Search

Background and Aims Glycoprotein VI (GPVI) is a platelet collagen/fibrin(ogen) receptor and an emerging pharmacological target for the treatment of thrombotic and thrombo-inflammatory diseases, notably ischaemic stroke. A first anti-human GPVI (hGPVI) antibody Fab-fragment (ACT017/glenzocimab, K D: 4.1 nM) recently passed a clinical phase 1b/2a study in patients with acute ischaemic stroke and was found to be well tolerated, safe, and potentially beneficial. In this study, a novel humanized anti-GPVI antibody Fab-fragment (EMA601; K D: 0.195 nM) was developed that inhibits hGPVI function with very high potency in vitro and in vivo. Methods Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6tg/tg) mouse platelets. The in vivo effect of Emf6.1Fab was assessed in a tail bleeding assay, an arterial thrombosis model and the transient middle cerebral artery occlusion (tMCAO) model of ischaemic stroke. Using complementary-determining region grafting, a humanized version of Emf6.1Fab (EMA601) was generated. Emf6.1Fab/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry. Results Emf6.1Fab (K D: 0.427 nM) blocked GPVI function in human and hGP6tg/tg mouse platelets in multiple assays in vitro at concentrations ≥5 µg/mL. Emf6.1Fab (4 mg/kg)-treated hGP6tg/tg mice showed potent hGPVI inhibition ex vivo and were profoundly protected from arterial thrombosis as well as from cerebral infarct growth after tMCAO, whereas tail-bleeding times remained unaffected. Emf6.1Fab binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (K D: 0.195 nM) and fully inhibited the receptor at 0.5 µg/mL, corresponding to a >50-fold potency compared with ACT017. Conclusions EMA601 is a conceptually novel and promising anti-platelet agent to efficiently prevent or treat arterial thrombosis and thrombo-inflammatory pathologies in humans at risk. [ABSTRACT FROM AUTHOR]

Aims: The aggregation and deposition of amyloid‐β (Aβ) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer's disease (AD). Aside from full‐length Aβ peptides starting with an aspartate residue in position 1, both N‐terminally truncated and elongated Aβ peptides are produced by various proteases from the amyloid precursor protein (APP) and have been detected in brain tissues and body fluids. Recently, we demonstrated that the particularly abundant N‐terminally truncated Aβ4–x peptides are generated by ADAMTS4, a secreted metalloprotease that is exclusively expressed in the oligodendrocyte cell population. In this study, we investigated whether ADAMTS4 might also be involved in the generation of N‐terminally elongated Aβ peptides. Methods: We used cell‐free and cell‐based assays in combination with matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOF) and electrochemiluminescence sandwich immunoassays to identify and quantify N‐terminally elongated Aβ peptide variants. Antibodies against these Aβ variants were characterised by peptide microarrays and employed for the immunohistochemical analyses of human brain samples. Results: In this study, we discovered additional ADAMTS4 cleavage sites in APP. These were located N‐terminal to Asp‐(1) in the Aβ peptide sequence between residues Glu‐(‐7) and Ile‐(‐6) as well as Glu‐(‐4) and Val‐(‐3), resulting in the release of N‐terminally elongated Aβ‐6‐x and Aβ‐3‐x peptides, of which the latter serve as a component in a promising Aβ‐based plasma biomarker. Aβ‐6/‐3‐40 peptides were detected in supernatants of various cell lines and in the cerebrospinal fluid (CSF), and ADAMTS4 enzyme activity promoted the release of Aβ‐6/‐3‐x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N‐terminally elongated Aβ‐6/‐3‐x peptides. Discussion: The current findings implicate ADAMTS4 in both the pathological process of Aβ peptide aggregation and in the early detection of amyloid pathology in AD. [ABSTRACT FROM AUTHOR]

Introduction: Subgroups of autoantibodies directed against voltage-gated potassium channel (Kv) complex components have been associated with immunotherapy-responsive clinical syndromes. The high prevalence and the role of autoantibodies directly binding Kv remain, however, controversial. Our objective was to determine Kv autoantibody binding requirements and to clarify their contribution to the observed immune response. Methods: Binding epitopes were studied in sera (n = 36) and cerebrospinal fluid (CSF) (n = 12) from a patient cohort positive for Kv1.2 but negative for 32 common neurological autoantigens and controls (sera n = 18 and CSF n = 5) by phospho and deep mutational scans. Autoantibody specificity and contribution to the observed immune response were resolved on recombinant cells, cerebellum slices, and nerve fibers. Results: 83% of the patients (30/36) within the studied cohort shared one out of the two major binding epitopes with Kv1.2-3 reactivity. Eleven percent (4/36) of the serum samples showed no binding. Fingerprinting resolved close to identical sequence requirements for both shared epitopes. Kv autoantibody response is directed against juxtaparanodal regions in peripheral nerves and the axon initial segment in central nervous system neurons and exclusively mediated by the shared epitopes. Discussion: Systematic mapping revealed two shared autoimmune responses, with one dominant Kv1.2-3 autoantibody epitope being unexpectedly prevalent. The conservation of the molecular binding requirements among these patients indicates a uniform autoantibody repertoire with monospecific reactivity. The enhanced sensitivity of the epitope-based (10/12) compared with that of the cellbased detection (7/12) highlights its use for detection. The determined immunodominant epitope is also the primary immune response visible in tissue, suggesting a diagnostic significance and a specific value for routine screening. [ABSTRACT FROM AUTHOR]

