Epitope-specific antibody fragments block aggregation of AGelD187N, an aberrant peptide in gelsolin amyloidosis.

Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of systemic amyloidosis. The amyloidogenic gelsolin fragment AGelD187N does not play any physiological role in the body, unlike most aggregating proteins related to other protein misfolding diseases. However, no therapeutic agents that specifically and effectively target and neutralize AGelD187N exist. We used phage display technology to identify novel single-chain variable fragments that bind to different epitopes in the monomeric AGelD187N that were further maturated by variable domain shuffling and converted to antigen-binding fragment (Fab) antibodies. The generated antibody fragments had nanomolar binding affinity for full-length AGelD187N, as evaluated by biolayer interferometry. Importantly, all four Fabs selected for functional studies efficiently inhibited the amyloid formation of full-length AGelD187N as examined by thioflavin fluorescence assay and transmission electron microscopy. Two Fabs, neither of which bound to the previously proposed fibril-forming region of AGelD187N, completely blocked the amyloid formation of AGelD187N. Moreover, no small soluble aggregates, which are considered pathogenic species in protein misfolding diseases, were formed after successful inhibition of amyloid formation by the most promising aggregation inhibitor, as investigated by size-exclusion chromatography combined with multiangle light scattering. We conclude that all regions of the full-length AGelD187N are important in modulating its assembly into fibrils and that the discovered epitope-specific anti-AGelD187N antibody fragments provide a promising starting point for a disease-modifying therapy for gelsolin amyloidosis, which is currently lacking.

Identification of isoaspartate-modified transthyretin as potential target for selective immunotherapy of transthyretin amyloidosis.

BACKGROUND: Numerous studies suggest a progressive accumulation of post-translationally modified peptides within amyloid fibrils, including isoaspartate (isoD) modifications. Here, we generated and characterised novel monoclonal antibodies targeting isoD-modified transthyretin (TTR). The antibodies were used to investigate the presence of isoD-modified TTR in deposits from transthyretin amyloidosis patients and to mediate antibody-dependent phagocytosis of TTR fibrils. METHODS: Monoclonal antibodies were generated by immunisation of mice using an isoD-modified peptide and subsequent hybridoma generation. The antibodies were characterised in terms of affinity and specificity to isoD-modified TTR using surface plasmon resonance, transmission electron microscopy and immunohistochemical staining of human cardiac tissue. The potential to elicit antibody-dependent phagocytosis of TTR fibrils was assessed using THP-1 cells. RESULTS: We developed two mouse monoclonal antibodies, 2F2 and 4D4, with high nanomolar affinity for isoD-modified TTR and strong selectivity over the unmodified epitope. Both antibodies show presence of isoD-modified TTR in human cardiac tissue, but not in freshly purified recombinant TTR, suggesting isoD modification only present in aged fibrillar deposits. Likewise, the antibodies only facilitated phagocytosis of TTR fibrils and not TTR monomers by THP-1 cells. CONCLUSIONS: These antibodies label aged, non-native TTR deposits, leaving native TTR unattended and thereby potentially enabling new therapeutic approaches.

Amyloid-beta antibody binding to cerebral amyloid angiopathy fibrils and risk for amyloid-related imaging abnormalities.

Therapeutic antibodies have been developed to target amyloid-beta (Aß), and some of these slow the progression of Alzheimer's disease (AD). However, they can also cause adverse events known as amyloid-related imaging abnormalities with edema (ARIA-E). We investigated therapeutic Aß antibody binding to cerebral amyloid angiopathy (CAA) fibrils isolated from human leptomeningeal tissue to study whether this related to the ARIA-E frequencies previously reported by clinical trials. The binding of Aß antibodies to CAA Aß fibrils was evaluated in vitro using immunoprecipitation, surface plasmon resonance, and direct binding assay. Marked differences in Aß antibody binding to CAA fibrils were observed. Solanezumab and crenezumab showed negligible CAA fibril binding and these antibodies have no reported ARIA-E cases. Lecanemab showed a low binding to CAA fibrils, consistent with its relatively low ARIA-E frequency of 12.6%, while aducanumab, bapineuzumab, and gantenerumab all showed higher binding to CAA fibrils and substantially higher ARIA-E frequencies (25-35%). An ARIA-E frequency of 24% was reported for donanemab, and its binding to CAA fibrils correlated with the amount of pyroglutamate-modified Aß present. The findings of this study support the proposal that Aß antibody-CAA interactions may relate to the ARIA-E frequency observed in patients treated with Aß-based immunotherapies.

