Monoclonal Autoantibodies Research Articles

Next-generation cell-penetrating antibodies for tumor targeting and RAD51 inhibition.

Monoclonal antibody therapies for cancer have demonstrated extraordinary clinical success in recent years. However, these strategies are thus far mostly limited to specific cell surface antigens, even though many disease targets are found intracellularly. Here we report studies on the humanization of a full-length, nucleic acid binding, monoclonal lupus-derived autoantibody, 3E10, which exhibits a novel mechanism of cell penetration and tumor specific targeting. Comparing humanized variants of 3E10, we demonstrate that cell uptake depends on the nucleoside transporter ENT2, and that faster cell uptake and superior in vivo tumor targeting are associated with higher affinity nucleic acid binding. We show that one human variant retains the ability of the parental 3E10 to bind RAD51, serving as a synthetically lethal inhibitor of homology-directed repair in vitro. These results provide the basis for the rational design of a novel antibody platform for therapeutic tumor targeting with high specificity following systemic administration.

Human cerebrospinal fluid monoclonal CASPR2 autoantibodies induce changes in electrophysiology, functional MRI, and behavior in rodent models

Anti-contactin associated protein receptor 2 (CASPR2) encephalitis is a severe autoimmune encephalitis with a variable clinical phenotype including behavioral abnormalities, cognitive decline, epileptic seizures, peripheral nerve hyperexcitability and neuropathic pain. The detailed mechanisms of how CASPR2 autoantibodies lead to synaptic dysfunction and clinical symptoms are largely unknown. Aiming for analyses from the molecular to the clinical level, we isolated antibody-secreting cells from the cerebrospinal fluid of two patients with CASPR2 encephalitis. From these we cloned four anti-CASPR2 human monoclonal autoantibodies (mAbs) with strong binding to brain and peripheral nerves. All were highly hypermutated and mainly of the IgG4 subclass. Mutagenesis studies determined selective binding to the discoidin domain of CASPR2. Surface plasmon resonance revealed affinities with dissociation constants KD in the pico- to nanomolar range. CASPR2 mAbs interrupted the interaction of CASPR2 with its binding partner contactin 2 in vitro and were internalized after binding to CASPR2-expressing cells. Electrophysiological recordings of rat hippocampal slices after stereotactic injection of CASPR2 mAbs showed characteristic afterpotentials following electrical stimulation. In vivo experiments with intracerebroventricular administration of human CASPR2 mAbs into mice and rats showed EEG-recorded brain hyperexcitability but no spontaneous recurrent seizures. Behavioral assessment of infused mice showed a subtle clinical phenotype, mainly affecting sociability. Mouse brain MRI exhibited markedly reduced resting-state functional connectivity without short-term structural changes. Together, the experimental data support the direct pathogenicity of CASPR2 autoantibodies. The minimally invasive EEG and MRI techniques applied here may serve as novel objective, quantifiable tools for improved animal models, in particular for subtle neuropsychiatric phenotypes or repeated measurements.

Abstract We110: Novel allosteric ligand designed to prevent Autoantibody targeting the Angiotensin Type 1 Receptor (AT1R) in cardiovascular diseases (CVD)

