Functional interplay between tumour cells and their neoplastic extracellular matrix plays a decisive role in malignant progression of carcinomas. Here we provide a comprehensive data set of the human ...HNSCC-associated fibroblast matrisome. Although much attention has been paid to the deposit of collagen, we identify oncofetal fibronectin (FN) as a major and obligate component of the matrix assembled by stromal fibroblasts from head and neck squamous cell carcinomas (HNSCC). FN overexpression in tumours from 435 patients corresponds to an independent unfavourable prognostic indicator. We show that migration of carcinoma collectives on fibrillar FN-rich matrices is achieved through αvβ6 and α9β1 engagement, rather than α5β1. Moreover, αvβ6-driven migration occurs independently of latent TGF-β activation and Smad-dependent signalling in tumour epithelial cells. These results provide insights into the adhesion-dependent events at the tumour-stroma interface that govern the collective mode of migration adopted by carcinoma cells to invade surrounding stroma in HNSCC.
Alzheimer's disease (AD) is characterized by deposition of amyloid-β (Aβ) plaques and neurofibrillary tangles in the brain, accompanied by synaptic dysfunction and neurodegeneration. Antibody-based ...immunotherapy against Aβ to trigger its clearance or mitigate its neurotoxicity has so far been unsuccessful. Here we report the generation of aducanumab, a human monoclonal antibody that selectively targets aggregated Aβ. In a transgenic mouse model of AD, aducanumab is shown to enter the brain, bind parenchymal Aβ, and reduce soluble and insoluble Aβ in a dose-dependent manner. In patients with prodromal or mild AD, one year of monthly intravenous infusions of aducanumab reduces brain Aβ in a dose- and time-dependent manner. This is accompanied by a slowing of clinical decline measured by Clinical Dementia Rating-Sum of Boxes and Mini Mental State Examination scores. The main safety and tolerability findings are amyloid-related imaging abnormalities. These results justify further development of aducanumab for the treatment of AD. Should the slowing of clinical decline be confirmed in ongoing phase 3 clinical trials, it would provide compelling support for the amyloid hypothesis.
Aggregation of α-synuclein (α-syn) is neuropathologically and genetically linked to Parkinson's disease (PD). Since stereotypic cell-to-cell spreading of α-syn pathology is believed to contribute to ...disease progression, immunotherapy with antibodies directed against α-syn is considered a promising therapeutic approach for slowing disease progression. Here we report the identification, binding characteristics, and efficacy in PD mouse models of the human-derived α-syn antibody BIIB054, which is currently under investigation in a Phase 2 clinical trial for PD. BIIB054 was generated by screening human memory B-cell libraries from healthy elderly individuals. Epitope mapping studies conducted using peptide scanning, X-ray crystallography, and mutagenesis show that BIIB054 binds to α-syn residues 1–10. BIIB054 is highly selective for aggregated forms of α-syn with at least an 800-fold higher apparent affinity for fibrillar versus monomeric recombinant α-syn and a strong preference for human PD brain tissue. BIIB054 discriminates between monomers and oligomeric/fibrillar forms of α-syn based on high avidity for aggregates, driven by weak monovalent affinity and fast binding kinetics. In efficacy studies in three different mouse models with intracerebrally inoculated preformed α-syn fibrils, BIIB054 treatment attenuated the spreading of α-syn pathology, rescued motor impairments, and reduced the loss of dopamine transporter density in dopaminergic terminals in striatum. The preclinical data reported here provide a compelling rationale for clinical development of BIIB054 for the treatment and prevention of PD.
•Identification of an aggregate selective human-derived antibody to α-synuclein.•BIIB054 shows ≥800-fold higher binding to aggregates than monomer.•Structure activity relationship of the BIIB054/α-synuclein complex.•BIIB054 treatment in mice reduces spread of pathology and improves motor function.
Amyloid-β (Aβ) deposits are a relatively late consequence of Aβ aggregation in Alzheimer's disease. When pathogenic Aβ seeds begin to form, propagate and spread is not known, nor are they ...biochemically defined. We tested various antibodies for their ability to neutralize Aβ seeds before Aβ deposition becomes detectable in Aβ precursor protein-transgenic mice. We also characterized the different antibody recognition profiles using immunoprecipitation of size-fractionated, native, mouse and human brain-derived Aβ assemblies. At least one antibody, aducanumab, after acute administration at the pre-amyloid stage, led to a significant reduction of Aβ deposition and downstream pathologies 6 months later. This demonstrates that therapeutically targetable pathogenic Aβ seeds already exist during the lag phase of protein aggregation in the brain. Thus, the preclinical phase of Alzheimer's disease-currently defined as Aβ deposition without clinical symptoms-may be a relatively late manifestation of a much earlier pathogenic seed formation and propagation that currently escapes detection in vivo.
