse hippocampal long-term potentiation. These findings identify a calcium-dependent method for purifying bioactive brain oligomeric amyloid-ß, at least some of which appears fibrillar.

biAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

SAR228810: an antibody for protofibrillar amyloid ß peptide designed to reduce the risk of amyloid-related imaging abnormalities (ARIA).

BACKGROUND: Anti-amyloid ß (Aß) immunotherapy represents a major area of drug development for Alzheimer's disease (AD). However, Aß peptide adopts multiple conformations and the pathological forms to be specifically targeted have not been identified. Aß immunotherapy-related vasogenic edema has also been severely dose limiting for antibodies with effector functions binding vascular amyloid such as bapineuzumab. These two factors might have contributed to the limited efficacy demonstrated so far in clinical studies. METHODS: To address these limitations, we have engineered SAR228810, a humanized monoclonal antibody (mAb) with limited Fc effector functions that binds specifically to soluble protofibrillar and fibrillar forms of Aß peptide and we tested it together with its murine precursor SAR255952 in vitro and in vivo. RESULTS: Unlike gantenerumab and BAN2401, SAR228810 and SAR255952 do not bind to Aß monomers, low molecular weight Aß oligomers or, in human brain sections, to Aß diffuse deposits which are not specific of AD pathology. Both antibodies prevent Aß42 oligomer neurotoxicity in primary neuronal cultures. In vivo, SAR255952, a mouse aglycosylated IgG1, dose-dependently prevented brain amyloid plaque formation and plaque-related inflammation with a minimal active dose of 3 mg/kg/week by the intraperitoneal route. No increase in plasma Aß levels was observed with SAR255952 treatment, in line with its lack of affinity for monomeric Aß. The effects of SAR255952 translated into synaptic functional improvement in ex-vivo hippocampal slices. Brain penetration and decoration of cerebral amyloid plaques was documented in live animals and postmortem. SAR255952 (up to 50 mg/kg/week intravenously) did not increase brain microhemorrhages and/or microscopic changes in meningeal and cerebral arteries in old APPSL mice while 3D6, the murine version of bapineuzumab, did. In immunotolerized mice, the clinical candidate SAR228810 demonstrated the same level of efficacy as the murine SAR255952. CONCLUSION: Based on the improved efficacy/safety profile in non-clinical models of SAR228810, a first-in-man single and multiple dose administration clinical study has been initiated in AD patients.

Reduction of inflammation and preservation of neurological function by anti-CD52 therapy in murine experimental autoimmune encephalomyelitis.

Alemtuzumab, a monoclonal antibody directed against human CD52, is used in the treatment of MS. To characterize the impact of anti-CD52 administration, a monoclonal antibody to mouse CD52 (anti-muCD52) was generated and evaluated in EAE mouse models of MS. A single course of anti-muCD52 provided a therapeutic benefit accompanied by a reduction in the frequency of autoreactive T lymphocytes and production of pro-inflammatory cytokines. Examination of the CNS revealed a decrease in infiltrating lymphocytes, demyelination and axonal loss. Electrophysiological assessment showed preservation of axonal conductance in the spinal cord. These findings suggest that anti-CD52 therapy may help preserve CNS integrity.

Capture-stabilize approach for membrane protein SPR assays.

Measuring the binding kinetics of antibodies to intact membrane proteins by surface plasmon resonance has been challenging largely because of the inherent difficulties in capturing membrane proteins on chip surfaces while retaining their native conformation. Here we describe a method in which His-tagged CXCR5, a GPCR, was purified and captured on a Biacore chip surface via the affinity tag. The captured receptor protein was then stabilized on the chip surface by limited cross-linking. The resulting chip surface retained ligand binding activity and was used for monoclonal antibody kinetics assays by a standard Biacore kinetics assay method with a simple low pH regeneration step. We demonstrate the advantages of this whole receptor assay when compared to available peptide-based binding assays. We further extended the application of the capture-stabilize approach to virus-like particles and demonstrated its utility analyzing antibodies against CD52, a GPI-anchored protein, in its native membrane environment. The results are the first demonstration of chemically stabilized chip surfaces for membrane protein SPR assays.

Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice.

