Patient-derived Antibodies Research Articles

Structural and functional mechanisms of anti-NMDAR autoimmune encephalitis.

Autoantibodies against neuronal membrane proteins can manifest in autoimmune encephalitis, inducing seizures, cognitive dysfunction and psychosis. Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most dominant autoimmune encephalitis; however, insights into how autoantibodies recognize and alter receptor functions remain limited. Here we determined structures of human and rat NMDARs bound to three distinct patient-derived antibodies using single-particle electron cryo-microscopy. These antibodies bind different regions within the amino-terminal domain of the GluN1 subunit. Through electrophysiology, we show that all three autoantibodies acutely and directly reduced NMDAR channel functions in primary neurons. Antibodies show different stoichiometry of binding and antibody-receptor complex formation, which in one antibody, 003-102, also results in reduced synaptic localization of NMDARs. These studies demonstrate mechanisms of diverse epitope recognition and direct channel regulation of anti-NMDAR autoantibodies underlying autoimmune encephalitis.

Interference of lupus anticoagulant causing antiprothrombin and anti–beta-2-glycoprotein I antibodies on international normalized ratio measurements: comparative analysis of international normalized ratio methods

BackgroundLife-long vitamin K antagonist (VKA) therapy is recommended as a standard of care in antiphospholipid syndrome (APS) patients with thrombosis. Concerns have been raised about the validity of international normalized ratio (INR) measurements in lupus anticoagulant (LA)-positive APS patients because LA may interfere with phospholipid-dependent coagulation tests and could elevate INR measurements. ObjectivesHere, we aimed to determine the interference of antigen-specific monoclonal and isolated patient antibodies with LA activity on INR measurements. MethodsPooled normal plasma and control plasma from patients on VKA (without LA) were incubated with monoclonal and isolated patient immunoglobulin G antiprothrombin and anti–beta-2-glycoprotein I antibodies that express LA activity. INR was determined before and after addition using 3 laboratory assays (Owren STA-Hepato Prest, Quick STA-NeoPTimal, and Quick STA-Neoplastine R) and 1 point-of-care test device (CoaguChek Pro II). ResultsAntiprothrombin and anti–beta-2-glycoprotein I antibodies with LA activity interfered with recombinant human thromboplastin reagents (Quick STA-Neoplastine R and CoaguChek Pro II), particularly when added to plasma of VKA-treated controls. This effect was most evident on point-of-care test INR measurements, while the recombinant Quick reagent exhibited a lesser degree of interference. In contrast, tissue-derived thromboplastin reagents (Owren STA-Hepato Prest and Quick STA-NeoPTimal) remained largely unaffected by these antibodies, both in pooled normal plasma and VKA anticoagulated control plasma. Among these reagents, the Owren INR reagent exhibited the lowest sensitivity to the influence of LA antibodies. This observed difference in sensitivity is independent of the plasma dilution factor or the presence of factor V or fibrinogen in Owren reagent. ConclusionINR reagents that utilize recombinant human thromboplastin are more sensitive to the presence of monoclonal and patient-derived antibodies with LA activity. Consequently, APS patients positive for LA should be monitored using tissue-derived thromboplastin reagents, given its reduced susceptibility to interference by LA-causing antibodies.

Neuronal nicotinic acetylcholine receptor antibodies in autoimmune central nervous system disorders.

Neuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the central nervous system (CNS), playing critical roles in brain function. Antigenicity of nAChRs has been well demonstrated with antibodies to ganglionic AChR subtypes (i.e., subunit α3 of α3β4-nAChR) and muscle AChR autoantibodies, thus making nAChRs candidate autoantigens in autoimmune CNS disorders. Antibodies to several membrane receptors, like NMDAR, have been identified in autoimmune encephalitis syndromes (AES), but many AES patients have yet to be unidentified for autoantibodies. This study aimed to develop of a cell-based assay (CBA) that selectively detects potentially pathogenic antibodies to subunits of the major nAChR subtypes (α4β2- and α7-nAChRs) and its use for the identification of such antibodies in "orphan" AES cases. The study involved screening of sera derived from 1752 patients from Greece, Turkey and Italy, who requested testing for AES-associated antibodies, and from 1203 "control" patients with other neuropsychiatric diseases, from the same countries or from Germany. A sensitive live-CBA with α4β2-or α7-nAChR-transfected cells was developed to detect antibodies against extracellular domains of nAChR major subunits. Flow cytometry (FACS) was performed to confirm the CBA findings and indirect immunohistochemistry (IHC) to investigate serum autoantibodies' binding to rat brain tissue. Three patients were found to be positive for serum antibodies against nAChR α4 subunit by CBA and the presence of the specific antibodies was quantitatively confirmed by FACS. We detected specific binding of patient-derived serum anti-nAChR α4 subunit antibodies to rat cerebellum and hippocampus tissue. No serum antibodies bound to the α7-nAChR-transfected or control-transfected cells, and no control serum antibodies bound to the transfected cells. All patients positive for serum anti-nAChRs α4 subunit antibodies were negative for other AES-associated antibodies. All three of the anti-nAChR α4 subunit serum antibody-positive patients fall into the AES spectrum, with one having Rasmussen encephalitis, another autoimmune meningoencephalomyelitis and another being diagnosed with possible autoimmune encephalitis. This study lends credence to the hypothesis that the major nAChR subunits are autoimmune targets in some cases of AES and establishes a sensitive live-CBA for the identification of such patients.

