Formation and clearance of TNF-TNF inhibitor complexes during TNF inhibitor treatment.

BACKGROUND AND PURPOSE: Millions of patients with inflammatory diseases are treated with tumour necrosis factor (TNF) inhibitors (TNFi). Individual treatment response varies, in part related to variable drug clearance. The role of TNF-TNFi complexes in clearance of the different TNFi is controversial. Moreover, mechanistic insight into the structural aspects and biological significance of TNF-TNFi complexes is lacking. We hypothesized a role for Fc-mediated clearance of TNF-TNFi immune complexes. Therefore, we investigated circulating TNF-TNFi complexes upon treatment with certolizumab-lacking Fc tails-in comparison with adalimumab, golimumab, infliximab and etanercept. EXPERIMENTAL APPROACH: Drug-tolerant ELISAs were developed and used to quantify TNF during adalimumab, golimumab, etanercept, certolizumab and infliximab treatment in patients with inflammatory arthritis or ulcerative colitis for a maximum follow-up of 1 year. Effects on in vitro TNF production and Fc-mediated uptake of TNF-TNFi complexes were investigated for all five TNFi. KEY RESULTS: Circulating TNF concentrations were >20-fold higher during certolizumab treatment compared with adalimumab, reaching up to 23.1 ng·ml-1 . Internalization of TNF-TNFi complexes by macrophages depended on Fc valency, with efficient uptake for the full antibody TNFi (three Fc tails), but little or no uptake for etanercept and certolizumab (one and zero Fc tail, respectively). TNF production was not affected by TNFi. Total TNF load did not affect clearance rate of total TNFi. CONCLUSIONS AND IMPLICATIONS: Differences in TNFi structure profoundly affect clearance of TNF, while it is unlikely that TNF itself significantly contributes to target-mediated drug disposition of TNFi.

Complement activation drives antibody-mediated transfusion-related acute lung injury via macrophage trafficking and formation of NETs.

ABSTRACT: Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and, to date, is without available therapies. Here, we investigated the role of the complement system in TRALI. Murine anti-major histocompatibility complex class I antibodies were used in TRALI mouse models, in combination with analyses of plasma samples from patients with TRALI. We found that in vitro complement activation was related to in vivo antibody-mediated TRALI induction, which was correlated with increased macrophage trafficking from the lungs to the blood in a fragment crystallizable region (Fc)-dependent manner and that this was dependent on C5. Human immunoglobulin G 1 variants of the murine TRALI-inducing antibody 34-1-2S, either unable to activate complement and/or bind to Fcγ receptors (FcγRs), revealed an essential role for the complement system, but not for FcγRs, in the onset of 34-1-2S-mediated TRALI in mice. In addition, we found high levels of complement activation in the plasma of patients with TRALI (n = 53), which correlated with elevated neutrophil extracellular trap (NET) markers. In vitro we found that NETs could be formed in a murine, 2-hit model, mimicking TRALI with lipopolysaccharide and C5a stimulation. Collectively, this reveals a critical role of Fc-mediated complement activation in TRALI, with a direct relation to macrophage trafficking from the lungs to the blood and an association with NET formation, suggesting that targeting the complement system may be an attractive therapeutic approach for combating TRALI.

CD5L is a canonical component of circulatory IgM.

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

Comparative analysis of spike-specific IgG Fc glycoprofiles elicited by adenoviral, mRNA, and protein-based SARS-CoV-2 vaccines.

IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps.

IgG Fab Glycans Hinder FcRn-Mediated Placental Transport.