Enzyme replacement therapy is often hampered by the rapid clearance and degradation of the administered enzyme, limiting its efficacy and requiring frequent dosing. Encapsulation of therapeutic molecules into red blood cells (RBCs) is a clinically proven approach to improve the pharmacokinetics and efficacy of biologics and small molecule drugs. Here we evaluated the ability of RBCs encapsulated with phenylalanine hydroxylase (PAH) to metabolize phenylalanine (Phe) from the blood and confer sustained enzymatic activity in the circulation. Significant quantities of PAH were successfully encapsulated within murine RBCs (PAH-RBCs) with minimal loss of endogenous hemoglobin. While intravenously administered free PAH enzyme was rapidly eliminated from the blood within a few hours, PAH-RBCs persisted in the circulation for at least 10days. A single injection of PAH-RBCs was able to decrease Phe levels by nearly 80% in normal mice. These results demonstrate the ability of enzyme-loaded RBCs to metabolize circulating amino acids and highlight the potential to treat disorders of amino acid metabolism.

LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.

beta-glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of the mannose-6-phosphate receptor. Affinity-chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of beta-glucocerebrosidase. This interaction involves a coiled-coil domain within the lumenal domain. beta-glucocerebrosidase activity and protein levels were severely decreased in LIMP-2-deficient mouse tissues. Analysis of fibroblasts and macrophages isolated from these mice indicated that the majority of beta-glucocerebrosidase was secreted. Missorting of beta-glucocerebrosidase was also evident in vivo, as protein and activity levels were significantly higher in sera from LIMP-2-deficient mice compared to wild-type. Reconstitution of LIMP-2 in LIMP-2-deficient fibroblasts led to a rescue of beta-glucocerebrosidase levels and distribution. LIMP-2 expression also led to lysosomal transport of a beta-glucocerebrosidase endoplasmic reticulum retention mutant. These data support a role for LIMP-2 as the mannose-6-phosphate-independent trafficking receptor for beta-glucocerebrosidase.

Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease.

Recombinant human glucocerebrosidase (imiglucerase, Cerezyme) is used in enzyme replacement therapy for Gaucher disease. Complex oligosaccharides present on Chinese hamster ovary cell-expressed glucocerebrosidase (GCase) are enzymatically remodeled into a mannose core, facilitating mannose receptor-mediated uptake into macrophages. Alternative expression systems could be used to produce GCase containing larger oligomannose structures, offering the possibility of an improvement in targeting to macrophages. A secondary advantage of these expression systems would be to eliminate the need for carbohydrate remodeling. Here, multiple expression systems were used to produce GCase containing primarily terminal oligomannose, from Man2 to Man9. GCase from these multiple expression systems was compared to Cerezyme with respect to affinity for mannose receptor and serum mannose-binding lectin (MBL), macrophage uptake, and intracellular half-life. In vivo studies comparing clearance and targeting of Cerezyme and the Man9 form of GCase were carried out in a Gaucher mouse model (D409V/null). Mannose receptor binding, macrophage uptake, and in vivo targeting were similar for all forms of GCase. Increased MBL binding was observed for all forms of GCase having larger mannose structures than those of Cerezyme, which could influence pharmacokinetic behavior. These studies demonstrate that although alternative cell expression systems are effective for producing oligomannose-terminated glucocerebrosidase, there is no biochemical or pharmacological advantage in producing GCase with an increased number of mannose residues. The display of alternative carbohydrate structures on GCase expressed in these systems also runs the risk of undesirable consequences, such as an increase in MBL binding or a possible increase in immunogenicity due to the presentation of non-mammalian glycans.

EPI64 regulates microvillar subdomains and structure.

EPI64 is a TBC domain-containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane. Uncoupling EPI64's binding to EBP50, expression of any construct mislocalizing its TBC domain, or knock down of EBP50 results in loss of microvilli. The TBC domain of EPI64 binds directly to Arf6-GTP. Overexpressing the TBC domain increases Arf6-GTP levels, and expressing dominant-active Arf6 results in microvillar loss. These data reveal that microvilli have distinct cytoskeletal subdomains and that EPI64 regulates microvillar structure.

Chemical chaperones and permissive temperatures alter localization of Gaucher disease associated glucocerebrosidase variants.