Vaccination with structurally adapted fungal protein fibrils induces immunity to Parkinson's disease.

The pathological misfolding and aggregation of soluble α-synuclein into toxic oligomers and insoluble amyloid fibrils causes Parkinson's disease, a progressive age-related neurodegenerative disease for which there is no cure. HET-s is a soluble fungal protein that can form assembled amyloid fibrils in its prion state. We engineered HET-s(218-298) to form four different fibrillar vaccine candidates, each displaying a specific conformational epitope present on the surface of α-synuclein fibrils. Vaccination with these four vaccine candidates prolonged the survival of immunized TgM83+/- mice challenged with α-synuclein fibrils by 8% when injected into the brain to model brain-first Parkinson's disease or by 21% and 22% when injected into the peritoneum or gut wall, respectively, to model body-first Parkinson's disease. Antibodies from fully immunized mice recognized α-synuclein fibrils and brain homogenates from patients with Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Conformation-specific vaccines that mimic epitopes present only on the surface of pathological fibrils but not on soluble monomers, hold great promise for protection against Parkinson's disease, related synucleinopathies and other amyloidogenic protein misfolding disorders.

Nanobodies counteract the toxicity of an amyloidogenic light chain by stabilizing a partially open dimeric conformation.

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 VL domain and stabilise an unexpected partially open LC dimer in which the two VL domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms.

Promising Alzheimer's therapies shrink brains.

Effects of antiamyloid antibody drugs worry some scientists, who call for more study.

The Ultrastructure of Tissue Damage by Amyloid Fibrils.

Amyloidosis is a group of diseases that includes Alzheimer's disease, prion diseases, transthyretin (ATTR) amyloidosis, and immunoglobulin light chain (AL) amyloidosis. The mechanism of organ dysfunction resulting from amyloidosis has been a topic of debate. This review focuses on the ultrastructure of tissue damage resulting from amyloid deposition and therapeutic insights based on the pathophysiology of amyloidosis. Studies of nerve biopsy or cardiac autopsy specimens from patients with ATTR and AL amyloidoses show atrophy of cells near amyloid fibril aggregates. In addition to the stress or toxicity attributable to amyloid fibrils themselves, the toxicity of non-fibrillar states of amyloidogenic proteins, particularly oligomers, may also participate in the mechanisms of tissue damage. The obscuration of the basement and cytoplasmic membranes of cells near amyloid fibrils attributable to an affinity of components constituting these membranes to those of amyloid fibrils may also play an important role in tissue damage. Possible major therapeutic strategies based on pathophysiology of amyloidosis consist of the following: (1) reducing or preventing the production of causative proteins; (2) preventing the causative proteins from participating in the process of amyloid fibril formation; and/or (3) eliminating already-deposited amyloid fibrils. As the development of novel disease-modifying therapies such as short interfering RNA, antisense oligonucleotide, and monoclonal antibodies is remarkable, early diagnosis and appropriate selection of treatment is becoming more and more important for patients with amyloidosis.

Innate immunity to prions: anti-prion systems turn a tsunami of prions into a slow drip.

The yeast prions (infectious proteins) [URE3] and [PSI+] are essentially non-functional (or even toxic) amyloid forms of Ure2p and Sup35p, whose normal function is in nitrogen catabolite repression and translation termination, respectively. Yeast has an array of systems working in normal cells that largely block infection with prions, block most prion formation, cure most nascent prions and mitigate the toxic effects of those prions that escape the first three types of systems. Here we review recent progress in defining these anti-prion systems, how they work and how they are regulated. Polymorphisms of the prion domains partially block infection with prions. Ribosome-associated chaperones ensure proper folding of nascent proteins, thus reducing [PSI+] prion formation and curing many [PSI+] variants that do form. Btn2p is a sequestering protein which gathers [URE3] amyloid filaments to one place in the cells so that the prion is often lost by progeny cells. Proteasome impairment produces massive overexpression of Btn2p and paralog Cur1p, resulting in [URE3] curing. Inversely, increased proteasome activity, by derepression of proteasome component gene transcription or by 60S ribosomal subunit gene mutation, prevents prion curing by Btn2p or Cur1p. The nonsense-mediated decay proteins (Upf1,2,3) cure many nascent [PSI+] variants by associating with Sup35p directly. Normal levels of the disaggregating chaperone Hsp104 can also cure many [PSI+] prion variants. By keeping the cellular levels of certain inositol polyphosphates / pyrophosphates low, Siw14p cures certain [PSI+] variants. It is hoped that exploration of the yeast innate immunity to prions will lead to discovery of similar systems in humans.