Abs can be glycosylated in both their Fc and Fab regions with marked effects on Ab function and binding. High levels of IgG Fab glycosylation are associated with malignant and autoimmune conditions, exemplified by rheumatoid arthritis and highly Fab-glycosylated (∼90%) anti-citrullinated protein Abs (ACPAs). Important properties of IgG, such as long half-life and placental transport, are facilitated by the human neonatal Fc receptor (hFcRn). Although it is known that glycosylation of Abs can affect binding to Fc receptors, little is known on the impact of IgG Fab glycosylation on hFcRn binding and transplacental transport. Therefore, we analyzed the interaction between hFcRn and IgG with and without Fab glycans in vitro with various methods as well as in vivo by studying placental transfer of Fab-glycosylated Abs from mothers to newborns. No effect of Fab glycosylation on IgG binding to hFcRn was found by surface plasmon resonance and hFcRn affinity chromatography. In contrast, studies in a cell membrane context revealed that Fab glycans negatively impacted IgG-hFcRn interaction. In line with this, we found that Fab-glycosylated IgGs were transported ∼20% less efficiently across the placenta. This appeared to be a general phenomenon, observed for ACPAs, non-ACPAs, as well as total IgG in rheumatoid arthritis patients and healthy controls. Our results suggest that, in a cellular context, Fab glycans inhibit IgG-hFcRn interaction and thus negatively affect the transplacental transfer of IgG. As Fab-glycosylated Abs are frequently associated with autoimmune and malignant disorders and may be potentially harmful, this might encompass a regulatory mechanism, limiting the half-life and transport of such Abs.

Phagocytosis of platelets opsonized with differently glycosylated anti-HLA hIgG1 by monocyte-derived macrophages.

Immune-mediated platelet refractoriness (PR) remains a significant problem in the setting of platelet transfusion and is predominantly caused by the presence of alloantibodies directed against class I human leukocyte antigens (HLA). Opsonization of donor platelets with these alloantibodies can result in rapid clearance after transfusion via multiple mechanisms, including antibody dependent cellular phagocytosis (ADCP). Interestingly, not all alloimmunized patients develop PR to unmatched platelet transfusions, suggesting variation in HLA-specific IgG responses between patients. Previously, we observed that the glycosylation profile of anti-HLA antibodies was highly variable between PR patients, especially with respect to Fc galactosylation, sialylation and fucosylation. In the current study, we investigated the effect of different Fc glycosylation patterns, with known effects on complement deposition and FcγR binding, on phagocytosis of opsonized platelets by monocyte-derived human macrophages. We found that the phagocytosis of antibody- and complement-opsonized platelets, by monocyte derived M1 macrophages, was unaffected by these qualitative IgG-glycan differences.

Altered Fc glycosylation of anti-HLA alloantibodies in hemato-oncological patients receiving platelet transfusions.

BACKGROUND: The formation of alloantibodies directed against class I human leukocyte antigens (HLA) continues to be a clinically challenging complication after platelet transfusions, which can lead to platelet refractoriness (PR) and occurs in approximately 5%-15% of patients with chronic platelet support. Interestingly, anti-HLA IgG levels in alloimmunized patients do not seem to predict PR, suggesting functional or qualitative differences among anti-HLA IgG. The binding of these alloantibodies to donor platelets can result in rapid clearance after transfusion, presumably via FcγR-mediated phagocytosis and/or complement activation, which both are affected by the IgG-Fc glycosylation. OBJECTIVES: To characterize the Fc glycosylation profile of anti-HLA class I antibodies formed after platelet transfusion and to investigate its effect on clinical outcome. PATIENTS/METHODS: We screened and captured anti-HLA class I antibodies (anti-HLA A2, anti-HLA A24, and anti-HLA B7) developed after platelet transfusions in hemato-oncology patients, who were included in the PREPAReS Trial. Using liquid chromatography-mass spectrometry, we analyzed the glycosylation profiles of total and anti-HLA IgG1 developed over time. Subsequently, the glycosylation data was linked to the patients' clinical information and posttransfusion increments. RESULTS: The glycosylation profile of anti-HLA antibodies was highly variable between patients. In general, Fc galactosylation and sialylation levels were elevated compared to total plasma IgG, which correlated negatively with the platelet count increment. Furthermore, high levels of afucosylation were observed for two patients. CONCLUSIONS: These differences in composition of anti-HLA Fc-glycosylation profiles could potentially explain the variation in clinical severity between patients.

At Critically Low Antigen Densities, IgM Hexamers Outcompete Both IgM Pentamers and IgG1 for Human Complement Deposition and Complement-Dependent Cytotoxicity.