Point mutations in the lysosomal hydrolase, glucocerebrosidase (GC), can cause Gaucher disease, a common lysosomal storage disease. Several clinically important GC mutations impede folding in the endoplasmic reticulum (ER) and target these enzymes for ER-associated degradation (ERAD). The removal of these misfolded proteins decreases the lysosomal concentration of GC, which results in glucosylceramide accumulation. The most common GC variant, N370S, and other clinically relevant variants, G202R and L444P, exhibit different cellular localization patterns in patient-derived fibroblasts. We show that these distributions can be altered by manipulation of the ER folding environment, either by chemical chaperones or by temperature shifts. N370S, L444P, and G202R GC are destabilized in the neutral pH environment of the ER, rendering them prone to ERAD. Fibroblasts harboring the G202R and L444P GC mutations grown at 30 degrees C localize the mutant proteins to the lysosome, and this increases total GC activity. Both of these temperature-sensitive mutants appear to be stable at 37 degrees C once they are trafficked to the low pH environment of the lysosome. Chemical chaperones correct the ER instability and significant ER retention of G202R GC. N370S is also destabilized under ER simulating conditions, a deficiency that is corrected by chemical chaperone binding. These data clearly show manipulating the ER environment with chemical chaperones increases the lysosomal concentration of partially active GC variants and suggest that small molecules could be used to treat Gaucher disease.

Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent.

Tumors associated with osteomalacia elaborate the novel factor(s), phosphatonin(s), which causes phosphaturia and hypophosphatemia by cAMP-independent pathways. We show that secreted frizzled-related protein-4 (sFRP-4), a protein highly expressed in such tumors, is a circulating phosphaturic factor that antagonizes renal Wnt-signaling. In cultured opossum renal epithelial cells, sFRP-4 specifically inhibited sodium-dependent phosphate transport. Infusions of sFRP-4 in normal rats over 2 hours specifically increased renal fractional excretion of inorganic phosphate (FEPi) from 14% +/- 2% to 34% +/- 5% (mean +/- SEM, P < 0.01). Urinary cAMP and calcium excretion were unchanged. In thyro-parathyroidectomized rats, sFRP-4 increased FEPi from 0.7% +/- 0.2% to 3.8% +/- 1.2% (P < 0.05), demonstrating that sFRP-4 inhibits renal inorganic phosphate reabsorption by PTH-independent mechanisms. Administration of sFRP-4 to intact rats over 8 hours increased FEPi, decreased serum phosphate (1.95 +/- 0.1 to 1.53 +/- 0.09 mmol/l, P < 0.05) but did not alter serum 1alpha, 25-dihydroxyvitamin D, renal 25-hydroxyvitamin D 1alpha-hydroxylase cytochrome P450, and sodium-phosphate cotransporter mRNA concentrations. Infusion of sFRP-4 antagonizes Wnt action as demonstrated by reduced renal beta-catenin and increased phosphorylated beta-catenin concentrations. The sFRP-4 is detectable in normal human serum and in the serum of a patient with tumor-induced osteomalacia. Thus, sFRP-4 displays phosphatonin-like properties, because it is a circulating protein that promotes phosphaturia and hypophosphatemia and blunts compensatory increases in 1alpha, 25-dihydroxyvitamin D.

Distinct cell type-specific expression of scaffolding proteins EBP50 and E3KARP: EBP50 is generally expressed with ezrin in specific epithelia, whereas E3KARP is not.

The ezrin/radixin/moesin (ERM) proteins are regulated microfilament membrane linking proteins. Previous tissue localization studies have revealed that the three related proteins show distinct tissue distributions, with ezrin being found predominantly in polarized epithelial cells, whereas moesin is enriched in endothelial cells and lymphocytes. EBP50 and E3KARP are two related scaffolding proteins that bind to the activated form of ERM proteins in vitro, and through their PDZ domains to the cytoplasmic domains of specific membrane proteins, including the Na+/H+ exchanger isoform (NHE3) present in kidney proximal tubules and the beta2-adrenergic receptor. Using specific antibodies to EBP50 and E3KARP for localization in murine tissues, we find that the cellular distribution of EBP50 and E3KARP is mutually exclusive. Epithelial cells expressing ezrin generally co-express EBP50, such as intestinal epithelial cells, gastric parietal cells, the epithelial cells of the kidney proximal tubule, the terminal bronchiole of the lung, and in mesothelia. This correlation is not absolute as cells of the mucous epithelium of the stomach and in the renal corpuscle, express ezrin but no detectable EBP50, whereas the bile canaliculi of hepatocytes express EBP50 and not ezrin. E3KARP has a restricted tissue distribution with the highest expression being found in lung. It is largely colocalized with moesin and radixin, especially in the alveoli of the lung, as well as being highly enriched in the renal corpuscle. These results document a preference for co-expression of EBP50, but not E3KARP, with ezrin in polarized epithelia. These results place constraints on the physiological roles that can be proposed for these scaffolding molecules.