Aducanumab, a human-derived antibody targeting amyloid-β (Aβ), is in Phase 3 clinical trials for the treatment of Alzheimer's disease. Biochemical and structural analyses show that aducanumab binds a ...linear epitope formed by amino acids 3-7 of the Aβ peptide. Aducanumab discriminates between monomers and oligomeric or fibrillar aggregates based on weak monovalent affinity, fast binding kinetics and strong avidity for epitope-rich aggregates. Direct comparative studies with analogs of gantenerumab, bapineuzumab and solanezumab demonstrate clear differentiation in the binding properties of these antibodies. The crystal structure of the Fab fragment of aducanumab bound to its epitope peptide reveals that aducanumab binds to the N terminus of Aβ in an extended conformation, distinct from those seen in structures with other antibodies that target this immunodominant epitope. Aducanumab recognizes a compact epitope that sits in a shallow pocket on the antibody surface. In silico analyses suggest that aducanumab interacts weakly with the Aβ monomer and may accommodate a variety of peptide conformations, further supporting its selectivity for Aβ aggregates. Our studies provide a structural rationale for the low affinity of aducanumab for non-pathogenic monomers and its greater selectivity for aggregated forms than is seen for other Aβ-targeting antibodies.
Calcium homeostasis plays a major role in maintaining neuronal function under physiological conditions. Amyloid-β (Aβ) initiates pathological processes that include disruption in intracellular ...calcium levels, so amelioration of the calcium alteration could serve as an indirect functional indicator of treatment efficacy. Therefore, calcium dynamics were used as a measure of functional outcome. We evaluated the effects of the anti-Aβ antibody aducanumab on calcium homeostasis and plaque clearance in aged Tg2576 mice with in vivo multiphoton imaging. Acute topical application of aducanumab to the brain resulted in clearance of amyloid plaques. Although chronic systemic administration of aducanumab in 22-month-old mice did not clear existing plaques, calcium overload was ameliorated over time. Therefore, this antibody likely restores neuronal network function that possibly underlies cognitive deficits, indicating promise as a clinical treatment. In addition, functional readouts such as calcium overload may be a more useful outcome measure to monitor treatment efficacy in models of Alzheimer's disease compared with amyloid burden alone.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is currently without a cure. Aducanumab is an anti-amyloid-β antibody being developed for the treatment of AD. Interim analyses of a phase 1b clinical trial have suggested potential beneficial effects on amyloid pathology and cognitive status in patients treated with aducanumab (Sevigny et al., 2016). Here, we show that a murine analog of aducanumab clears amyloid plaques in an acute setting and restores calcium homeostasis disrupted in a mouse model of AD upon chronic treatment. Therefore, we demonstrate that aducanumab reverses a functional outcome measure reflective of neural network activity.
Characterization of tau binding by gosuranemab Sopko, Richelle; Golonzhka, Olga; Arndt, Joseph ...
Neurobiology of disease,
December 2020, 2020-12-00, 2020-12-01, Letnik:
146, Številka:
C
Journal Article
Recenzirano
Odprti dostop
Deposition of tau aggregates in the brain is a pathological hallmark of several neurodegenerative diseases, termed tauopathies, such as Alzheimer's disease (AD), corticobasal degeneration, and ...progressive supranuclear palsy (PSP). As transcellular spread of pathological tau aggregates has been implicated in disease progression, immunotherapy is being considered as a treatment for tauopathies. Here we report a detailed biochemical and biophysical characterization of the tau-binding properties of gosuranemab, a humanized monoclonal antibody directed against N-terminal tau that is currently being investigated as a treatment for AD. Binding experiments showed that gosuranemab exhibited high affinity for tau monomer, tau fibrils, and insoluble tau from different tauopathies. Epitope mapping studies conducted using X-ray crystallography and mutagenesis showed that gosuranemab bound to human tau residues 15–22. Immunodepletion of pathological human brain homogenates and transgenic mouse interstitial fluid (ISF) with gosuranemab resulted in reduced tau aggregation in tau biosensor cells. Preincubation of seed-competent AD-tau with gosuranemab significantly inhibited tau aggregation in mouse primary cortical neurons. Gosuranemab also significantly reduced unbound N-terminal tau in cerebrospinal fluid (CSF) from individuals with PSP and AD, and in ISF and CSF of treated transgenic mice. These results are consistent with the >90% target engagement observed in the CSF of some clinical trial dosing cohorts and support the evaluation of gosuranemab as a potential treatment for AD.
Display omitted
•Gosuranemab, an anti-tau antibody, is being studied for Alzheimer's disease.•Gosuranemab bound to tau with high affinity.•Gosuranemab inhibited pathologic tau–induced tau aggregation in cells.•Structure/activity of gosuranemab–tau complex showed that the epitope was tau15–22.•Gosuranemab bound extracellular tau in interstitial and cerebrospinal fluid.
