Human Monoclonal Autoantibodies Research Articles

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

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.

Cryo-electron microscopy structure of full length TSH receptor in complex with TSH receptor blocking human monoclonal autoantibody K1-70TM

Cryo-electron microscopy structure of full length TSH receptor in complex with TSH receptor blocking human monoclonal autoantibody K1-70TM

Human monoclonal autoantibodies in autoimmune basal ganglia encephalitis target dopamine receptors and act as drug agonists

Abstract Neuropsychiatric diseases in autoimmune basal ganglia encephalitis (BGE) are associated with anti-neuronal autoantibodies which target the basal ganglia. BGE includes the group A streptococcal sequelae Sydenham chorea (SC) and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). In SC, autoantibodies against dopamine receptors 1 and 2 (D1R and D2R) are elevated, and IgG in SC sera and cerebrospinal fluid (CSF) induce excessive dopaminergic D2R signaling and dopamine release. D1R autoantibody reactivity targeting the basal ganglia is not defined. Here we demonstrate specific IgG targeting of D1R in multiple neuropsychiatric PANDAS cohorts by serum, CSF, and human-derived mAbs. Anti-D1R human mAbs and serum autoantibodies induced D1R second messenger signaling in a dose-dependent manner. The results suggest that D1R autoantibodies enhanced antibody-mediated IgG signaling activity in the presence of the neurotransmitter dopamine. D1R autoantibodies sensitized D1R and exacerbated endogenous dopamine signaling in disease. Agonistic D1R signaling was inhibited by synthetic D1R peptide epitopes derived from human D1R extracellular loops which identified the immunodominant epitopes and antibody specificity. Taken together, D2R autoantibodies in BGE lead to excessive dopamine release and chorea, while D1R autoantibodies act like dopamine on receptors leading to allosteric enhancement and neuropsychiatric disease. Collectively, our novel findings suggest dopamine receptor autoantibodies in basal ganglia disorders act like drugs and mediate hyperactive dopaminergic receptor signaling.

DDX5-targeting fully human monoclonal autoantibody inhibits proliferation and promotes differentiation of acute promyelocytic leukemia cells by increasing ROS production

Acute promyelocytic leukemia (APL) therapy involves the compounds cytotoxic to both malignant tumor and normal cells. Relapsed APL is resistant to subsequent chemotherapy. Novel agents are in need to kill APL cells selectively with minimal toxicity. DDX5 has been recognized to be a novel target to suppress acute myeloid leukemia (AML). However, the role of DDX5 remains elusive in APL. Here a DDX5-targeting fully human monoclonal autoantibody named after 2F5 was prepared. It is demonstrated that 2F5 selectively inhibited APL cell proliferation without toxicity to normal neutrophil and tissues. Moreover, 2F5 was confirmed to induce G0/G1 phase arrest in APL cells, and promote APL cell differentiation combined with decreased DDX5 expression and increased reactive oxygen species (ROS) production. Knockdown of DDX5 by siRNA also inhibited proliferation, promoted cell differentiation and enhanced ROS production in APL cells. However, the ROS inhibitor reversed the effects of 2F5 on DDX5 and ROS in APL cells. Thus, we conclude that DDX5-targeting 2F5 inhibits APL cell proliferation, and promotes cell differentiation via induction of ROS. 2F5 showed the therapeutic value of fully human monoclonal autoantibody in APL, which provides a novel and valid approach for treatment of relapse/refractory APL.

Distinctive binding properties of human monoclonal LGI1 autoantibodies determine pathogenic mechanisms.

Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus. In live cell-based assays, LGI1 epitope recognition was examined with patient sera (n = 31), CSFs (n = 11), longitudinal serum samples (n = 15), and using mAbs (n = 14) generated from peripheral B cells of two patients. All sera and 9/11 CSFs bound both the leucine-rich repeat (LRR) and the epitempin repeat (EPTP) domains of LGI1, with stable ratios of LRR:EPTP antibody levels over time. By contrast, the mAbs derived from both patients recognized either the LRR or EPTP domain. mAbs against both domain specificities showed varied binding strengths, and marked genetic heterogeneity, with high mutation frequencies. LRR-specific mAbs recognized LGI1 docked to its interaction partners, ADAM22 and ADAM23, bound to rodent brain sections, and induced internalization of the LGI1-ADAM22/23 complex in both HEK293T cells and live hippocampal neurons. By contrast, few EPTP-specific mAbs bound to rodent brain sections or ADAM22/23-docked LGI1, but all inhibited the docking of LGI1 to ADAM22/23. After intrahippocampal injection, and by contrast to the LRR-directed mAbs, the EPTP-directed mAbs showed far less avid binding to brain tissue and were consistently detected in the serum. Post-injection, both domain-specific mAbs abrogated long-term potentiation induction, and LRR-directed antibodies with higher binding strengths induced memory impairment. Taken together, two largely dichotomous populations of LGI1 mAbs with distinct domain binding characteristics exist in the affinity matured peripheral autoantigen-specific memory pools of individuals, both of which have pathogenic potential. In human autoantibody-mediated diseases, the detailed characterization of patient mAbs provides a valuable method to dissect the molecular mechanisms within polyclonal populations.

Practical applications of studies on the TSH receptor and TSH receptor autoantibodies

Studies on the TSH receptor (TSHR) have numerous practical applications in vitro and in vivo. For example human monoclonal autoantibodies (MAbs) to the TSHR are useful reagents for in vitro diagnostics. Measurement of TSHR autoantibodies (TRAbs) is helpful in diagnosis and management of autoimmune thyroid disease. Currently available highly sensitive and specific assays to measure TRAbs use the human TSHR MAb M22 instead of the TSH. Furthermore, preparations of the human TSHR MAb M22 are useful as the World Health Organisation International Standard for thyroid stimulating antibody and for calibration of the assays for measuring TRAbs. Preparations of thermostabilised TSHR extracellular domain have recently become available and this is likely to have an impact on improvements in specificity testing for TRAb assays. In addition the stable TSHR preparations have practical application for specific immunoadsorption of patient serum TRAbs. Human TSHR MAbs also have promising prospects as new therapeutics. Autoantibodies with TSHR antagonistic activities are “natural” inhibitors of TSHR stimulation and are expected to be helpful in controlling TSHR activity in patients with Graves’ disease, Graves’ ophthalmopathy and thyroid cancer.

Monovalent IgG4 autoantibodies require self-antigen driven affinity maturation to acquire pathogenic capacity

Abstract INTRODUCTION The mechanisms underlying B cell-mediated autoimmune disease and the origin of autoreactive B cells are not well understood. Human monoclonal autoantibodies (mAbs) are valuable tools for investigating both. In this study, we used mAbs derived from patients with the autoimmune disorder, myasthenia gravis (MG). A subset of patients with MG have pathogenic autoantibodies that recognize muscle-specific tyrosine kinase (MuSK). The autoantibodies of MuSK MG are predominantly of the IgG4 subclass and functionally monovalent as a result of Fab-arm exchange. OBJECTIVE To gain insight into the origin and development of these unique autoantibodies. METHODS AND RESULTS We examined MG patient-derived mAbs, their corresponding germline-encoded unmutated common ancestors (UCA) and monovalent antigen-binding fragments (Fabs) to investigate how antigen-driven affinity maturation contributes to both binding and immunopathology. Mature mAbs, their UCA counterparts and mature monovalent Fabs bound to the MuSK autoantigen and retained their pathogenic capacity. However, monovalent UCA Fabs, which still bound the autoantigen, lost their pathogenic capacity. The mature Fabs were characterized by very high affinity (sub-nanomolar) driven by a rapid on-rate and slow off-rate. However, the UCA affinity was approximately 100-fold less than the mature Fabs, which was driven by a rapid off-rate. SUMMARY/CONCLUSION These findings indicate that the autoantigen initiates the autoimmune response in MuSK MG and drives autoimmunity through the accumulation of somatic hypermutations such that monovalent IgG4 Fab-arm exchanged MG autoantibodies reach a high affinity threshold required for pathogenic capacity.