IgM is secreted as a pentameric polymer containing a peptide called the joining chain (J chain). However, integration of the J chain is not required for IgM assembly and in its absence IgM predominantly forms hexamers. The conformations of pentameric and hexameric IgM are remarkably similar with a hexagonal arrangement in solution. Despite these similarities, hexameric IgM has been reported to be a more potent complement activator than pentameric IgM, but reported relative potencies vary across different studies. Because of these discrepancies, we systematically investigated human IgM-mediated complement activation. We recombinantly generated pentameric and hexameric human IgM (IgM+J and IgM-J, respectively) mAbs and measured their ability to induce complement deposition and complement-dependent cytotoxicity when bound to several Ags at varying densities. At high Ag densities, hexameric and pentameric IgM activate complement to a similar extent as IgG1. However, at low densities, hexameric IgM outcompeted pentameric IgM and even more so IgG1. These differences became progressively more pronounced as antigenic density became critically low. Our findings highlight that the differential potency of hexameric and pentameric IgM for complement activation is profoundly dependent on the nature of its interactions with Ag. Furthermore, it underscores the importance of IgM in immunity because it is a more potent complement activator than IgG1 at low Ag densities.

Sodium stibogluconate and CD47-SIRPα blockade overcome resistance of anti-CD20-opsonized B cells to neutrophil killing.

Anti-CD20 antibodies such as rituximab are broadly used to treat B-cell malignancies. These antibodies can induce various effector functions, including immune cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Neutrophils can induce ADCC toward solid cancer cells by trogoptosis, a cytotoxic mechanism known to be dependent on trogocytosis. However, neutrophils seem to be incapable of killing rituximab-opsonized B-cell lymphoma cells. Nevertheless, neutrophils do trogocytose rituximab-opsonized B-cell lymphoma cells, but this only reduces CD20 surface expression and is thought to render tumor cells therapeutically resistant to further rituximab-dependent destruction. Here, we demonstrate that resistance of B-cell lymphoma cells toward neutrophil killing can be overcome by a combination of CD47-SIRPα checkpoint blockade and sodium stibogluconate (SSG), an anti-leishmaniasis drug and documented inhibitor of the tyrosine phosphatase SHP-1. SSG enhanced neutrophil-mediated ADCC of solid tumor cells but enabled trogoptotic killing of B-cell lymphoma cells by turning trogocytosis from a mechanism that contributes to resistance into a cytotoxic anti-cancer mechanism. Tumor cell killing in the presence of SSG required both antibody opsonization of the target cells and disruption of CD47-SIRPα interactions. These results provide a more detailed understanding of the role of neutrophil trogocytosis in antibody-mediated destruction of B cells and clues on how to further optimize antibody therapy of B-cell malignancies.

IgG regulation through FcRn blocking: A novel mechanism for the treatment of myasthenia gravis.

The neonatal Fc receptor (FcRn) is an MHC class I-like molecule that is widely distributed in mammalian organs, tissues, and cells. FcRn is critical to maintaining immunoglobulin G (IgG) and albumin levels through rescuing these molecules from lysosomal degradation. IgG autoantibodies are associated with many autoimmune diseases, including myasthenia gravis (MG), a rare neuromuscular autoimmune disease that causes debilitating and, in its generalized form (gMG), potentially life-threatening muscle weakness. IgG autoantibodies are directly pathogenic in MG and target neuromuscular junction proteins, causing neuromuscular transmission failure. Treatment approaches that reduce autoantibody levels, such as therapeutic plasma exchange and intravenous immunoglobulin, have been shown to be effective for gMG patients but are not indicated as ongoing maintenance therapies and can be associated with burdensome side effects. Agents that block FcRn-mediated recycling of IgG represent a rational and promising approach for the treatment of gMG. Blocking FcRn allows targeted reduction of all IgG subtypes without decreasing concentrations of other Ig isotypes; therefore, FcRn blocking could be a safe and effective treatment strategy for a broad population of gMG patients. Several FcRn-blocking antibodies and one antibody Fc fragment have been developed and are currently in various stages of clinical development. This article describes the mechanism of FcRn blockade as a novel approach for IgG-mediated disease therapy and reviews promising clinical data using such FcRn blockers for the treatment of gMG.

Afucosylated IgG Targets FcγRIV for Enhanced Tumor Therapy in Mice.