Background: The clinical data suggest an association of autoantibodies (AAb) targeting angiotensin II type1 receptor (AT1R) and outcomes in heart failure (HF) and preeclampsia. Notably, patient’s risk increases 2 to 4-fold for heart diseases and hypertension. Although AT1R blockers (ARBs) have emerged as a key component in the therapeutic arsenal against CVD, we believe that rather than completely blocking AT1R signaling, maintaining a balanced signaling is crucial for cardiovascular homeostasis. Therefore, we posit that reducing AT1R overactivation and blocking AAb-mediated activation could benefit cardiac health and attenuate the progression of CVD. In our previous study (PMID:36440576), we identified small molecules capable of inhibiting AAb-induced AT1R signaling. Leveraging these observations, we have developed a novel AT1R allosteric ligand that inhibits autoantibody binding to AT1R while still retaining normal AT1R signaling. Methods: Using computational and medicinal chemistry approaches, we designed a series of novel AT1R ligands and obtained compounds with >98% purity through chemical synthesis. Monoclonal antibodies mimicking AT1R-AAb have been generated. Serum samples from dilated cardiomyopathy (DCM) and preeclampsia (PE) patient cohorts were collected and presence of AT1R-AAb was determined using ELISA and epitope-peptide competition studies. Pharmacological characterization of compounds was performed using calcium mobilization, 125I -AngII binding, ex vivo vasoconstriction, and ELISA assays. Results: We identified an AT1R allosteric compound that reduced AngII-mediated calcium signaling with a 5-fold shift in EC 50 . The IC 50 was determined to be 18 µM. It reduces the AngII-mediated vasoconstriction while blocking both monoclonal and patient-derived autoantibody binding with an IC 50 of 5 µM. Conclusion: We have identified a novel AT1R allosteric ligand with negative allosteric modulator (NAM) properties, which has the potential to block autoantibodies and could serve as a new generation of ARBs. Uniquely, it retains beneficial AT1R signaling without competing with AngII, and its allosteric ligand property can be tailored to achieve optimal AT1R signaling.

A Comparison of Natural and Therapeutic Anti-IgE Antibodies.

Immunoglobulin E (IgE) plays a critical role for the immune system, fighting against parasites, toxins, and cancer. However, when it reacts to allergens without proper regulation, it can cause allergic reactions, including anaphylaxis, through a process initiated by effector cells such as basophils and mast cells. These cells display IgE on their surface, bound to the high-affinity IgE receptor FcεRI. A cross-linking antigen then triggers degranulation and the release of inflammatory mediators from the cells. Therapeutic monoclonal anti-IgE antibodies such as omalizumab, disrupt this process and are used to manage IgE-related conditions such as severe allergic asthma and chronic spontaneous urticaria. Interestingly, naturally occurring anti-IgE autoantibodies circulate at surprisingly high levels in healthy humans and mice and may thus be instrumental in regulating IgE activity. Although many open questions remain, recent studies have shed new light on their role as IgE regulators and their mechanism of action. Here, we summarize the latest insights on natural anti-IgE autoantibodies, and we compare their functional features to therapeutic monoclonal anti-IgE autoantibodies.

Converging synaptic and network dysfunctions in distinct autoimmune encephalitis

Psychiatric and neurological symptoms, as well as cognitive deficits, represent a prominent phenotype associated with variable forms of autoimmune encephalitis, regardless of the neurotransmitter receptor targeted by autoantibodies. The mechanistic underpinnings of these shared major neuropsychiatric symptoms remain however unclear. Here, we investigate the impacts of patient-derived monoclonal autoantibodies against the glutamatergic NMDAR (NMDAR mAb) and inhibitory GABAaR (GABAaR mAb) signalling in the hippocampal network. Unexpectedly, both excitatory and inhibitory synaptic receptor membrane dynamics, content and transmissions are altered by NMDAR or GABAaR mAb, irrespective of the affinity or antagonistic effect of the autoantibodies. The effect of NMDAR mAb on inhibitory synapses and GABAaR mAb on excitatory synapses requires neuronal activity and involves protein kinase signalling. At the cell level, both autoantibodies increase the excitation/inhibition balance of principal cell inputs. Furthermore, NMDAR or GABAaR mAb leads to hyperactivation of hippocampal networks through distinct alterations of principal cell and interneuron properties. Thus, autoantibodies targeting excitatory NMDAR or inhibitory GABAaR trigger convergent network dysfunctions through a combination of shared and distinct mechanisms.

