Quantification and characterization of anti-interferon beta antibodies in multiple sclerosis patients undergoing interferon beta-1b therapy

The increasing recognition of antibody induction in patients undergoing treatment with biologically active recombinant proteins, makes these antibodies of concern. In the case of Relapsing Remitting Multiple Sclerosis (RRMS), administration of interferon beta (IFNβ) - lb can lead to the development of anti- IFN β antibodies in some patients. These antibodies may attenuate the effects of IFNβ, resulting in resistance to therapy or to disease relapses. On the other hand, anti-IFNP antibodies may have carrier or stabilizing functions. The purpose of the current study was the quantification of anti- IFN β antibodies in MS patients' sera, and to further characterize the frequency and serial profile of the antibody response. This was achieved by a sandwich enzyme-linked immunosorbent assay (ELISA) specifically developed for this study. In the process of developing the sandwich ELISA, an anti- IFN β antibody reference pool was established by screening serum samples from 50 RRMS patients undergoing IFNβ therapy, using a qualitative direct ELISA method. Ten serum samples, identified as anti- IFN β antibody positive, were selected for the reference pool which was arbitrarily assigned an anti- IFNP antibody concentration of 100 Laboratory Units / mL. Serial serum samples of 20 patients on IFNβ -1β therapy were assayed using a standard curve generated from the reference pool, and each assay included a set of sera from 5 healthy individuals. The intraassay coefficient of variation (CV) was 6-10% and inter-assay CV was 3-8%. The validity of the assay was assured through binding specificity, recovery and inhibition tests. Of 20 patients treated for 1-24 months, approximately 16 developed antibodies within the first three months. Two antibody profiles were observed: one profile consisted of a rise and gradual decline of antibodies to pretreatment levels, and a second profile characterized by a rise which was followed by a decline to lower antibody levels that were maintained over the course of treatment. Additionally, supernatants of cultured lymphocytes were assayed, using a biotin-streptavidin amplified version of the sandwich-ELISA, and results indicate that some patients secrete anti- IFNβ antibodies in vitro. These findings confirm that IFNβ -1b is immunogenic in MS patients and that the appearance of anti- IFNβ ' antibodies is an early phenomenon. The sandwich ELISA is a specific, sensitive and reproducible method for the quantification of anti- IFNβ ' antibodies, and can be easily adapted in any laboratory for the large-scale throughput monitoring of anti- IFNP antibodies. The assay can also be employed as a tool to study the effects of anti-IFNP antibodies on the therapeutic efficacy of IFNJ3 in MS patients, and the mode of action of IFNp.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Evolution of the anti-interferon beta (IFNβ) antibody response in multiple sclerosis patients : IgG subclass distribution, affinity maturation and clinical correlates

Multiple Sclerosis (MS), a chronic degenerative disease of the central nervous system, is characterized by demyelination, axonal damage, and inflammatory lesions in the white matter. Symptoms include neurological deficits, relapses and progressive disability. Three recombinant interferon beta (IFNβ) products and glatiramer acetate are licensed for treatment. They have been shown to reduce the frequency and severity of relapses and slow disease progression in about 30% of treated patients. Long-term administration of IFNβ can result in the development of anti- (IFNβ) antibodies. Binding antibodies (BAbs) bind (IFNβ) and neutralizing antibodies (NAbs) prevent interaction with its receptor, reducing IFNβ bioavailability and clinical efficacy. The detection and characterization of anti-IFNβ antibodies does not adhere to any internationally recommended standards. A comprehensive strategy is required to elucidate the antibody properties that play a role in the immune response against IFNβ. To this extent, our objectives were: first, to investigate the IgG subclass-specificities of BAbs over time; second, to ascertain the affinity maturation pattern of BAbs and NAbs; and third, to investigate the effects of NAbs on clinical efficacy. We used an enzyme-linked-immunosorbent assay (ELISA) to measure relative distribution of IgG subclass-specific BAbs and found that subclasses not only change over time, but their distribution varies between subcutaneous (SC) IFNβ-la and SC IFNβ-lb. We also found that NAb+ patients tend to have higher levels of IgG4 subclass-specific BAbs than NAb- patients. To investigate the affinity maturation of anti-IFNβ antibodies, we utilized BiacoreTM, a biosensor device based on the optical phenomenon of Surface Plasmon Resonance (SPR). Our results indicate that relative antibody affinities, as reflected by antibody dissociation rates, improve over time in NAb+ patients. Furthermore, we found a close parallel between antibody affinity and NAb levels. Our investigation showed that the effects of NAbs on clinical efficacy are delayed, with an increase in relapse rates being more evident in NAb+ patients than in NAb- patients at year 3 (IFNβ-1b), and at year 3 and 4 (IFNβ-la). We conclude that there is a need for a quantitative and qualitative framework for monitoring anti-IFNβ antibodies that could prove valuable for better management of IFNβ-treated MS patients.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