Preclinical studies on the toxicology, pharmacokinetics and safety of K1-70TM a human monoclonal autoantibody to the TSH receptor with TSH antagonist activity

BackgroundThe human monoclonal autoantibody K1-70™ binds to the TSH receptor (TSHR) with high affinity and blocks TSHR cyclic AMP stimulation by TSH and thyroid stimulating autoantibodies.MethodsThe preclinical toxicology assessment following weekly intravenous (IV) or intramuscular (IM) administration of K1-70™ in rats and cynomolgus monkeys for 29 days was carried out. An assessment of delayed onset toxicity and/or reversibility of toxicity was made during a further 4 week treatment free period. The pharmacokinetic parameters of K1-70™ and the effects of different doses of K1-70™ on serum thyroid hormone levels in the study animals were determined in rats and primates after IV and IM administration.ResultsLow serum levels of T3 and T4 associated with markedly elevated levels of TSH were observed in the study animals following IV and IM administration of K1-70™. The toxicological findings were attributed to the pharmacology of K1-70™ and were consistent with the hypothyroid state. The no observable adverse effect level (NOAEL) could not be established in the rat study while in the primate study it was 100 mg/kg/dose for both males and females.ConclusionsThe toxicology, pharmacodynamic and pharmacokinetic data in this preclinical study were helpful in designing the first in human study with K1-70™ administered to subjects with Graves’ disease.

Experimental Reproduction of Dynamic Fluctuation of TSH Receptor-Binding Antibodies Between Stimulation and Inhibition.

ContextHyperthyroidism in Graves disease (GD) is caused by autoantibody stimulation of the TSH receptor (TSHR). TSHR autoantibody (TSHR-Ab) activity is measured routinely by inhibition of labeled ligand (TSH or M22) binding to the TSHR [TSH-binding inhibitory immunoglobulins (TBIIs)] or by stimulation of cAMP production in isolated cells [TSH receptor–stimulating antibodies (TSAbs)]. Usually, measurements of TSHR-Abs by TBIIs agree reasonably well with TSAb values at least in the setting of hyperthyroidism, and both measurements tend to change in parallel during treatment with some exceptions. In this study, we describe three unusual cases, which illustrate nearly pure stimulating, blocking, or neutral properties of TSHR-Abs.ObjectiveWhether patient serum TSHR-Abs can be reproduced by mixtures of human monoclonal autoantibodies to the TSHR was studied because the sera in most patients show moderate properties having both of TBII and TSAb activities.DesignWe compared the TBII and TSAb activities of serum from four unusual patients in detail with mixtures of human monoclonal TSHR-Abs (mAbs) M22 (stimulating), K1-18 (stimulating), and K1-70 (blocking).ResultsCharacteristic of a patient’s serum was similar to M22 or K1-18, another was similar to K1-70, whereas another was similar to a mixture of K1-70 and M22 (or K1-18). Additionally, some patients seemed to have neutral TSHR-Abs in their sera.ConclusionsOur studies suggest that the characteristics of TSHR-Abs in the patient serum can be mimicked by mixtures of human mAbs to the TSHR, stimulating, blocking, and neutral if any.

Characterization of pathogenic monoclonal autoantibodies derived from muscle-specific kinase myasthenia gravis patients.

Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by pathogenic autoantibodies that bind to membrane proteins at the neuromuscular junction. Most patients have autoantibodies against the acetylcholine receptor (AChR), but a subset of patients have autoantibodies against muscle-specific tyrosine kinase (MuSK) instead. MuSK is an essential component of the pathway responsible for synaptic differentiation, which is activated by nerve-released agrin. Through binding MuSK, serum-derived autoantibodies inhibit agrin-induced MuSK autophosphorylation, impair clustering of AChRs, and block neuromuscular transmission. We sought to establish individual MuSK autoantibody clones so that the autoimmune mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from 6 MuSK MG patients using a fluorescently tagged MuSK antigen multimer, then generated a panel of human monoclonal autoantibodies (mAbs) from these cells. Here we focused on 3 highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells, and at mouse neuromuscular junctions, where they colocalized with AChRs. These 3 IgG isotype mAbs (2 IgG4 and 1 IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The mAbs inhibited AChR clustering, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation, which suggests an alternative mechanism for inhibiting AChR clustering.

Exosomes Expressing Thyrotropin Receptor Attenuate Autoantibody-Mediated Stimulation of Cyclic Adenosine Monophosphate Production.