The Role and Mechanism of Autoantibodies in Dysimmunity Related Neurological Diseases

Autoantibodies are antibodies produced by B cells capable of attacking their own tissues or organs after loss of immune tolerance. A large body of literature shows that autoantibodies play an important role in neurological diseases associated with immune abnormalities. Nowadays, neurological disorders are an important cause of disability and death in populations around the world, so it is important to study the role and mechanisms of autoantibodies in neurological disorders associated with immune abnormalities. The blood-brain barrier is biologically important for maintaining the normal physiological state of the central nervous system, and we have previously found that monoclonal autoantibodies of SLE origin can inhibit the expression of tight junction proteins in the blood-brain barrier. Our findings provide clues for the study of the role of autoantibodies in neurological disorders and greatly attract us to conduct a more in-depth and systematic review and synthesis. In this paper, we summarize the functions of autoantibodies in autoimmune and non-autoimmune diseases and focus on the roles and mechanisms of autoantibodies in immune-associated neurological disorders by taking central and peripheral neurological diseases as the entry points, which can provide new molecular targets and new theoretical basis for the specific diagnosis and targeted therapy of neurological diseases.

TSH receptor specific monoclonal autoantibody K1-70TM targeting of the TSH receptor in subjects with Graves' disease and Graves' orbitopathy-Results from a phase I clinical trial

TSH receptor specific monoclonal autoantibody K1-70TM targeting of the TSH receptor in subjects with Graves' disease and Graves' orbitopathy-Results from a phase I clinical trial

Regulatory T-cell deficiency leads to features of autoimmune liver disease overlap syndrome in scurfy mice.

Scurfy mice have a complete deficiency of functional regulatory T cells (Treg) due to a frameshift mutation in the Foxp3 gene. The impaired immune homeostasis results in a lethal lymphoproliferative disorder affecting multiple organs, including the liver. The autoimmune pathology in scurfy mice is in part accompanied by autoantibodies such as antinuclear antibodies (ANA). ANA are serological hallmarks of several autoimmune disorders including autoimmune liver diseases (AILD). However, the underlying pathogenesis and the role of Treg in AILD remain to be elucidated. The present study therefore aimed to characterize the liver disease in scurfy mice. Sera from scurfy mice were screened for ANA by indirect immunofluorescence assay (IFA) and tested for a wide range of AILD-associated autoantibodies by enzyme-linked immunosorbent assay, line immunoassay, and addressable laser bead immunoassay. CD4+ T cells of scurfy mice were transferred into T cell-deficient B6/nude mice. Monoclonal autoantibodies from scurfy mice and recipient B6/nude mice were tested for ANA by IFA. Liver tissue of scurfy mice was analyzed by conventional histology. Collagen deposition in scurfy liver was quantified via hepatic hydroxyproline content. Real-time quantitative PCR was used to determine fibrosis-related hepatic gene expression. Hepatic immune cells were differentiated by flow cytometry. All scurfy mice produced ANA. AILD-associated autoantibodies, predominantly antimitochondrial antibodies, were detected at significantly higher levels in scurfy sera. CD4+ T cells from scurfy mice were sufficient to induce anti-dsDNA autoantibodies and ANA with an AILD-related nuclear envelope staining pattern. Liver histology revealed portal inflammation with bile duct damage and proliferation, as in primary biliary cholangitis (PBC), and interface hepatitis with portal-parenchymal necroinflammation, as found in autoimmune hepatitis (AIH). In scurfy liver, TNFα and fibrosis-related transcripts including Col1a1, Timp1, Acta2, Mmp2, and Mmp9 were upregulated. The level of proinflammatory monocytic macrophages (Ly-6Chi) was increased, while M2-type macrophages (CD206+) were downregulated compared to wildtype controls. Despite severe hepatic inflammation, fibrosis did not develop within 25 days, which is close to the lifespan of scurfy mice. Our findings suggest that Treg-deficient scurfy mice spontaneously develop clinical, serological, and immunopathological characteristics of AILD with overlapping features of PBC and AIH.