De Novo Design of a β‑Helix Tau Protein Scaffold: An Oligomer-Selective Vaccine Immunogen Candidate for Alzheimer’s Disease

Tau pathology is associated with many neurodegenerative disorders, including Alzheimer’s disease (AD), where the spatio–temporal pattern of tau neurofibrillary tangles strongly correlates with disease progression, which motivates therapeutics selective for misfolded tau. Here, we introduce a new avidity-enhanced, multi-epitope approach for protein-misfolding immunogen design, which is predicted to mimic the conformational state of an exposed epitope in toxic tau oligomers. A predicted oligomer-selective tau epitope 343KLDFK347 was scaffolded by designing a β-helix structure that incorporated multiple instances of the 16-residue tau fragment 339VKSEKLDFKDRVQSKI354. Large-scale conformational ensemble analyses involving Jensen–Shannon Divergence and the embedding depth D showed that the multi-epitope scaffolding approach, employed in designing the β-helix scaffold, was predicted to better discriminate toxic tau oligomers than other “monovalent” strategies utilizing a single instance of an epitope for vaccine immunogen design. Using Rosetta, 10,000 sequences were designed and screened for the linker portions of the β-helix scaffold, along with a C-terminal stabilizing α-helix that interacts with the linkers, to optimize the folded structure and stability of the scaffold. Structures were ranked by energy, and the lowest 1% (82 unique sequences) were verified using AlphaFold. Several selection criteria involving AlphaFold are implemented to obtain a lead-designed sequence. The structure was further predicted to have free energetic stability by using Hamiltonian replica exchange molecular dynamics (MD) simulations. The synthesized β-helix scaffold showed direct binding in surface plasmon resonance (SPR) experiments to several antibodies that were raised to the structured epitope using a designed cyclic peptide. Moreover, the strength of binding of these antibodies to in vitro tau oligomers correlated with the strength of binding to the β-helix construct, suggesting that the construct presents an oligomer-like conformation and may thus constitute an effective oligomer-selective immunogen

Molecular interactions between monoclonal oligomer-specific antibody 5E3 and its amyloid beta cognates.

Oligomeric amyloid β (Aβ) is currently considered the most neurotoxic form of the Aβ peptide implicated in Alzheimer's disease (AD). The molecular structures of the oligomers have remained mostly unknown due to their transient nature. As a result, the molecular mechanisms of interactions between conformation-specific antibodies and their Aβ oligomer (AβO) cognates are not well understood. A monoclonal conformation-specific antibody, m5E3, was raised against a structural epitope of Aβ oligomers. m5E3 binds to AβOs with high affinity, but not to Aβ monomers or fibrils. In this study, a computational model of the variable fragment (Fv) of the m5E3 antibody (Fv5E3) is introduced. We further employ docking and molecular dynamics simulations to determine the molecular details of the antibody-oligomer interactions, and to classify the AβOs as Fv5E3-positives and negatives, and to provide a rationale for the low affinity of Fv5E3 for fibrils. This information will help us to perform site-directed mutagenesis on the m5E3 antibody to improve its specificity and affinity toward oligomeric Aβ species. We also provide evidence for the possible capability of the m5E3 antibody to disaggregate AβOs and to fragment protofilaments