Memory loss and aberrant neurogenesis in mice exposed to patient anti-N-methyl-d-aspartate receptor antibodies

ObjectiveAnti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis results in chronic epilepsy and permanent cognitive impairment. One of the possible causes of cognitive impairment in anti-NMDAR could be aberrant neurogenesis, an established contributor to memory loss in idiopathic drug-resistant epilepsy. We developed a mouse model of anti-NMDAR encephalitis and showed that mice exposed to patient anti-NMDAR antibodies for 2 weeks developed seizures and memory loss. In the present study, we assessed the delayed effects of patient-derived antibodies on cognitive phenotype and examined the corresponding changes in hippocampal neurogenesis. MethodsMonoclonal anti-NMDAR antibodies or control antibodies were continuously infused into the lateral ventricle of male C56BL/6J mice (8–12 weeks) via osmotic minipumps for 2 weeks. The motor and anxiety phenotypes were assessed using the open field paradigm, and hippocampal memory and learning were assessed using the object location, Y maze, and Barnes maze paradigms during weeks 1 and 3–4 of antibody washout. The numbers of newly matured granule neurons (Prox-1+) and immature progenitor cells (DCX+) as well as their spatial distribution within the hippocampus were assessed at these time points. Bromodeoxyuridine (BrdU, 50 mg/kg, i.p., daily) was injected on days 2–12 of the infusion, and proliferating cell immunoreactivity was compared in antibody-treated mice and control mice during week 4 of the washout. ResultsMice infused with anti-NMDAR antibodies demonstrated spatial memory impairment during week 1 of antibody washout (p = 0.02, t-test; n = 9–11). Histological analysis of hippocampal sections from these mice revealed an increased ectopic displacement of Prox-1+ cells in the dentate hilus compared to the control-antibody-treated mice (p = 0.01; t-test). Mice exposed to anti-NMDAR antibodies also had an impairment of spatial memory and learning during weeks 3–4 of antibody washout (object location: p = 0.009; t-test; Y maze: p = 0.006, t-test; Barnes maze: p = 0.008, ANOVA; n = 8–10). These mice showed increased ratios of the low proliferating (bright) to fast proliferating (faint) BrdU+ cell counts and decreased number of DCX+ cells in the hippocampal dentate gyrus (p = 0.006 and p = 0.04, respectively; t-tests) suggesting ectopic migration and delayed cell proliferation. SignificanceThese findings suggest that memory and learning impairments induced by patient anti-NMDAR antibodies are sustained upon removal of antibodies and are accompanied by aberrant hippocampal neurogenesis. Interventions directed at the manipulation of neuronal plasticity in patients with encephalitis and cognitive loss may be protective and therapeutically relevant.

Cooperativity and induced oligomerization control the interaction of SARS-CoV-2 with its cellular receptor and patient-derived antibodies

Cooperativity and induced oligomerization control the interaction of SARS-CoV-2 with its cellular receptor and patient-derived antibodies

Low ectonucleotidase activity and increased neutrophil-platelet aggregates in patients with antiphospholipid syndrome

AbstractMany patients with antiphospholipid syndrome had decreased ectonucleotidase activity on neutrophils and platelets, which enabled extracellular nucleotides to trigger neutrophil-platelet aggregates. This phenotype was replicated by treating healthy neutrophils and platelets with patient-derived antiphospholipid antibodies or ectonucleotidase inhibitors.

Development of bioactive short fiber-reinforced printable hydrogels with tunable mechanical and osteogenic properties for bone repair.