The mechanisms of immunopathology underlying B cell depletion therapy-mediated remission and relapse in patients with MuSK MG

The application of reverse translational medicine allows for the understanding of immune pathogenesis via therapeutic intervention. We applied this approach to the MuSK subtype of myasthenia gravis. Treatment with the CD20-specific B cell depletion therapy (BCDT) demonstrated that MuSK MG patients respond remarkably well; the majority invariably reach remission accompanied by a remarkable drop in autoantibody levels. Circulating antibodies are primarily produced by bone marrow resident plasma cells, which do not express CD20. So, how does BCDT diminish MuSK autoantibodies and induce rapid remission? We developed a mechanistic model, which hypothesized that plasmablasts, which are short-lived antibody secreting B cell populations, produce MuSK-specific autoantibodies. Anti-CD20-mediated BCDT is expected to deplete CD20-expressing plasmablasts or CD20 expressing memory cells that supply the plasmablast population. To test this hypothesis, we performed a series of investigations, which were reported over the last seven years and are summarized in this review. First, we isolated plasmablasts from patients and generated human recombinant monoclonal autoantibodies (mAb) which bound MuSK and had pathogenic capacity, demonstrating that MuSK autoantibodies can be produced by this specific cell population. The characterization of the mAbs showed that MuSK autoantibodies can include unique properties including unusually high antigen binding affinity, and an elevated frequency of N-linked glycosylation in their binding domains. Further characterization suggested that MuSK autoantibody-producing cells may form in the early stages of B cell development due to defective tolerance mechanisms. Finally, we sought to determine how these pathogenic B cell clones behave over time. High throughput B cell receptor sequencing was applied to investigate longitudinally collected samples from patients treated with anti-CD20-mediated BCDT. MuSK-specific clonal variants were detected at multiple timepoints spanning more than five years and reemerged after BCDT-induced remission, predating disease relapse by several months. These collective investigations provide a more detailed mechanistic understanding that MuSK MG, the key features of which include production of autoantibodies by circulating plasmablasts that can be targeted by CD20-specific BCDT, and that pathogenic clones can survive BCDT and reemerge prior to manifestation of clinical relapse.

Unraveling Mechanisms of Autoimmune Skin Blistering: Applying Single-Cell Transcriptomics to Pemphigus B Cells

Unraveling Mechanisms of Autoimmune Skin Blistering: Applying Single-Cell Transcriptomics to Pemphigus B Cells

Microglia actively remove NR1 autoantibody-bound NMDA receptors and associated post-synaptic proteins in neuron microglia co-cultures.

Autoantibodies against the NR1 subunit of NMDA receptors (NMDARs) have been shown to promote crosslinking and internalization of bound receptors in NMDAR encephalitis (NMDARE). This internalization-mediated loss of NMDARs is thought to be the major mechanism leading to pathogenic outcomes in patients. However, the role of bound autoantibody in engaging the resident immune cells, microglia, remains poorly understood. Here, using a patient-derived monoclonal NR1 autoantibody (hNR1-mAb) and a co-culture system of microglia and neurons, we could show that hNR1-mAb bound to hippocampal neurons led to microglia-mediated removal of hNR1-mAb bound NMDARs. These complexes were found to accumulate inside endo-lysosomal compartments of microglia. Utilizing another patient isolated monoclonal autoantibody, against the α1-subunit of GABAA receptors (α1-GABAA -mAb), such removal of receptors was found to be specific to the antibody-bound receptor targets. Interestingly, along with receptor removal, we also observed a reduction in synapse number, more specifically in the numbers of post-synaptic proteins like PSD95 and Homer 1, when microglia were present in the culture. Importantly, mutations in the Fc region of hNR1-mAb, blocking its Fcγ receptor (FcγR) and complement binding, attenuated hNR1-mAb driven loss of NMDARs and synapses, indicating that microglia engagement by bound hNR1-mAb is critical for receptor and synapse loss. Our data argues for an active involvement of microglia in removal of NMDARs and other receptors in individuals with autoimmune encephalitis, thereby contributing to the etiology of these diseases.

Comparative Effects of Domain-Specific Human Monoclonal Antibodies Against LGI1 on Neuronal Excitability.