Background: Exosomes or small extracellular vesicles secreted from cells are nanovesicles with a diameter of 40-150 nm, which play a number of roles in both physiologic and pathologic processes. In Graves' disease (GD), autoantibodies bind to the thyrotropin receptor (TSHR) on the surface of thyroid follicular epithelial cells and stimulate thyroid growth and thyroid hormone synthesis and secretion via cyclic adenosine monophosphate (cAMP) production. The present study aimed to confirm the existence of TSHR in exosomes secreted from thyroid cells and to define the role of TSHR exosomes in GD. Methods: Exosomes were isolated by differential centrifugation from the culture medium of the human thyroid follicular epithelial cell line (NTHY-ori 3-1) and thyroid carcinoma cell lines (8305C, 8505C, and FTC-133). TSHR expression in cell lysates and exosomes was evaluated by Western blot analysis. In order to study the function of TSHR exosomes, human embryonic kidney (HEK) 293 cells stably expressing TSHR (HEK/TSHR) were established. Using exosomes isolated from both HEK and HEK/TSHR cells, the binding capacity of the M22 human monoclonal autoantibody to TSHR exosomes and their effect on M22-mediated stimulation of cAMP production in HEK/TSHR cells were evaluated. As a positive control for the functional assay, human recombinant TSHR chimera protein capable of binding to TSH was used. Results: TSHR was detected in exosomes from cancer cells as well as normal epithelial cells. An in vitro binding assay showed that alkaline phosphatase-labeled M22 bound to TSHR exosomes in a dose-dependent manner. M22 dose-dependently stimulated intracellular cAMP production in HEK/TSHR cells. The addition of exosomes from HEK/TSHR cells but not those from parental HEK cells significantly ameliorated cAMP production stimulated by treatment with M22 in HEK/TSHR cells. A decoy effect similar to TSHR exosomes was observed for human recombinant TSHR chimera. Conclusions: The results suggest that exosomes expressing TSHR may be secreted from normal and cancerous thyroid cells. In the thyroid gland of patients with GD, TSHR exosomes may exert a decoy effect by sequestering autoantibody, thereby ameliorating autoantibody-mediated activation of thyroid function.

N-methyl-D-aspartate receptor dysfunction by unmutated human antibodies against the NR1 subunit.

Anti–N‐methyl‐D‐aspartate receptor (NMDAR) encephalitis is the most common autoimmune encephalitis related to autoantibody‐mediated synaptic dysfunction. Cerebrospinal fluid–derived human monoclonal NR1 autoantibodies showed low numbers of somatic hypermutations or were unmutated. These unexpected germline‐configured antibodies showed weaker binding to the NMDAR than matured antibodies from the same patient. In primary hippocampal neurons, germline NR1 autoantibodies strongly and specifically reduced total and synaptic NMDAR currents in a dose‐ and time‐dependent manner. The findings suggest that functional NMDAR antibodies are part of the human naïve B cell repertoire. Given their effects on synaptic function, they might contribute to a broad spectrum of neuropsychiatric symptoms. Ann Neurol 2019;85:771–776

Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain.

The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22-260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.

An Assay to Determine Mechanisms of Rapid Autoantibody-Induced Neurotransmitter Receptor Endocytosis and Vesicular Trafficking in Autoimmune Encephalitis.

N-Methyl-D-aspartate (NMDA) receptors (NMDARs) are among the most important excitatory neurotransmitter receptors in the human brain. Autoantibodies to the human NMDAR cause the most frequent form of autoimmune encephalitis involving autoantibody-mediated receptor cross-linking and subsequent internalization of the antibody-receptor complex. This has been deemed to represent the predominant antibody effector mechanism depleting the NMDAR from the synaptic and extra-synaptic neuronal cell membrane. To assess in detail the molecular mechanisms of autoantibody-induced NMDAR endocytosis, vesicular trafficking, and exocytosis we transiently co-expressed rat GluN1-1a-EGFP and GluN2B-ECFP alone or together with scaffolding postsynaptic density protein 95 (PSD-95), wild-type (WT), or dominant-negative (DN) mutant Ras-related in brain (RAB) proteins (RAB5WT, RAB5DN, RAB11WT, RAB11DN) in HEK 293T cells. The cells were incubated with a pH-rhodamine-labeled human recombinant monoclonal GluN1 IgG1 autoantibody (GluN1-aAbpH−rhod) genetically engineered from clonally expanded intrathecal plasma cells from a patient with anti-NMDAR encephalitis, and the pH-rhodamine fluorescence was tracked over time. We show that due to the acidic luminal pH, internalization of the NMDAR-autoantibody complex into endosomes and lysosomes increases the pH-rhodamine fluorescence. The increase in fluorescence allows for mechanistic assessment of endocytosis, vesicular trafficking in these vesicular compartments, and exocytosis of the NMDAR-autoantibody complex under steady state conditions. Using this method, we demonstrate a role for PSD-95 in stabilization of NMDARs in the cell membrane in the presence of GluN1-aAbpH−rhod, while RAB proteins did not exert a significant effect on vertical trafficking of the internalized NMDAR autoantibody complex in this heterologous expression system. This novel assay allows to unravel molecular mechanisms of autoantibody-induced receptor internalization and to study novel small-scale specific molecular-based therapies for autoimmune encephalitis syndromes.