Personalized bone-regenerative materials have attracted substantial interest in recent years. Modern clinical settings demand the use of engineered materials incorporating patient-derived cells, cytokines, antibodies, and biomarkers to enhance the process of regeneration. In this work, we formulated short microfiber-reinforced hydrogels with platelet-rich fibrin (PRF) to engineer implantable multi-material core-shell bone grafts. By employing 3D bioprinting technology, we fabricated a core-shell bone graft from a hybrid composite hydroxyapatite-coated poly(lactic acid) (PLA) fiber-reinforced methacryolyl gelatin (GelMA)/alginate hydrogel. The overall concept involves 3D bioprinting of long bone mimic microstructures that resemble a core-shell cancellous-cortical structure, with a stiffer shell and a softer core with our engineered biomaterial. We observed a significantly enhanced stiffness in the hydrogel scaffold incorporated with hydroxyapatite (HA)-coated PLA microfibers compared to the pristine hydrogel construct. Furthermore, HA non-coated PLA microfibers were mixed with PRF and GelMA/alginate hydrogel to introduce a slow release of growth factors which can further enhance cell maturation and differentiation. These patient-specific bone grafts deliver cytokines and growth factors with distinct spatiotemporal release profiles to enhance tissue regeneration. The biocompatible and bio-responsive bone mimetic core-shell multi-material structures enhance osteogenesis and can be customized to have materials at a specific location, geometry, and material combination.

Impaired functional connectivity of the hippocampus in translational murine models of NMDA-receptor antibody associated neuropsychiatric pathology

Decreased hippocampal connectivity and disruption of functional networks are established resting-state functional MRI (rs-fMRI) features that are associated with neuropsychiatric symptom severity in human anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. However, the underlying pathophysiology of NMDAR encephalitis remains poorly understood. Application of patient-derived monoclonal antibodies against the NR1 (GluN1) subunit of the NMDAR now allows for the translational investigation of functional connectivity in experimental murine NMDAR antibody disease models with neurodevelopmental disorders. Using rs-fMRI, we studied functional connectivity alterations in (1) adult C57BL/6 J mice that were intrathecally injected with a recombinant human NR1 antibody over 14 days (n = 10) and in (2) a newly established mouse model with in utero exposure to a human recombinant NR1 antibody (NR1-offspring) at the age of (2a) 8 weeks (n = 15) and (2b) 10 months (n = 14). Adult NR1-antibody injected mice showed impaired functional connectivity within the left hippocampus compared to controls, resembling impaired connectivity patterns observed in human NMDAR encephalitis patients. Similarly, NR1-offspring showed significantly reduced functional connectivity in the hippocampus after 8 weeks, and impaired connectivity in the hippocampus was likewise observed in NR1-offspring at the age of 10 months. We successfully reproduced functional connectivity changes within the hippocampus in different experimental murine systems that were previously observed in human NMDAR encephalitis patients. Translational application of this method within a combined imaging and histopathological framework will allow future experimental studies to identify the underlying biological mechanisms and may eventually facilitate non-invasive monitoring of disease activity and treatment responses in autoimmune encephalitis.

Structure of pathogenic anti-PF4 autoantibodies reveals the mechanism of immune response formation in vaccine-induced thrombotic thrombocytopenia (VITT).

Abstract Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe, but rare, side effect of adenoviral-vectored COVID-19 vaccines. It is attributed to formation of autoantibodies recognizing Platelet Factor 4 (PF4) and capable of triggering platelet activation, which leads to thrombosis. We used mass spectrometry (MS) to determine the structure of a VITT patient-derived anti-PF4 antibodies (VITT IgG) and identified their unique properties underlying pathogenicity. The VITT IgGs studied in this work were extracted from the plasma of a ChAdOx1 vaccine recipient, who developed VITT. Intact-mass MS analysis of these antibodies revealed one major clone, and its complete structural characterization was carried out using MS-based de novo sequencing. This allowed us to establish its subclass (IgG2), determine the amino acid sequence, and identify an N-glycan in the variable region. This information was used to build a 3D model of the VITT IgG, which revealed the presence of a large polyanionic patch within the paratope involving four CDR regions. Molecular dynamics simulations highlight the role of electrostatics as the major driver of the VITT IgG binding to PF4. The large equatorial belt of the positive charge circumscribing the PF4 tetramer serves as a distributed epitope, allowing it to cross-link up to three VITT IgG molecules, giving rise to large immune complexes capable of FcyRIIa-mediated platelet activation. The molecular mechanism of VITT pathogenesis emerging from this work explains how VITT IgGs and PF4 form large immune complexes in the absence of heparin, which is essential to for platelet activation in a similar disease, heparin-induced thrombocytopenia, and sheds light on the etiology of this devastating condition. Supported by grant from NIH R01 GM112666

NMDA-receptor-Fc-fusion constructs neutralize anti-NMDA receptor antibodies.