Introduction: Subgroups of autoantibodies directed against voltage-gated potassium channel (K v ) complex components have been associated with immunotherapy-responsive clinical syndromes. The high prevalence and the role of autoantibodies directly binding K v remain, however, controversial. Our objective was to determine K v autoantibody binding requirements and to clarify their contribution to the observed immune response., Methods: Binding epitopes were studied in sera (n = 36) and cerebrospinal fluid (CSF) (n = 12) from a patient cohort positive for K v 1.2 but negative for 32 common neurological autoantigens and controls (sera n = 18 and CSF n = 5) by phospho and deep mutational scans. Autoantibody specificity and contribution to the observed immune response were resolved on recombinant cells, cerebellum slices, and nerve fibers., Results: 83% of the patients (30/36) within the studied cohort shared one out of the two major binding epitopes with K v 1.2-3 reactivity. Eleven percent (4/36) of the serum samples showed no binding. Fingerprinting resolved close to identical sequence requirements for both shared epitopes. K v autoantibody response is directed against juxtaparanodal regions in peripheral nerves and the axon initial segment in central nervous system neurons and exclusively mediated by the shared epitopes., Discussion: Systematic mapping revealed two shared autoimmune responses, with one dominant K v 1.2-3 autoantibody epitope being unexpectedly prevalent. The conservation of the molecular binding requirements among these patients indicates a uniform autoantibody repertoire with monospecific reactivity. The enhanced sensitivity of the epitope-based (10/12) compared with that of the cell-based detection (7/12) highlights its use for detection. The determined immunodominant epitope is also the primary immune response visible in tissue, suggesting a diagnostic significance and a specific value for routine screening., Competing Interests: MS, RM, ID and LK are employees of the Euroimmun AG, a company that develops, produces, and manufactures immunoassays for the detection of disease-associated antibodies. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The remaining authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Talucci, Arlt, Kreissner, Nasouti, Wiessler, Miske, Mindorf, Dettmann, Moniri, Bayer, Broegger Christensen, Ayzenberg, Kraft, Endres, Komorowski, Villmann, Doppler, Prüss and Maric.)

Background and Objectives: Stiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyRα subunit-binding autoantibodies with subsequent changes in function and surface numbers were reported. GlyR autoantibodies have also been described in patients with focal epilepsy. Autoimmune reactivity against the GlyRβ subunits has not yet been shown. Autoantibodies against GlyRα1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyRβ making it not unlikely that GlyRβ-specific autoantibody (aAb) exist and contribute to the disease pathology., Methods: In this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyRβ. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyRβ binding and define aAb binding to distinct protein regions. Preadsorption approaches of aAbs using living cells and the purified extracellular receptor domain were further used. Finally, functional consequences for inhibitory neurotransmission upon GlyRβ aAb binding were resolved by whole-cell patch-clamp recordings., Results: Among 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyRβ aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyRβ colocalized with the scaffold protein gephyrin independent of the presence of GlyRα1. At the functional level, binding of GlyRβ aAb from both patients to its target impair glycine efficacy., Discussion: Our study establishes GlyRβ as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyRα1-positive sera, which alter glycine potency, aAbs against GlyRβ impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyRβ aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization.

Antibody-mediated neurological diseases constitute an emerging clinical entity that remains to be fully explored. Recent studies identified autoantibodies that directly confer pathogenicity, and it was shown that in these cases immunotherapies can result in profound positive patient responses. These advances highlight the urgent need for improved means to effectively screen patient samples for novel autoantibodies (aAbs) and their subsequent characterization. Here, we discuss challenges and opportunities for peptide microarrays to contribute to the identification, mapping, and characterization of the underlying monospecific disease-defining binding surfaces. We outline control experiments, workflow modifications and bioinformatic filtering methods that enhance the predictive power of array-based studies. Further, we highlight experimental and computer-based display approaches that have the potential to expand the use of synthetic microarrays over the detection of discontinuous epitopes. Knowledge over the autoantibody epitopes in neurological disease will enhance our understanding of the pathological mechanisms and thereby potentially contribute to novel diagnostic approaches or even innovative antigen-specific treatments that avoid the serious adverse effects seen with currently used immunosuppressive therapies., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)