Promising strategies for maximizing IgG effector functions rely on the introduction of natural and non-immunogenic modifications. The Fc domain of IgG antibodies contains an N-linked oligosaccharide at position 297. Human IgG antibodies lacking the core fucose in this glycan have enhanced binding to human (FcγR) IIIa/b, resulting in enhanced antibody dependent cell cytotoxicity and phagocytosis through these receptors. However, it is not yet clear if glycan-enhancing modifications of human IgG translate into more effective treatment in mouse models. We generated humanized hIgG1-TA99 antibodies with and without core-fucose. C57Bl/6 mice that were injected intraperitoneally with B16F10-gp75 mouse melanoma developed significantly less metastasis outgrowth after treatment with afucosylated hIgG1-TA99 compared to mice treated with wildtype hhIgG1-TA99. Afucosylated human IgG1 showed stronger interaction with the murine FcγRIV, the mouse orthologue of human FcγRIIIa, indicating that this glycan change is functionally conserved between the species. In agreement with this, no significant differences were observed in tumor outgrowth in FcγRIV-/- mice treated with human hIgG1-TA99 with or without the core fucose. These results confirm the potential of using afucosylated therapeutic IgG to increase their efficacy. Moreover, we show that afucosylated human IgG1 antibodies act across species, supporting that mouse models can be suitable to test afucosylated antibodies.

C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases.

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody's constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in the absence and presence of C1r2s2 In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

High titers and low fucosylation of early human anti-SARS-CoV-2 IgG promote inflammation by alveolar macrophages.

Patients diagnosed with coronavirus disease 2019 (COVID-19) become critically ill primarily around the time of activation of the adaptive immune response. Here, we provide evidence that antibodies play a role in the worsening of disease at the time of seroconversion. We show that early-phase severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) spike protein-specific immunoglobulin G (IgG) in serum of critically ill COVID-19 patients induces excessive inflammatory responses by human alveolar macrophages. We identified that this excessive inflammatory response is dependent on two antibody features that are specific for patients with severe COVID-19. First, inflammation is driven by high titers of anti-spike IgG, a hallmark of severe disease. Second, we found that anti-spike IgG from patients with severe COVID-19 is intrinsically more proinflammatory because of different glycosylation, particularly low fucosylation, of the antibody Fc tail. Low fucosylation of anti-spike IgG was normalized in a few weeks after initial infection with SARS-CoV-2, indicating that the increased antibody-dependent inflammation mainly occurs at the time of seroconversion. We identified Fcγ receptor (FcγR) IIa and FcγRIII as the two primary IgG receptors that are responsible for the induction of key COVID-19-associated cytokines such as interleukin-6 and tumor necrosis factor. In addition, we show that anti-spike IgG-activated human macrophages can subsequently break pulmonary endothelial barrier integrity and induce microvascular thrombosis in vitro. Last, we demonstrate that the inflammatory response induced by anti-spike IgG can be specifically counteracted by fostamatinib, an FDA- and EMA-approved therapeutic small-molecule inhibitor of Syk kinase.

C-Reactive Protein Enhances IgG-Mediated Cellular Destruction Through IgG-Fc Receptors in vitro.

Antibody-mediated blood disorders ensue after auto- or alloimmunization against blood cell antigens, resulting in cytopenia. Although the mechanisms of cell destruction are the same as in immunotherapies targeting tumor cells, many factors are still unknown. Antibody titers, for example, often do not strictly correlate with clinical outcome. Previously, we found C-reactive protein (CRP) levels to be elevated in thrombocytopenic patients, correlating with thrombocyte counts, and bleeding severity. Functionally, CRP amplified antibody-mediated phagocytosis of thrombocytes by phagocytes. To investigate whether CRP is a general enhancer of IgG-mediated target cell destruction, we extensively studied the effect of CRP on in vitro IgG-Fc receptor (FcγR)-mediated cell destruction: through respiratory burst, phagocytosis, and cellular cytotoxicity by a variety of effector cells. We now demonstrate that CRP also enhances IgG-mediated effector functions toward opsonized erythrocytes, in particular by activated neutrophils. We performed a first-of-a-kind profiling of CRP binding to all human FcγRs and IgA-Fc receptor I (FcαRI) using a surface plasmon resonance array. CRP bound these receptors with relative affinities of FcγRIa = FcγRIIa/b = FcγRIIIa > FcγRIIIb = FcαRI. Furthermore, FcγR blocking (in particular FcγRIa) abrogated CRP's ability to amplify IgG-mediated neutrophil effector functions toward opsonized erythrocytes. Finally, we observed that CRP also amplified killing of breast-cancer tumor cell line SKBR3 by neutrophils through anti-Her2 (trastuzumab). Altogether, we provide for the first time evidence for the involvement of specific CRP-FcγR interactions in the exacerbation of in vitro IgG-mediated cellular destruction; a trait that should be further evaluated as potential therapeutic target e.g., for tumor eradication.

