The outbreak of the novel coronavirus disease (COVID‐19) quickly spread all over China and to more than 20 other countries. Although the virus (severe acute respiratory syndrome ...coronavirus SARS‐Cov‐2) nucleic acid real‐time polymerase chain reaction (PCR) test has become the standard method for diagnosis of SARS‐CoV‐2 infection, these real‐time PCR test kits have many limitations. In addition, high false‐negative rates were reported. There is an urgent need for an accurate and rapid test method to quickly identify a large number of infected patients and asymptomatic carriers to prevent virus transmission and assure timely treatment of patients. We have developed a rapid and simple point‐of‐care lateral flow immunoassay that can detect immunoglobulin M (IgM) and IgG antibodies simultaneously against SARS‐CoV‐2 virus in human blood within 15 minutes which can detect patients at different infection stages. With this test kit, we carried out clinical studies to validate its clinical efficacy uses. The clinical detection sensitivity and specificity of this test were measured using blood samples collected from 397 PCR confirmed COVID‐19 patients and 128 negative patients at eight different clinical sites. The overall testing sensitivity was 88.66% and specificity was 90.63%. In addition, we evaluated clinical diagnosis results obtained from different types of venous and fingerstick blood samples. The results indicated great detection consistency among samples from fingerstick blood, serum and plasma of venous blood. The IgM‐IgG combined assay has better utility and sensitivity compared with a single IgM or IgG test. It can be used for the rapid screening of SARS‐CoV‐2 carriers, symptomatic or asymptomatic, in hospitals, clinics, and test laboratories.
Complement is an ancient danger-sensing system that contributes to host defense, immune surveillance and homeostasis. C5a and its G protein–coupled receptor mediate many of the proinflammatory ...properties of complement. Despite the key role of C5a in allergic asthma, autoimmune arthritis, sepsis and cancer, knowledge about its regulation is limited. Here we demonstrate that IgG1 immune complexes (ICs), the inhibitory IgG receptor FcγRIIB and the C-type lectin–like receptor dectin-1 suppress C5a receptor (C5aR) functions. IgG1 ICs promote the association of FcγRIIB with dectin-1, resulting in phosphorylation of Src homology 2 domain–containing inositol phosphatase (SHIP) downstream of FcγRIIB and spleen tyrosine kinase downstream of dectin-1. This pathway blocks C5aR-mediated ERK1/2 phosphorylation, C5a effector functions in vitro and C5a-dependent inflammatory responses in vivo, including peritonitis and skin blisters in experimental epidermolysis bullosa acquisita. Notably, high galactosylation of IgG N-glycans is crucial for this inhibitory property of IgG1 ICs, as it promotes the association between FcγRIIB and dectin-1. Thus, galactosylated IgG1 and FcγRIIB exert anti-inflammatory properties beyond their impact on activating FcγRs.
Immunoglobulin G (IgG) deficiency increases the risk of acute exacerbations and mortality in chronic obstructive pulmonary disease (COPD). However, the impact of IgG subclass deficiency on mortality ...in COPD is unknown. Here, we determined which IgG subclass, if any, is associated with increased risk of mortality in COPD.
We measured serum IgG subclass concentrations of 489 hospitalized patients with COPD who were enrolled in the Rapid Transition Program (clinicaltrials.gov identifier NCT02050022). To evaluate the impact of IgG subclass deficiency on 1-year mortality, Cox proportional hazards regression analyses were performed with adjustments for potential confounders.
Deficiencies in IgG1, IgG2, IgG3, and IgG4 were present in 1.8%, 12.1%, 4.3%, and 11.2% of patients, respectively. One-year mortality was 56% in patients with IgG1 deficiency, 27% in IgG2 deficiency, 24% in IgG3 deficiency, and 31% in IgG4 deficiency. Cox proportional modeling showed that IgG1 and IgG4 deficiencies increased the 1-year mortality risk with an adjusted hazard ratio of 3.92 (95% confidence interval CI = 1.55-9.87) and 1.74 (95% CI = 1.02-2.98), respectively. Neither IgG2 nor IgG3 deficiency significantly increased 1-year mortality. Two or more IgG subclass deficiencies were observed in 5.3%. Patients with 2 or more IgG subclass deficiencies had a higher 1-year mortality than those without any deficiencies (46.2% vs. 19.7%, p < 0.001), with an adjusted hazard ratio of 2.22 (95% CI = 1.18-4.17).
IgG1 and IgG4 deficiency was observed in 1.8% and 11.2% of hospitalized patients with COPD, respectively, and these deficiencies were associated with a significantly increased risk of 1-year mortality.