Rational Generation of Monoclonal Antibodies Selective for Pathogenic Forms of Alpha-Synuclein

Misfolded toxic forms of alpha-synuclein (α-Syn) have been implicated in the pathogenesis of synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The α-Syn oligomers and soluble fibrils have been shown to mediate neurotoxicity and cell-to-cell propagation of pathology. To generate antibodies capable of selectively targeting pathogenic forms of α-Syn, computational modeling was used to predict conformational epitopes likely to become exposed on oligomers and small soluble fibrils, but not on monomers or fully formed insoluble fibrils. Cyclic peptide scaffolds reproducing these conformational epitopes exhibited neurotoxicity and seeding activity, indicating their biological relevance. Immunization with the conformational epitopes gave rise to monoclonal antibodies (mAbs) with the desired binding profile showing selectivity for toxic α-Syn oligomers and soluble fibrils, with little or no reactivity with monomers, physiologic tetramers, or Lewy bodies. Recognition of naturally occurring soluble α-Syn aggregates in brain extracts from DLB and MSA patients was confirmed by surface plasmon resonance (SPR). In addition, the mAbs inhibited the seeding activity of sonicated pre-formed fibrils (PFFs) in a thioflavin-T fluorescence-based aggregation assay. In neuronal cultures, the mAbs protected primary rat neurons from toxic α-Syn oligomers, reduced the uptake of PFFs, and inhibited the induction of pathogenic phosphorylated aggregates of endogenous α-Syn. Protective antibodies selective for pathogenic species of α-Syn, as opposed to pan α-Syn reactivity, are expected to provide enhanced safety and therapeutic potency by preserving normal α-Syn function and minimizing the diversion of active antibody from the target by the more abundant non-toxic forms of α-Syn in the circulation and central nervous system.Medicine, Faculty ofScience, Faculty ofNon UBCBiochemistry and Molecular Biology, Department ofPhysics and Astronomy, Department ofReviewedFacultyResearche

CHIMERA repetitive mild traumatic brain injury induces chronic behavioural and neuropathological phenotypes in wild-type and APP/PS1 mice

Background: The annual incidence of traumatic brain injury (TBI) in the United States is over 2.5 million, with approximately 3–5 million people living with chronic sequelae. Compared with moderate-severe TBI, the long-term effects of mild TBI (mTBI) are less understood but important to address, particularly for contact sport athletes and military personnel who have high mTBI exposure. The purpose of this study was to determine the behavioural and neuropathological phenotypes induced by the Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA) model of mTBI in both wild-type (WT) and APP/PS1 mice up to 8 months post-injury. Methods: Male WT and APP/PS1 littermates were randomized to sham or repetitive mild TBI (rmTBI; 2 × 0.5 J impacts 24 h apart) groups at 5.7 months of age. Animals were assessed up to 8 months post-injury for acute neurological deficits using the loss of righting reflex (LRR) and Neurological Severity Score (NSS) tasks, and chronic behavioural changes using the passive avoidance (PA), Barnes maze (BM), elevated plus maze (EPM) and rotarod (RR) tasks. Neuropathological assessments included white matter damage; grey matter inflammation; and measures of Aβ levels, deposition, and aducanumab binding activity. Results: The very mild CHIMERA rmTBI conditions used here produced no significant acute neurological or motor deficits in WT and APP/PS1 mice, but they profoundly inhibited extinction of fear memory specifically in APP/PS1 mice over the 8-month assessment period. Spatial learning and memory were affected by both injury and genotype. Anxiety and risk-taking behaviour were affected by injury but not genotype. CHIMERA rmTBI induced chronic white matter microgliosis, axonal injury and astrogliosis independent of genotype in the optic tract but not the corpus callosum, and it altered microgliosis in APP/PS1 amygdala and hippocampus. Finally, rmTBI did not alter long-term tau, Aβ or amyloid levels, but it increased aducanumab binding activity. Conclusions: CHIMERA is a useful model to investigate the chronic consequences of rmTBI, including behavioural abnormalities consistent with features of post-traumatic stress disorder and inflammation of both white and grey matter. The presence of human Aβ greatly modified extinction of fear memory after rmTBI.Applied Science, Faculty ofMedicine, Faculty ofOther UBCMechanical Engineering, Department ofMedicine, Department ofNeurology, Division ofPathology and Laboratory Medicine, Department ofReviewedFacult