Augmented antibody-based anticancer therapeutics boost neutrophil cytotoxicity.

Most clinically used anticancer mAbs are of the IgG isotype, which can eliminate tumor cells through NK cell-mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI) but are unable to activate NK cells and have poorer half-life. Here, we combined the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-EGFR TrisomAb but were less efficient in mediating killing in the presence of IgG anti-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide potential alternatives for cancer patients who do not benefit from current IgG mAb therapy.

Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity.

Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.

IgG Subclasses Shape Cytokine Responses by Human Myeloid Immune Cells through Differential Metabolic Reprogramming.

IgG Abs are crucial for various immune functions, including neutralization, phagocytosis, and Ab-dependent cellular cytotoxicity. In this study, we identified another function of IgG by showing that IgG immune complexes elicit distinct cytokine profiles by human myeloid immune cells, which are dependent on FcγR activation by the different IgG subclasses. Using monoclonal IgG subclasses with identical Ag specificity, our data demonstrate that the production of Th17-inducing cytokines, such as TNF, IL-1ß, and IL-23, is particularly dependent on IgG2, whereas type I IFN responses are controlled by IgG3, and IgG1 is able to regulate both. In addition, we identified that subclass-specific cytokine production is orchestrated at the posttranscriptional level through distinct glycolytic reprogramming of human myeloid immune cells. Combined, these data identify that IgG subclasses provide pathogen- and cell type-specific immunity through differential metabolic reprogramming by FcγRs. These findings may be relevant for future design of Ab-related therapies in the context of infectious diseases, chronic inflammation, and cancer.

Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a large impact on global health, travel, and economy. Therefore, preventative and therapeutic measures are urgently needed. Here, we isolated monoclonal antibodies from three convalescent coronavirus disease 2019 (COVID-19) patients using a SARS-CoV-2 stabilized prefusion spike protein. These antibodies had low levels of somatic hypermutation and showed a strong enrichment in VH1-69, VH3-30-3, and VH1-24 gene usage. A subset of the antibodies was able to potently inhibit authentic SARS-CoV-2 infection at a concentration as low as 0.007 micrograms per milliliter. Competition and electron microscopy studies illustrate that the SARS-CoV-2 spike protein contains multiple distinct antigenic sites, including several receptor-binding domain (RBD) epitopes as well as non-RBD epitopes. In addition to providing guidance for vaccine design, the antibodies described here are promising candidates for COVID-19 treatment and prevention.

FcγR Binding and ADCC Activity of Human IgG Allotypes.

Antibody dependent cellular cytotoxicity (ADCC) is an Fc-dependent effector function of IgG important for anti-viral immunity and anti-tumor therapies. NK-cell mediated ADCC is mainly triggered by IgG-subclasses IgG1 and IgG3 through the IgG-Fc-receptor (FcγR) IIIa. Polymorphisms in the immunoglobulin gamma heavy chain gene likely form a layer of variation in the strength of the ADCC-response, but this has never been studied in detail. We produced all 27 known IgG allotypes and assessed FcγRIIIa binding and ADCC activity. While all IgG1, IgG2, and IgG4 allotypes behaved similarly within subclass, large allotype-specific variation was found for IgG3. ADCC capacity was affected by residues 291, 292, and 296 in the CH2 domain through altered affinity or avidity for FcγRIIIa. Furthermore, allotypic variation in hinge length affected ADCC, likely through altered proximity at the immunological synapse. Thus, these functional differences between IgG allotypes have important implications for therapeutic applications and susceptibility to infectious-, allo- or auto-immune diseases.