Exploring the sequence features determining amyloidosis in human antibody light chains.

The light chain (AL) amyloidosis is caused by the aggregation of light chain of antibodies into amyloid fibrils. There are plenty of computational resources available for the prediction of short aggregation-prone regions within proteins. However, it is still a challenging task to predict the amyloidogenic nature of the whole protein using sequence/structure information. In the case of antibody light chains, common architecture and known binding sites can provide vital information for the prediction of amyloidogenicity at physiological conditions. Here, in this work, we have compared classical sequence-based, aggregation-related features (such as hydrophobicity, presence of gatekeeper residues, disorderness, ß-propensity, etc.) calculated for the CDR, FR or VL regions of amyloidogenic and non-amyloidogenic antibody light chains and implemented the insights gained in a machine learning-based webserver called "VLAmY-Pred" ( https://web.iitm.ac.in/bioinfo2/vlamy-pred/ ). The model shows prediction accuracy of 79.7% (sensitivity: 78.7% and specificity: 79.9%) with a ROC value of 0.88 on a dataset of 1828 variable region sequences of the antibody light chains. This model will be helpful towards improved prognosis for patients that may likely suffer from diseases caused by light chain amyloidosis, understanding origins of aggregation in antibody-based biotherapeutics, large-scale in-silico analysis of antibody sequences generated by next generation sequencing, and finally towards rational engineering of aggregation resistant antibodies.

Rational affinity maturation of anti-amyloid antibodies with high conformational and sequence specificity.

The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate high-quality conformational antibodies in a systematic and site-specific manner due to the properties of protein aggregates (hydrophobic, multivalent, and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer's Aß fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to mediate high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aß42 aggregates, and identified promising clones using deep sequencing. The resulting antibodies displayed similar or higher affinities than clinical-stage Aß antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retained high conformational specificity for Aß aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinity-maturated antibodies displayed extremely low levels of nonspecific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers.

Molecular mechanism of amyloidogenic mutations in hypervariable regions of antibody light chains.

Systemic light chain (AL) amyloidosis is a fatal protein misfolding disease in which excessive secretion, misfolding, and subsequent aggregation of free antibody light chains eventually lead to deposition of amyloid plaques in various organs. Patient-specific mutations in the antibody VL domain are closely linked to the disease, but the molecular mechanisms by which certain mutations induce misfolding and amyloid aggregation of antibody domains are still poorly understood. Here, we compare a patient VL domain with its nonamyloidogenic germline counterpart and show that, out of the five mutations present, two of them strongly destabilize the protein and induce amyloid fibril formation. Surprisingly, the decisive, disease-causing mutations are located in the highly variable complementarity determining regions (CDRs) but exhibit a strong impact on the dynamics of conserved core regions of the patient VL domain. This effect seems to be based on a deviation from the canonical CDR structures of CDR2 and CDR3 induced by the substitutions. The amyloid-driving mutations are not necessarily involved in propagating fibril formation by providing specific side chain interactions within the fibril structure. Rather, they destabilize the VL domain in a specific way, increasing the dynamics of framework regions, which can then change their conformation to form the fibril core. These findings reveal unexpected influences of CDR-framework interactions on antibody architecture, stability, and amyloid propensity.

Antibody Fragments as Tools for Elucidating Structure-Toxicity Relationships and for Diagnostic/Therapeutic Targeting of Neurotoxic Amyloid Oligomers.

The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.

Targeting Pathological Amyloid Aggregates with Conformation-Sensitive Antibodies.