Autoantibodies to leucine-rich glioma inactivated protein 1 (LGI1) cause an autoimmune limbic encephalitis with frequent focal seizures and anterograde memory dysfunction. LGI1 is a neuronal secreted linker protein with 2 functional domains: the leucine-rich repeat (LRR) and epitempin (EPTP) regions. LGI1 autoantibodies are known to interfere with presynaptic function and neuronal excitability; however, their epitope-specific mechanisms are incompletely understood. We used patient-derived monoclonal autoantibodies (mAbs), which target either LRR or EPTP domains of LGI1 to investigate long-term antibody-induced alteration of neuronal function. LRR- and EPTP-specific effects were evaluated by patch-clamp recordings in cultured hippocampal neurons and compared with biophysical neuron modeling. Kv1.1 channel clustering at the axon initial segment (AIS) was quantified by immunocytochemistry and structured illumination microscopy techniques. Both EPTP and LRR domain-specific mAbs decreased the latency of first somatic action potential firing. However, only the LRR-specific mAbs increased the number of action potential firing together with enhanced initial instantaneous frequency and promoted spike-frequency adaptation, which were less pronounced after the EPTP mAb. This also led to an effective reduction in the slope of ramp-like depolarization in the subthreshold response, suggesting Kv1 channel dysfunction. A biophysical model of a hippocampal neuron corroborated experimental results and suggests that an isolated reduction of the conductance of Kv1-mediated K+ currents largely accounts for the antibody-induced alterations in the initial firing phase and spike-frequency adaptation. Furthermore, Kv1.1 channel density was spatially redistributed from the distal toward the proximal site of AIS under LRR mAb treatment and, to a lesser extant, under EPTP mAb. These findings indicate an epitope-specific pathophysiology of LGI1 autoantibodies. The pronounced neuronal hyperexcitability and SFA together with dropped slope of ramp-like depolarization after LRR-targeted interference suggest disruption of LGI1-dependent clustering of K+ channel complexes. Moreover, considering the effective triggering of action potentials at the distal AIS, the altered spatial distribution of Kv1.1 channel density may contribute to these effects through impairing neuronal control of action potential initiation and synaptic integration.

Monoclonal human auto-antibodies: novel tools for the understanding and treatment of neuropsychiatric symptoms

Monoclonal human auto-antibodies: novel tools for the understanding and treatment of neuropsychiatric symptoms

Cryo-electron microscopy structures of human thyroid peroxidase (TPO) in complex with TPO antibodies.

Determination of the structure of the extracellular domain of human thyroid peroxidase (hTPO) by cryo-electron microscopy (cryo-EM) is described. TPO, purified to homogeneity was complexed with the human TPO monoclonal autoantibody 2G4 Fab and also with a mouse monoclonal TPO antibody 4F5 Fab (which competes with autoantibody binding to TPO). Both complexes were analysed by cryo-EM. The two structures (global resolution 3.92Å and 3.4Å for the 2G4 complex and 4F5 complex respectively) show TPO as a monomer with 4 domains; the N-terminal domain, the peroxidase domain (POD), the complement control protein like (CCP) domain and the epidermal growth factor like (EGF) domain which are all visible in the structures. The relative positions of the domains are fixed with a disulphide bond between cysteine residues Cys146 in the POD and Cys756 in the CCP domain preventing significant flexibility of the molecule. The entrance to the enzyme active site, the haem group and the calcium binding site are clearly visible on the opposite side of the TPO molecule from the 2G4 and 4F5 binding sites. Extensive interactions are seen between TPO and the two antibodies which both bind to distinct epitopes on the POD domain, including some residues in the immunodominant region B (IDR-B) mainly via different residues. However the epitopes of the two antibodies contain three shared TPO residues. This is the first high resolution structure of TPO to be reported and it should help guide development of new inhibitors of TPO enzyme activity for therapeutic applications.