The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of ...monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity.
Abstract Immune globulin preparations such as intravenous immunoglobulin (IVIG) and monoclonal antibodies are widely used in clinics as effective therapeutic agents for the treatment of a number of ...autoimmune diseases, cancer, inflammations and other pathologies. Significant amounts of IgG aggregates have been found in the highly concentrated solutions of therapeutic immune proteins. The IgG self-aggregation that appears especially after prolonged storage increases the immunogenicity of the preparations and also modifies their physical properties, first of all producing the high viscosity. The attractive IgG–IgG interactions pose a significant problem for the clinical usage of the immune proteins. During last decades intensive studies of the IgG self-association were performed. The presence of IgG dimers was demonstrated in pooled preparations. These complexes are the result of idiotype–anti-idiotype interactions. In concentrated solutions of immune globulins and monoclonal antibodies self-associated IgG molecules formed a network, increasing the viscosity. The forces responsible for the IgG association are characteristic of the protein–protein interactions in general. The amino acid residues of the Fab and Fc portions participate in the IgG–IgG contacts. Recently contact residues were modified by the site-directed mutagenesis in order to decrease the formation of the IgG self-aggregates. The mutant IgG antibodies were characterized by enhanced stability as compared with the non-modified antibody molecules. Peptic pFc′ fragment and the CH 3 domain were shown to be capable of interacting with Fc regions, thus preventing IgG aggregation. In perspective both approaches could improve the formulation of immune globulin preparations. Removal of IgG aggregates could be achieved by chromatography on hydroxyapatite.
Effector CD8+ T cells are important mediators of adaptive immunity, and receptor-ligand interactions that regulate their survival may have therapeutic potential. Here, we identified a subset of ...effector CD8+ T cells that expressed the inhibitory fragment crystallizable (Fc) receptor FcγRIIB following activation and multiple rounds of division. CD8+ T cell-intrinsic genetic deletion of Fcgr2b increased CD8+ effector T cell accumulation, resulting in accelerated graft rejection and decreased tumor volume in mouse models. Immunoglobulin G (IgG) antibody was not required for FcγRIIB-mediated control of CD8+ T cell immunity, and instead, the immunosuppressive cytokine fibrinogen-like 2 (Fgl2) was a functional ligand for FcγRIIB on CD8+ T cells. Fgl2 induced caspase-3/7-mediated apoptosis in Fcgr2b+, but not Fcgr2b−/−, CD8+ T cells. Increased expression of FcγRIIB correlated with freedom from rejection following withdrawal from immunosuppression in a clinical trial of kidney transplant recipients. Together, these findings demonstrate a cell-intrinsic coinhibitory function of FcγRIIB in regulating CD8+ T cell immunity.
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•Inhibitory Fc receptor FcγRIIB is expressed on multipotent effector CD8+ T cells•FcγRIIB on CD8+ T cells functions in a cell-intrinsic manner to limit T cell survival•Fcgr2b−/− CD8+ T cells confer increased allograft rejection and tumor immunity•Fgl2 induces FcγRIIB-mediated CD8+ T cell apoptosis via activation of caspase-3/7
It is thought that Fc receptors are not expressed on T cells. Morris et al. report that a subset of potent CD8+ effector T cells express and are regulated by the inhibitory Fc receptor FcγRIIB. Ligation of FcγRIIB with the immunosuppressive cytokine Fgl2, rather than IgG, functions to induce caspase-3/7-mediated apoptosis and limit CD8+ T cell immunity.
Although most vaccines are administered i.m., little is known about the dendritic cells (DCs) that are present within skeletal muscles. In this article, we show that expression of CD64, the ...high-affinity IgG receptor FcγRI, distinguishes conventional DCs from monocyte-derived DCs (Mo-DCs). By using such a discriminatory marker, we defined the distinct DC subsets that reside in skeletal muscles and identified their migratory counterparts in draining lymph nodes (LNs). We further used this capability to analyze the functional specialization that exists among muscle DCs. After i.m. administration of Ag adsorbed to alum, we showed that alum-injected muscles contained large numbers of conventional DCs that belong to the CD8α(+)- and CD11b(+)-type DCs. Both conventional DC types were capable of capturing Ag and of migrating to draining LNs, where they efficiently activated naive T cells. In alum-injected muscles, Mo-DCs were as numerous as conventional DCs, but only a small fraction migrated to draining LNs. Therefore, alum by itself poorly induces Mo-DCs to migrate to draining LNs. We showed that addition of small amounts of LPS to alum enhanced Mo-DC migration. Considering that migratory Mo-DCs had, on a per cell basis, a higher capacity to induce IFN-γ-producing T cells than conventional DCs, the addition of LPS to alum enhanced the overall immunogenicity of Ags presented by muscle-derived DCs. Therefore, a full understanding of the role of adjuvants during i.m. vaccination needs to take into account the heterogeneous migratory and functional behavior of muscle DCs and Mo-DCs revealed in this study.