N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most common subtype of autoimmune encephalitis characterized by a complex neuropsychiatric syndrome usually including memory impairment. Patients develop an intrathecal immune response against NMDARs with antibodies that presumably bind to the amino-terminal domain of the GluN1 subunit. The therapeutic response to immunotherapy is often delayed. Therefore, new therapeutic approaches for fast neutralization of NMDAR antibodies are needed. Here, we developed fusion constructs consisting of the Fc part of immunoglobulin G and the amino-terminal domains of either GluN1 or combinations of GluN1 with GluN2A or GluN2B. Surprisingly, both GluN1 and GluN2 subunits were required to generate high-affinity epitopes. The construct with both subunits efficiently prevented NMDAR binding of patient-derived monoclonal antibodies and of patient CSF containing high-titre NMDAR antibodies. Furthermore, it inhibited the internalization of NMDARs in rodent dissociated neurons and human induced pluripotent stem cell-derived neurons. Finally, the construct stabilized NMDAR currents recorded in rodent neurons and rescued memory defects in passive-transfer mouse models using intrahippocampal injections. Our results demonstrate that both GluN1 and GluN2B subunits contribute to the main immunogenic region of the NMDAR and provide a promising strategy for fast and specific treatment of NMDAR encephalitis, which could complement immunotherapy.

Peanut allergen inhibition prevents anaphylaxis in a humanized mouse model.

Peanut-induced allergy is an immunoglobulin E (IgE)-mediated type I hypersensitivity reaction that manifests symptoms ranging from local edema to life-threatening anaphylaxis. Although there are treatments for symptoms in patients with allergies resulting from allergen exposure, there are few preventive therapies other than strict dietary avoidance or oral immunotherapy, neither of which are successful in all patients. We have previously designed a covalent heterobivalent inhibitor (cHBI) that binds in an allergen-specific manner as a preventive for allergic reactions. Building on previous in vitro testing, here, we developed a humanized mouse model to test cHBI efficacy in vivo. Nonobese diabetic-severe combined immunodeficient γc-deficient mice expressing transgenes for human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3 developed mature functional human mast cells in multiple tissues and displayed robust anaphylactic reactions when passively sensitized with patient-derived IgE monoclonal antibodies specific for peanut Arachis hypogaea 2 (Ara h 2). The allergic response in humanized mice was IgE dose dependent and was mediated by human mast cells. Using this humanized mouse model, we showed that cHBI prevented allergic reactions for more than 2 weeks when administered before allergen exposure. cHBI also prevented fatal anaphylaxis and attenuated allergic reactions when administered shortly after the onset of symptoms. cHBI impaired mast cell degranulation in vivo in an allergen-specific manner. cHBI rescued the mice from lethal anaphylactic responses during oral Ara h 2 allergen-induced anaphylaxis. Together, these findings suggest that cHBI has the potential to be an effective preventative for peanut-specific allergic responses in patients.

Patient-derived monoclonal antibodies to SARS-CoV-2 nucleocapsid protein N-terminal and C-terminal domains cross-react with their counterparts of SARS-CoV, but not other human betacoronaviruses.