The clinical progression of Alzheimer disease (AD) is associated with the accumulation of tau neurofibrillary tangles, which may spread throughout the cortex by interneuronal tau transfer. If so, ...targeting extracellular tau species may slow the spreading of tau pathology and possibly cognitive decline. To identify suitable target epitopes, we tested the effects of a panel of tau antibodies on neuronal uptake and aggregation in vitro. Immunodepletion was performed on brain extract from tau-transgenic mice and postmortem AD brain and added to a sensitive fluorescence resonance energy transfer–based tau uptake assay to assess blocking efficacy. The antibodies reduced tau uptake in an epitope-dependent manner: N-terminal (Tau13) and middomain (6C5 and HT7) antibodies successfully prevented uptake of tau species, whereas the distal C-terminal–specific antibody (Tau46) had little effect. Phosphorylation-dependent (40E8 and p396) and C-terminal half (4E4) tau antibodies also reduced tau uptake despite removing less total tau by immunodepletion, suggesting specific interactions with species involved in uptake. Among the seven antibodies evaluated, 6C5 most efficiently blocked uptake and subsequent aggregation. More important, 6C5 also blocked neuron-to-neuron spreading of tau in a unique three-chamber microfluidic device. Furthermore, 6C5 slowed down the progression of tau aggregation even after uptake had begun. Our results imply that not all antibodies/epitopes are equally robust in terms of blocking tau uptake of human AD-derived tau species.
Most human neurodegenerative diseases share a phenotype of neuronal protein aggregation. In Amyotrophic Lateral Sclerosis (ALS), the abundant protein superoxide dismutase (SOD1) or the TAR-DNA ...binding protein TDP-43 can aggregate in motor neurons. Recently, numerous studies have highlighted the ability of aggregates to spread from neuron to neuron in a prion-like fashion. These studies have typically focused on the use of neuron-like cell lines or neurons that are not normally affected by the specific aggregated protein being studied. Here, we have investigated the uptake of pre-formed SOD1 aggregates by cultures containing pluripotent stem cell-derived human motor neurons. We found that all cells take up aggregates by a process resembling fluid-phase endocytosis, just as found in earlier studies. However, motor neurons, despite taking up smaller amounts of SOD1, were much more vulnerable to the accumulating aggregates. Thus, the propagation of disease pathology depends less on selective uptake than on selective response to intracellular aggregates. We further demonstrate that anti-SOD1 antibodies, being considered as ALS therapeutics, can act by blocking the uptake of SOD1, but also by blocking the toxic effects of intracellular SOD1. This work demonstrates the importance of using disease relevant cells even in studying phenomena such as aggregate propagation.
Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive disease with poor survival rates and limited treatment options. Upregulation of αvβ6 integrins within the alveolar epithelial cells ...is a characteristic feature of IPF and correlates with poor patient survival. The pro-fibrotic cytokine TGFβ1 can upregulate αvβ6 integrin expression but the molecular mechanisms driving this effect have not previously been elucidated. We confirm that stimulation with exogenous TGFβ1 increases expression of the integrin β6 subunit gene (ITGB6) and αvβ6 integrin cell surface expression in a time- and concentration-dependent manner. TGFβ1-induced ITGB6 expression occurs via transcriptional activation of the ITGB6 gene, but does not result from effects on ITGB6 mRNA stability. Basal expression of ITGB6 in, and αvβ6 integrins on, lung epithelial cells occurs via homeostatic αvβ6-mediated TGFβ1 activation in the absence of exogenous stimulation, and can be amplified by TGFβ1 activation. Fundamentally, we show for the first time that TGFβ1-induced ITGB6 expression occurs via canonical Smad signalling since dominant negative constructs directed against Smad3 and 4 inhibit ITGB6 transcriptional activity. Furthermore, disruption of a Smad binding site at -798 in the ITGB6 promoter abolishes TGFβ1-induced ITGB6 transcriptional activity. Using chromatin immunoprecipitation we demonstrate that TGFβ1 stimulation of lung epithelial cells results in direct binding of Smad3, and Smad4, to the ITGB6 gene promoter within this region. Finally, using an adenoviral TGFβ1 over-expression model of pulmonary fibrosis we demonstrate that Smad3 is crucial for TGFβ1-induced αvβ6 integrin expression within the alveolar epithelium in vivo. Together, these data confirm that a homeostatic, autocrine loop of αvβ6 integrin activated TGFβ1-induced ITGB6 gene expression regulates epithelial basal αvβ6 integrin expression, and demonstrates that this occurs via Smad-dependent transcriptional regulation at a single Smad binding site in the promoter of the β6 subunit gene. Active TGFβ1 amplifies this pathway both in vitro and in vivo, which may promote fibrosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