•Exploratory statistical analysis of data from 54 manufactured batches of an IgG1 therapeutic antibody (mAb1) suggests that ADCC activity is not only modulated by afucose but also by galactose.•We ...enzymatically modulate the galactose levels of four different batches of mAb1 and confirm that the presence of terminal galactose enhances ADCC activity.•The ADCC enhancing effect of galactose is observed in 4 additional monoclonal antibodies derived using standard CHO-based manufacturing processes.•In contrast, the ADCC activity of IgG1 monoclonal antibody glycoengineered to contain a high degree of afucosylation remains unchanged by the addition of galactose, suggesting that while the levels of galactose have some influence on ADCC activity, the impact of afucose is still predominant.
The therapeutic activity of monoclonal antibodies can involve immune cell mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC), an activity that is modulated by the structure of Fc-glycans, and in particular the lack of core fucose. The heterogeneity of these glycostructures and the inherent variability of traditional PBMC-based in vitro ADCC assays, have made it challenging to quantitatively assess the impact of other glycostructures on ADCC activity. We applied a quantitative NK cell based assay to generate a database consisting of Fc-glycostructure and ADCC data from 54 manufacturing batches of a CHO-derived monoclonal antibody. Explorative analysis of the data indicated that, apart from afucosylation, galactosylation levels could influence ADCC activity. We confirmed this hypothesis by demonstrating enhanced ADCC upon enzymatic hypergalactosylation of four different monoclonal antibodies derived using standard CHO manufacturing processes. Furthermore we quantitatively compare the effects of galactosylation and afucosylation in the context of glycan heterogeneity and demonstrate that while galactose can influence ADCC activity, afucosylation remains the primary driver of this activity.
Summary
IgG antibodies are actively produced in response to antigenic challenge or passively administered as an effective form of immunotherapy to confer immunity against foreign antigens. Their ...protective activity is mediated through their bifunctional nature: a variable Fab domain mediates antigen‐binding specificity, whereas the constant Fc domain engages Fcγ receptors (FcγRs) expressed on the surface of leukocytes to mediate effector functions. While traditionally considered the invariant domain of an IgG molecule, the Fc domain displays remarkable structural heterogeneity determined primarily by differences in the amino acid sequence of the various IgG subclasses and by the composition of the complex, Fc‐associated biantennary N‐linked glycan. These structural determinants regulate the conformational flexibility of the IgG Fc domain and affect its capacity to interact with distinct types of FcγRs (type I or type II FcγRs). FcγR engagement activates diverse downstream immunomodulatory pathways with pleiotropic functional consequences including cytotoxicity and phagocytosis of IgG‐coated targets, differentiation and activation of antigen presenting cells, modulation of T‐cell activation, plasma cell survival, and regulation of antibody responses. These functions highlight the importance of FcγR‐mediated pathways in the modulation of adaptive immune responses and suggest a central role for IgG–FcγR interactions during active and passive immunization.
Allogeneic cell therapeutics for cancer therapy or regenerative medicine are susceptible to antibody-mediated killing, which diminishes their efficacy. Here we report a strategy to protect cells from ...antibody-mediated killing that relies on engineered overexpression of the IgG receptor CD64. We show that human and mouse iPSC-derived endothelial cells (iECs) overexpressing CD64 escape antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity from IgG antibodies in vitro and in ADCC-enabled mice. When CD64 expression was combined with hypoimmune genetic modifications known to protect against cellular immunity, B2M
CIITA
CD47/CD64-transgenic iECs were resistant to both IgG antibody-mediated and cellular immune killing in vitro and in humanized mice. Mechanistic studies demonstrated that CD64 or its intracellularly truncated analog CD64t effectively capture monomeric IgG and occupy their F
, and the IgG bind and occupy their target antigens. In three applications of the approach, human CD64t-engineered thyroid epithelial cells, pancreatic beta cells and CAR T cells withstood clinically relevant levels of graft-directed antibodies and fully evaded antibody-mediated killing.
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