SARS-CoV-2 nucleocapsid (N) protein plays a key role in multiple stages of the viral life cycle such as viral replication and assembly. This protein is more conserved than the Spike protein of the virus and can induce both humoral and cell-mediated immune responses, thereby becoming a target for clinical diagnosis and vaccine development. However, the immunogenic characteristics of this protein during natural infection are still not completely understood. Patient-derived monoclonal antibodies (mAbs) against SARS-CoV-2 N protein were generated from memory B cells in the PBMCs using the antigen-specific B cell approach. For epitope mapping of the isolated hmAbs, a panel of series-truncated N proteins were used , which covered the N-terminal domain (NTD, aa 46-174 ) and C-terminal domain (CTD, aa 245-364 ), as well as the flanking regions of NTD and CTD. NTD- or CTD-specific Abs in the plasma from COVID-19 patients were also tested by ELISA method. Cross-binding of hmAbs or plasma Abs in COVID-19 patients to other human β-CoV N proteins was determined using the capture ELISA. We isolated five N-specific monoclonal antibodies (mAbs) from memory B cells in the peripheral blood of two convalescent COVID-19 patients. Epitope mapping revealed that three of the patient-derived mAbs (N3, N5 and N31) targeted the C-terminal domain (CTD), whereas two of the mAbs (N83 and 3B7) targeted the N-terminal domain (NTD) of SARS-CoV-2 N protein. All five patient-derived mAbs were cross-reactive to the N protein of SARS-CoV but showed little to no cross-reactivity to the N proteins of other human beta coronaviruses (β-CoVs). We also tested 52 plasma samples collected from convalescent COVID-19 patients for Abs against the N proteins of human β-CoVs and found that 78.8% of plasma samples showed detectable Abs against the N proteins of SARS-CoV-2 and SARS-CoV. No plasma sample had cross-reactive Abs to the N protein of MERS-CoV. Cross-reactive Abs to the N proteins of OC43 and HKU1 were detected in 36.5% (19/52) and 19.2% (10/52) of plasma samples, respectively. These results suggest that natural SARS-CoV-2 infection elicits cross-reactive Abs to the N protein of SARS-CoV and that the five patient-derived mAbs to SARS-CoV-2 N protein NTD and CTD cross-react with their counterparts of SARS-CoV, but not other human β-CoVs. Thus, these five patient-derived mAbs can potentially be used for developing the next generation of COVID-19 At-Home Test kits for rapid and specific screening of SARS-CoV-2 infection.

Longitudinal characterization of circulating neutrophils uncovers phenotypes associated with severity in hospitalized COVID-19 patients

Mechanisms of neutrophil involvement in severe COVID-19 remain incompletely understood. Here we collect longitudinal blood samples from 306 hospitalized COVID-19+ patients and 86 controls, and perform bulk RNA-sequencing of enriched neutrophils, plasma proteomics, and high-throughput antibody profiling to investigate relationships between neutrophil states and disease severity. We identify dynamic switches between 6 distinct neutrophil subtypes. At days 3 and 7 post-hospitalization, patients with severe disease display a granulocytic myeloid-derived suppressor cell-like gene expression signature, while patients with resolving disease show a neutrophil progenitor-like signature. Humoral responses are identified as potential drivers of neutrophil effector functions, with elevated SARS-CoV-2-specific IgG1-to-IgA1 ratios in plasma of severe patients who survived. In vitro experiments confirm that while patient-derived IgG antibodies induce phagocytosis in healthy donor neutrophils, IgA antibodies predominantly induce neutrophil cell death. Overall, our study demonstrates a dysregulated myelopoietic response in severe COVID-19 and a potential role for IgA-dominant responses contributing to mortality.

Patient-derived monoclonal antibody neutralizes HCV infection in vitro and vivo without generating escape mutants.

In recent years, new direct-acting antivirals for hepatitis C virus (HCV) have been approved, but hepatitis C continues to pose a threat to human health. It is important to develop neutralizing anti-HCV antibodies to prevent medical and accidental infection, such as might occur via liver transplantation of chronic HCV patients and needle-stick accidents in the clinic. In this study, we sought to obtain anti-HCV antibodies using phage display screening. Phages displaying human hepatocellular carcinoma patient-derived antibodies were screened by 4 rounds of biopanning with genotype-1b and -2a HCV envelope E2 protein adsorbed to magnetic beads. The three antibodies obtained from this screen had reactivity against E2 proteins derived from both genotype-1b and -2a strains. However, in epitope analysis, these antibodies did not recognize linear peptides from an overlapping E2 epitope peptide library, and did not bind to denatured E2 protein. In addition, these antibodies showed cross-genotypic neutralizing activity against genotype-1a, -1b, -2a, and -3a cell culture-generated infectious HCV particles (HCVcc). Moreover, emergence of viral escape mutants was not observed after repeated rounds of passaging of HCV-infected cells in the presence of one such antibody, e2d066. Furthermore, injection of the e2d066 antibody into human hepatocyte-transplanted immunodeficient mice inhibited infection by J6/JFH-1 HCVcc. In conclusion, we identified conformational epitope-recognizing, cross-genotypic neutralizing antibodies using phage display screening. Notably, e2d066 antibody did not select for escape mutant emergence in vitro and demonstrated neutralizing activity in vivo. Our results suggested that these antibodies may serve as prophylactic and therapeutic agents.

An Epitope-Specific LGI1-Autoantibody Enhances Neuronal Excitability by Modulating Kv1.1 Channel.