ADAMTS13 activity testing: evaluation of commercial platforms for diagnosis and monitoring of thrombotic thrombocytopenic purpura

BackgroundADAMTS13 activity is one of the key investigations needed to diagnose thrombotic thrombocytopenic purpura, and there are a number of different assays available to measure it. HemosIL AcuStar, a chemiluminescent immunoassay, was developed and used as a quicker, automated test. In clinical practice, discrepancies between AcuStar and the gold standard FRETS-VWF73 have been documented in a manner that would affect diagnosis and treatment. ObjectivesWe aimed to identify and highlight clinical situations where this discrepancy occurs and to attempt to determine the cause. MethodTherefore, we undertook a study to compare the FRETS-VWF73 assay with AcuStar, the Technozym Activity ELISA, and Ceveron FRET assays using a mixture of 94 retrospective and prospective patient samples. ResultsWe found that although the concordance between FRETS-VWF73 and the other methods was generally very good, discrepancies were found in a small number tested on AcuStar affecting diagnosis (5 of 32) and follow-up (7 of 51). A Wilcoxon test comparing FRETS-VWF73 to the AcuStar results suggested that the AcuStar results were significantly lower in 42 samples tested on all 4 platforms. We investigated potential causes for this difference by testing the impact of high vWF levels and addition of a monoclonal ADAMTS13 autoantibody (3H9) to samples. We found no impact of high vWF levels on interassay variability but found that 3H9 reduced ADAMTS13 activity levels much more in AcuStar and ELISA assays than in FRETS assays. ConclusionBased on our findings, we would suggest that when AcuStar is used upfront to guide management, a second testing method should be used in patients with an atypical thrombotic thrombocytopenic purpura presentation or unexpectedly slow ADAMTS13 recovery.

Nanoimmunosensor for the electrochemical detection of oncostatin M receptor and monoclonal autoantibodies in systemic sclerosis

Systemic sclerosis (SSc) is a chronic, autoimmune disease that primarily affects connective tissue. SSc can be classified into limited cutaneous (lSSc) and diffuse cutaneous (dSSc). Oncostatin M receptor (sOSMR) is an important inflammatory biomarker expressed in the serum of patients with autoimmune diseases. A nanoengineered immunosensor surface was developed. The biosensor was composed of a conductive layer of polypyrrole, electrodeposited gold nanoparticles, and sOSMR protein for anti-human OSMR monoclonal antibody biorecognition. The electrochemical response evaluated by cyclic voltammetry and electrochemical impedance spectroscopy indicated the detection of the target analyte present in clinical samples from lSSc and dSSc patients. The voltammetric anodic shift for lSSc specimens was 82.7% ± 0.9–93.6% ± 3.2, and dSSc specimens was 118.7 ± 2.6 to 379.6 ± 2.6, revealing a differential diagnostic character for SSc subtypes. The sensor platform was adapted for identifying sOSMR, using anti-OSMR antibodies as bioreceptors. With a linear response range estimated from 0.005 to 500 pg mL−1 and a limit of detection of 0.42 pg mL−1, the sensing strategy demonstrated high sensitivity in identifying the human OSMR protein in clinical samples. The proposed biosensor is a promising and innovative tool for SSc-related biomarker research.

Unexpected Crosslinking Effects of a Human Thyroid Stimulating Monoclonal Autoantibody, M22, with IGF1 on Adipogenesis in 3T3L-1 Cells.