High-density lipoproteins suppress Aβ-induced PBMC adhesion to human endothelial cells in bioengineered vessels and in monoculture

Background: Alzheimer’s Disease (AD), characterized by accumulation of beta-amyloid (Aβ) plaques in the brain, can be caused by age-related failures to clear Aβ from the brain through pathways that involve the cerebrovasculature. Vascular risk factors are known to increase AD risk, but less is known about potential protective factors. We hypothesize that high-density lipoproteins (HDL) may protect against AD, as HDL have vasoprotective properties that are well described for peripheral vessels. Epidemiological studies suggest that HDL is associated with reduced AD risk, and animal model studies support a beneficial role for HDL in selectively reducing cerebrovascular amyloid deposition and neuroinflammation. However, the mechanism by which HDL may protect the cerebrovascular endothelium in the context of AD is not understood. Methods: We used peripheral blood mononuclear cell adhesion assays in both a highly novel three dimensional (3D) biomimetic model of the human vasculature composed of primary human endothelial cells (EC) and smooth muscle cells cultured under flow conditions, as well as in monolayer cultures of ECs, to study how HDL protects ECs from the detrimental effects of Aβ. Results: Following Aβ addition to the abluminal (brain) side of the vessel, we demonstrate that HDL circulated within the lumen attenuates monocyte adhesion to ECs in this biofidelic vascular model. The mechanism by which HDL suppresses Aβ-mediated monocyte adhesion to ECs was investigated using monotypic EC cultures. We show that HDL reduces Aβ-induced PBMC adhesion to ECs independent of nitric oxide (NO) production, miR-233 and changes in adhesion molecule expression. Rather, HDL acts through scavenger receptor (SR)-BI to block Aβ uptake into ECs and, in cell-free assays, can maintain Aβ in a soluble state. We confirm the role of SR-BI in our bioengineered human vessel. Conclusion: Our results define a novel activity of HDL that suppresses Aβ-mediated monocyte adhesion to the cerebrovascular endothelium.Medicine, Faculty ofScience, Faculty ofOther UBCMedicine, Department ofNeurology, Division ofPathology and Laboratory Medicine, Department ofZoology, Department ofReviewedFacult

A Rational Structured Epitope Defines a Distinct Subclass of Toxic Amyloid-beta Oligomers

Oligomers of amyloid-β (AβO) are deemed key in synaptotoxicity and amyloid seeding of Alzheimer’s disease (AD). However, the heterogeneous and dynamic nature of AβO and inadequate markers for AβO subtypes have stymied effective AβO identification and therapeutic targeting <i>in vivo</i>. We identified an AβO-subclass epitope defined by differential solvent orientation of the lysine 28 side chain in a constrained loop of serine–asparagine–lysine (cSNK), rarely displayed in molecular dynamics simulations of monomer and fibril ensembles. A mouse monoclonal antibody targeting AβO^cSNK recognizes ∼50–60 kDa SDS-resistant soluble Aβ assemblages in AD brain and prolongs the lag phase of Aβ aggregation <i>in vitro</i>. Acute peripheral infusion of a murine IgG1 anti-AβO^cSNK in two AD mouse models reduced soluble brain Aβ aggregates by 20–30%. Chronic cSNK peptide immunization of APP/PS1 mice engendered an anti-AβO^cSNK IgG1 response without epitope spreading to Aβ monomers or fibrils and was accompanied by preservation of global PSD95 expression and improved cued fear memory. Our data indicate that the oligomer subtype AβO^cSNK participates in synaptotoxicity and propagation of Aβ aggregation <i>in vitro</i> and <i>in vivo</i>