BACKGROUND: The pathogenesis of Alzheimer's disease (AD) is not directly caused by the presence of senile plaques but rather by the detrimental effects exerted on neuronal cells by toxic soluble oligomers. Such species are formed early during the aggregation process of the Aß1-42 peptide or can be released from mature fibrils. Nowadays, efficient tools for an early diagnosis, as well as pharmaceutical treatments targeting the harmful agents in samples of AD patients, are still missing. OBJECTIVE: By integrating in vitro immunochemical assay with in vivo neuronal models of toxicity, we aim to understand and target the principles that drive toxicity in AD. METHODS: We evaluated the specificity and sensitivity of A11 and OC conformational antibodies to target a range of pathologically relevant amyloid conformers and rescue their cytotoxic effects in neuronal culture models using a number of cellular readouts. RESULTS: We demonstrated the peculiar ability of conformational antibodies to label pathologically relevant Aß1-42 oligomers and fibrils and to prevent their detrimental effects on neuronal cells. CONCLUSION: Our results substantially improve our knowledge on the role of toxic assemblies in neurodegenerative diseases, thus suggesting new and more effective diagnostic and therapeutic tools for AD.

Domain Interactions Determine the Amyloidogenicity of Antibody Light Chain Mutants.

In antibody light chain amyloidosis (AL), mutant light chains (LCs) or their variable domains (VLs) form fibrils, which accumulate in organs and lead to their failure. The molecular mechanism of this disease is still poorly understood. One of the key open issues is whether the mutant VLs and LCs differ in fibril formation. We addressed this question studying the effects of the VL mutations S20N and R61A within the isolated VL domain and in the full-length LC scaffold. Both VL variants readily form fibrils. Here, we find that in the LC context, the S20N variant is protected from fibril formation while for LC R61A fibril formation is even accelerated compared to VL R61A. Our analyses revealed that the partially unfolded state of the VL R61A domain destabilizes the CL domain by non-native interactions, in turn leading to a further unfolding of the VL domain. In contrast, the folded mutant VL S20N and VL wt form native interactions with CL. These are beneficial for LC stability and promote amyloid resistance. Thus the effects of specific mutations on the VL fold can have opposing effects on LC domain interactions, stability and amyloidogenicity.

Development of Conformational Antibodies to Detect Bcl-xL's Amyloid Aggregates in Metal-Induced Apoptotic Neuroblastoma Cells.

Bcl-xL, a member of the Bcl-2 family, is a pro-survival protein involved in apoptosis regulation. We have previously reported the ability of Bcl-xL to form various types of fibers, from native to amyloid conformations. Here, we have mimicked the effect of apoptosis-induced caspase activity on Bcl-xL by limited proteolysis using trypsin. We show that cleaved Bcl-xL (ΔN-Bcl-xL) forms fibers that exhibit the features of amyloid structures (BclxLcf37). Moreover, three monoclonal antibodies (mAbs), produced by mouse immunization and directed against ΔN-Bcl-xL or Bcl-xL fibers, were selected and characterized. Our results show that these mAbs specifically target ΔN-Bcl-xL in amyloid fibers in vitro. Upon metal-stress-induced apoptosis, these mAbs are able to detect the presence of Bcl-xL in amyloid aggregates in neuroblastoma SH-SY5Y cell lines. In conclusion, these specific mAbs directed against amyloidogenic conformations of Bcl-xL constitute promising tools for studying, in vitro and in cellulo, the contribution of Bcl-xL in apoptosis. These mAbs may further help in developing new diagnostics and therapies, considering Bcl-xL as a strategic target for treating brain lesions relevant to stroke and neurodegenerative diseases.

Salmonella Typhimurium biofilm disruption by a human antibody that binds a pan-amyloid epitope on curli.

Bacterial biofilms, especially those associated with implanted medical devices, are difficult to eradicate. Curli amyloid fibers are important components of the biofilms formed by the Enterobacteriaceae family. Here, we show that a human monoclonal antibody with pan-amyloid-binding activity (mAb 3H3) can disrupt biofilms formed by Salmonella enterica serovar Typhimurium in vitro and in vivo. The antibody disrupts the biofilm structure, enhancing biofilm eradication by antibiotics and immune cells. In mice, 3H3 injections allow antibiotic-mediated clearance of catheter-associated S. Typhimurium biofilms. Thus, monoclonal antibodies that bind a pan-amyloid epitope have potential to prevent or eradicate bacterial biofilms.