To study the effects of the crosslinking of IGF1 and/or the human thyroid-stimulating monoclonal autoantibody (TSmAb), M22 on mouse adipocytes, two- and three-dimensional (2D or 3D) cultures of 3T3-L1 cells were prepared. Each sample was then subjected to the following analyses: (1) lipid staining, (2) a real-time cellular metabolic analysis, (3) analysis of the mRNA expression of adipogenesis-related genes and extracellular matrix (ECM) molecules including collagen (Col) 1, 4 and 6, and fibronectin (Fn), and (4) measurement of the size and physical properties of the 3D spheroids with a micro-squeezer. Upon adipogenic differentiation (DIF+), lipid staining and the mRNA expression of adipogenesis-related genes in the 2D- or 3D-cultured 3T3-L1 cells substantially increased. On adding IGF1 but not M22 to DIF+ cells, a significant enhancement in lipid staining and gene expressions of adipogenesis-related genes was detected in the 2D-cultured 3T3-L1 cells, although some simultaneous suppression or enhancement effects by IGF1 and M22 against lipid staining or Fabp4 expression, respectively, were detected in the 3D 3T3-L1 spheroids. Real-time metabolic analyses indicated that monotherapy with IGF1 or M22 shifted cellular metabolism toward energetic states in the 2D 3T3-L1 cells upon DIF+, although no significant metabolic changes were induced by DIF+ alone in 2D cultures. In addition, some synergistical effects on cellular metabolism by IGF1 and M22 were also observed in the 2D 3T3-L1 cells as well as in cultured non-Graves' orbitopathy-related human orbital fibroblasts (n-HOFs), but not in Graves' orbitopathy-related HOFs (GHOFs). In terms of the physical properties of the 3D 3T3-L1 spheroids, (1) their sizes significantly increased upon DIF+, and this increase was significantly enhanced by the presence of both IGF1 and M22 despite downsizing by monotreatment, and (2) their stiffness increased substantially, and no significant effects by IGF-1 and/or M22 were observed. Regarding the expression of ECM molecules, (1) upon DIF+, significant downregulation or upregulation of Col1 and Fn (3D), or Col4 and 6 (2D and 3D) were observed, and (2) in the presence of IGF-1 and/or M22, the mRNA expression of Col4 was significantly downregulated by M22 (2D and 3D), but the expression of Col1 was modulated in different manners by monotreatment (upregulation) or the combined treatment (downregulation) (3D). These collective data suggest that the human-specific TSmAb M22 induced some unexpected simultaneous crosslinking effects with IGF-1 with respect to the adipogenesis of 2D-cultured 3T3-L1 cells and the physical properties of 3D 3T3-L1 spheroids.

Structure of full-length TSH receptor in complex with antibody K1-70™.

Determination of the full-length thyroid-stimulating hormone receptor (TSHR) structure by cryo-electron microscopy (cryo-EM) is described. The TSHR complexed with human monoclonal TSHR autoantibody K1-70™ (a powerful inhibitor of TSH action) was detergent solubilised, purified to homogeneity and analysed by cryo-EM. The structure (global resolution 3.3 Å) is a monomer with all three domains visible: leucine-rich domain (LRD), hinge region (HR) and transmembrane domain (TMD). The TSHR extracellular domain (ECD, composed of the LRD and HR) is positioned on top of the TMD extracellular surface. Extensive interactions between the TMD and ECD are observed in the structure, and their analysis provides an explanation of the effects of various TSHR mutations on TSHR constitutive activity and on ligand-induced activation. K1-70™ is seen to be well clear of the lipid bilayer. However, superimposition of M22™ (a human monoclonal TSHR autoantibody which is a powerful stimulator of the TSHR) on the cryo-EM structure shows that it would clash with the bilayer unless the TSHR HR rotates upwards as part of the M22™ binding process. This rotation could have an important role in TSHR stimulation by M22™ and as such provides an explanation as to why K1-70™ blocks the binding of TSH and M22™ without activating the receptor itself.

Molecular disease mechanisms of human antineuronal monoclonal autoantibodies.

Autoantibodies targeting brain antigens can mediate a wide range of neurological symptoms ranging from epileptic seizures to psychosis to dementia. Although earlier experimental work indicated that autoantibodies can be directly pathogenic, detailed studies on disease mechanisms, biophysical autoantibody properties, and target interactions were hampered by the availability of human material and the paucity of monospecific disease-related autoantibodies. The emerging generation of patient-derived monoclonal autoantibodies (mAbs) provides a novel platform for the detailed characterization of immunobiology and autoantibody pathogenicity in vitro and in animal models. This Feature Review focuses on recent advances in mAb generation and discusses their potential as powerful scientific tools for high-resolution imaging, antigenic target identification, atomic-level structural analyses, and the development of antibody-selective immunotherapies.

Subdoligranulum chews up joints: how a gut pathobiont can instigate arthritis

The microbiota has been implicated in triggering certain autoimmune diseases. In rheumatoid arthritis (RA), the ‘mucosal origins’ hypothesis suggests that such a trigger can instigate systemic autoimmune responses that lead to synovial inflammation. Chriswell et al. recently identified a human gut commensal bound by monoclonal autoantibodies and eliciting autoantibody-mediated, transferable arthritis in gnotobiotic mouse models.