Affinities of human NMDA receptor autoantibodies: implications for disease mechanisms and clinical diagnostics

Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a common autoimmune encephalitis presenting with psychosis, dyskinesias, autonomic dysfunction and seizures. The underlying autoantibodies against the NR1 subunit are directly pathogenic by disrupting synaptic NMDAR currents. However, antibody titers correlate only partially with the clinical outcome, suggesting the relevance of other factors such as antibody affinity. We thus determined the binding curves of human monoclonal autoantibodies and patients’ cerebrospinal fluid (CSF) against NR1-expressing HEK293 cells using flow cytometry. Antibody affinity was highly variable with binding constants (half-maximal concentration, c50) ranging from 1 to 74 µg/ml for monoclonal antibodies. Comparing values of individual monoclonal antibodies with human CSF samples suggested that the CSF signal is predominantly represented by higher-affinity antibodies, potentially in a concentration range of NR1 antibodies between 0.1 and 5 µg/ml, roughly reflecting 1–10% of total CSF IgG in NMDAR encephalitis. Binding curves further depended on the CSF composition which must be considered when interpreting established clinical routine assays. Normalization of measurements using reference samples allowed high reproducibility. Accurate and reproducible measurement of NR1 antibody binding suggested that biophysical properties of the antibody might contribute to disease severity. Normalization of the data can be an elegant way to allow comparable inter-laboratory quantification of CSF NR1 antibody titers in autoimmune encephalitis patients, a prerequisite for use as surrogate markers in clinical trials. Based on our calculations, low-affinity antibodies can easily remain undetected in routine cell-based assays, indicating that their relation to clinical symptoms should be analyzed in future studies.

Molecular mimicry and signaling by human monoclonal autoantibody derived from human myocarditis and heart failure may contribute to fibrosis and remodeling in cardiomyopathy

Abstract Previous studies in animal models of myocarditis suggest that immune responses against cardiac myosin may lead to remodeling in the heart and dilated cardiomyopathy. In human myocarditis and dilated cardiomyopathy, anti-cardiac myosin autoantibodies significantly associated with myocarditis in cohorts of adults and children and reacted significantly with both human cardiac myosin (HCM) and the β-adrenergic receptors (βAR). We produced a human monoclonal antibody (mAb) derived from human myocarditis which reacted with HCM and specific peptides from pathogenic regions of HCM. Autoantibodies against HCM and βAR from myocarditis and dilated cardiomyopathy including the human myocarditis-derived mAb targeted heart cells by signaling protein kinase A (PKA) in a primary heart cell line. To further define potential pathogenic mechanisms of the human mAb and serum autoantibodies from myocarditis and cardiomyopathy and to test our hypothesis that autoantibodies may alter gene expression related to extracellular matrix, remodeling and fibrosis, we investigated genes differentially expressed by RNA sequence analysis of a primary heart cell line after treatment with the human mAb and sera from both children and adults with myocarditis. The overall heatmap of differentially expressed genes in heart cells demonstrated a significant difference between the control sera group and both human mAb and myocarditis sera treatment groups. We identified upregulated genes related to fibrosis, apoptosis, inflammation and cardiomyopathy, suggesting that the myocarditis- and cardiomyopathy-derived autoantibodies may induce signaling in primary heart cells and potentially contribute to fibrosis and remodeling in the heart.