Type I interferon response drives neuroinflammation and synapse loss in Alzheimer disease.

Type I interferon (IFN) is a key cytokine that curbs viral infection and cell malignancy. Previously, we demonstrated a potent IFN immunogenicity of nucleic acid-containing (NA-containing) amyloid fibrils in the periphery. Here, we investigated whether IFN is associated with ß-amyloidosis inside the brain and contributes to neuropathology. An IFN-stimulated gene (ISG) signature was detected in the brains of multiple murine Alzheimer disease (AD) models, a phenomenon also observed in WT mouse brain challenged with generic NA-containing amyloid fibrils. In vitro, microglia innately responded to NA-containing amyloid fibrils. In AD models, activated ISG-expressing microglia exclusively surrounded NA+ amyloid ß plaques, which accumulated in an age-dependent manner. Brain administration of rIFN-ß resulted in microglial activation and complement C3-dependent synapse elimination in vivo. Conversely, selective IFN receptor blockade effectively diminished the ongoing microgliosis and synapse loss in AD models. Moreover, we detected activated ISG-expressing microglia enveloping NA-containing neuritic plaques in postmortem brains of patients with AD. Gene expression interrogation revealed that IFN pathway was grossly upregulated in clinical AD and significantly correlated with disease severity and complement activation. Therefore, IFN constitutes a pivotal element within the neuroinflammatory network of AD and critically contributes to neuropathogenic processes.

Study of the complement activation by amyloid aggregates of smooth muscle titin in vitro.

The giant muscle protein, titin, is the third most abundant protein in muscle (after myosin and actin). It was shown previously that smooth muscle titin (SMT) with a molecular mass of 500 kDa can form in vitro amorphous amyloid aggregates in two conditions: in solution of low ionic strength (0.15 M Glycine-KOH, pH 7.0) (SMT(Gly) aggregates) and in solution with ionic strength in the physiological range (0.2 M KCl, 20 mM imidazole, pH 7.2-7.4) (SMT(KCl) aggregates). Such aggregation in vivo, which may play a pathological or functional role, is not excluded. In view of the fact that some pathological amyloids can activate the classical and alternative pathways of complement system, we investigated the binding of complement component C1q and C3b to smooth muscle titin amyloid aggregates. The binding of С1q and C3b to SMT aggregates was not observed with ELISA assay. Since SMT aggregates do not activate the complement system, they are hardly implicated in the inflammatory process caused by muscle damage in amyloidoses.Abbreviations: SMT: smooth muscle titin; SMT(KCl) aggregates: SMT aggregates in solution containing 0.2 M KCl, 10 mM imidazole, pH 7.0; SMT(Gly) aggregates: SMT aggregates in solution containing 0.15 M glycine-KOH, pH 7.2-7.4; MAC: membrane attack complex; DLS: dynamic light scattering; NHS: Normal Human Serum.

Differential Roles of Plasma Protein Corona on Immune Cell Association and Cytokine Secretion of Oligomeric and Fibrillar Beta-Amyloid.

Alzheimer's disease (AD) is a primary neurological disease with no effective cure. A hallmark of AD is the presence of intracellular tangles and extracellular plaques derived from the aberrant aggregation of tau- and beta-amyloid (Aß). Aß presents in the brain as well as in cerebrospinal fluid and the circulation, and Aß toxicity has been attributed to amyloidosis and inflammation, among other causes. In this study, the effects of the plasma protein corona have been investigated with regard to the blood cell association and cytokine secretion of oligomeric (Aßo) and fibrillar Aß1-42(Aßf), two major forms of the peptide aggregates. Aßo displayed little change in membrane association in whole blood or washed blood (i.e., cells in the absence of plasma proteins) at 37 °C, while Aßf showed a clear preference for binding with all cell types sans plasma proteins. Immune cells exposed to Aßo, but not to Aßf, resulted in significant expression of cytokines IL-6 and TNF measured in real-time by a localized surface plasmon resonance sensor. These observations indicate greater immune cell association and cytokine stimulation of Aßo than Aßf and shed new light on the contrasting toxicities of Aßo and Aßf resulting from their differential capacities in acquiring a plasma protein corona. These results further implicate a close connection between Aß amyloidosis and immunopathology in AD.