Leucine-rich Glioma-Inactivated protein 1 (LGI1) is expressed in the central nervous system and its genetic loss of function is associated with epileptic disorders. Additionally, patients with LGI1-directed autoantibodies have frequent focal seizures as a key feature of their disease. LGI1 is composed of a Leucine-Rich Repeat (LRR) and an Epitempin (EPTP) domain. These domains are reported to interact with different members of the transsynaptic complex formed by LGI1 at excitatory synapses, including presynaptic Kv1 potassium channels. Patient-derived recombinant monoclonal antibodies (mAbs) are ideal reagents to study whether domain-specific LGI1-autoantibodies induce epileptiform activities in neurons and their downstream mechanisms. We measured the intrinsic excitability of CA3 pyramidal neurons in organotypic cultures from rat hippocampus treated with either an LRR- or an EPTP-reactive patient-derived mAb, or with IgG from control patients. We found an increase in intrinsic excitability correlated with a reduction of the sensitivity to a selective Kv1.1-channel blocker in neurons treated with the LRR mAb, but not in neurons treated with the EPTP mAb. Our findings suggest LRR mAbs are able to modulate neuronal excitability that could account for epileptiform activity observed in patients.

Patient-derived antibodies reveal the subcellular distribution and heterogeneous interactome of LGI1.

Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1's neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro-oligodendrocytic and astro-microglial protein complexes. Proteomic data support the presence of LGI1-Kv1-MAGUK complexes, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.

Tumor-reactive antibodies evolve from non-binding and autoreactive precursors

The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.

Spike-Dependent Opsonization Indicates Both Dose-Dependent Inhibition of Phagocytosis and That Non-Neutralizing Antibodies Can Confer Protection to SARS-CoV-2.

Spike-specific antibodies are central to effective COVID19 immunity. Research efforts have focused on antibodies that neutralize the ACE2-Spike interaction but not on non-neutralizing antibodies. Antibody-dependent phagocytosis is an immune mechanism enhanced by opsonization, where typically, more bound antibodies trigger a stronger phagocyte response. Here, we show that Spike-specific antibodies, dependent on concentration, can either enhance or reduce Spike-bead phagocytosis by monocytes independently of the antibody neutralization potential. Surprisingly, we find that both convalescent patient plasma and patient-derived monoclonal antibodies lead to maximum opsonization already at low levels of bound antibodies and is reduced as antibody binding to Spike protein increases. Moreover, we show that this Spike-dependent modulation of opsonization correlate with the outcome in an experimental SARS-CoV-2 infection model. These results suggest that the levels of anti-Spike antibodies could influence monocyte-mediated immune functions and propose that non-neutralizing antibodies could confer protection to SARS-CoV-2 infection by mediating phagocytosis.

A systematic study on the binding affinity of SARS-CoV-2 spike protein to antibodies.

The COVID-19 pandemic has caused a worldwide health crisis and economic recession. Effective prevention and treatment methods are urgently required to control the pandemic. However, the emergence of novel SARS-CoV-2 variants challenges the effectiveness of currently available vaccines and therapeutic antibodies. In this study, through the assessment of binding free energies, we analyzed the mutational effects on the binding affinity of the coronavirus spike protein to neutralizing antibodies, patient-derived antibodies, and artificially designed antibody mimics. We designed a scoring method to assess the immune evasion ability of viral variants. We also evaluated the differences between several targeting sites on the spike protein of antibodies. The results presented herein might prove helpful in the development of more effective therapies in the future.

A humanized mouse model of chronic COVID-19.

COVID-19 is an infectious disease; in some it presents as an uncontrolled, hyperactive immune response, causing severe immunological injury. Existing rodent models do not recapitulate the sustained immunopathology of patients with severe disease. Here, we describe a humanized mouse model of COVID-19 that uses adeno-associated virus to deliver human ACE2 to the lungs of humanized MISTRG6 mice. This model recapitulates innate and adaptive human immune responses to SARS-CoV-2 infection up to 28 days post-infection, with key features of chronic COVID-19 including weight loss, persistent viral RNA, lung pathology with fibrosis, a human inflammatory macrophage response, a persistent interferon-stimulated gene signature, and T-cell lymphopenia. We used this to study two therapeutics on immunopathology: patient-derived antibodies and steroids; We found that the same inflammatory macrophages crucial to containing early infection, later drove immunopathology. This model will enable evaluation of COVID-19 disease mechanisms and treatments.