Allergen-induced late airway response offers important pharmacodynamic targets, including T helper 2 (TH2) biomarkers. However, detection of inflammatory markers has been limited in dithiothreitol-processed sputum.

The binding strength between IgG and FcγR is influenced by the composition of the N-linked glycan at position N297 in the Fc-domain of IgG. Particularly, afucosylation increases the binding affinity of human IgG1 to human FcγRIIIa up to ∼20 fold, and additional galactosylation of the afucosylated IgG increases the affinity up to ∼40 fold. The increase in affinity for afucosylated IgG has previously been shown to depend on direct carbohydrate-carbohydrate interactions between the IgG-Fc glycan with an N-linked glycan at position 162 unique to hFcγRIIIa and hFcγRIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse FcγR, mFcγRIV. The N162-glycan in mFcγRIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFcγRIIIa. However, unlike hFcγRIIIa, mFcγRIV did not bind more avidly to IgG with increased galactose and reduced fucose. Overall, these results suggest the N162-glycan in the human FcγRIII family and its orthologous mouse FcγRIV to be functionally conserved.

The complement factor H-related (FHR) proteins are hypothesized to fine-tune the regulatory role of complement factor H (FH) in the alternative pathway of the complement system. Moreover, FHR-1, FHR-2, and FHR-5 have been proposed to be dimers, which further complicates accurate analysis. As FHRs are highly similar among themselves and toward FH, obtaining specific reagents for quantification of serum levels and functional analysis is challenging. In this study, we generated antibodies and developed ELISAs to measure FHR-1, FHR-2, and FHR-5 in serum. We used both recombinant and serum-derived proteins to show that four dimers occur in human circulation: homodimers of FHR-1, FHR-2, and FHR-5, as well as FHR-1/FHR-2 heterodimers. Heterodimers containing FHR-5 were not found. In individuals with homozygous CFHR1 deletions or compound heterozygous CFHR2 missense/nonsense mutations identified in this study, the respective FHR-1 and FHR-2 homo- and heterodimers were absent. Using FRET, we found that recombinant FHR dimers exchange monomers rapidly. This was confirmed ex vivo, using FHR-1- and FHR-2-deficient sera. Of all FHR dimers, FHR-5/5 homodimers demonstrated strong binding affinity toward heparin. Specific ELISAs demonstrated that serum levels of FHR-1/1, FHR-1/2, FHR-2/2, and FHR-5/5 dimers were low compared to FH, which circulates at a 10- to 200-fold molar excess. In summary, FHR-1, FHR-2, and FHR-5 homodimerize, with FHR-1 and FHR-2 forming heterodimers as well, and equilibrate quickly in plasma.

Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.

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.

Complement is activated as part of the innate immune defense against invading pathogens. Also, it helps to remove apoptotic debris and immune complexes from the circulation. Impaired complement function due to aberrant plasma levels of complement proteins may be indicative for complement-mediated diseases or can be involved in susceptibility for infections. To determine whether plasma levels are abnormal, reference intervals (RIs) are used from adult healthy donors. Since many complement-mediated diseases have an onset during childhood, it is important to know whether these RIs can be extrapolated to children. RIs of Factor H (FH), the crucial fluid-phase regulator, and the FH-related proteins (FHRs), its homologous counterparts, are unknown in healthy children. While FH is measured to diagnose and monitor therapy of patients with atypical hemolytic uremic syndrome, recent studies also implicated increased plasma levels of FHRs in disease. Here, we investigated the levels of FH and FHRs in healthy children using recently developed specific ELISAs. We found that levels of FH, FHR-2, and FHR-3 were equal to those found in healthy adults. Levels of FHR-4A and FHR-5 were lower in children than in adults. However, only the FHR-5 levels associated with age. The RIs of these FH family proteins now serve to support the interpretation of plasma levels in prospective and retrospective studies that can be used for routine diagnostic and monitoring purposes including pediatric patient samples.

It is currently thought that most-but not all-individuals infected with SARS-CoV-2 develop symptoms, but that the infectious period starts on average two days before the first overt symptoms appear. It is estimated that pre- and asymptomatic individuals are responsible for more than half of all transmissions. By detecting infected individuals before they have overt symptoms, wearable devices could potentially and significantly reduce the proportion of transmissions by pre-symptomatic individuals. Using laboratory-confirmed SARS-CoV-2 infections (detected via serology tests [to determine if there are antibodies against the SARS-CoV-2 in the blood] or SARS-CoV-2 infection tests such as polymerase chain reaction [PCR] or antigen tests) as the gold standard, we will determine the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the following two algorithms to detect first time SARS-CoV-2 infection including early or asymptomatic infection: the algorithm using Ava bracelet data when coupled with self-reported Daily Symptom Diary data (Wearable + Symptom Data Algo; experimental condition) the algorithm using self-reported Daily Symptom Diary data alone (Symptom Only Algo; control condition) In addition, we will determine which of the two algorithms has superior performance characteristics for detecting SARS-CoV-2 infection including early or asymptomatic infection as confirmed by SARS-CoV-2 virus testing.

Dual enkephalinase inhibitors (DENKIs) are involved in the regulation of nociception via opioid receptors. The novel compound STR-324 belongs to the DENKI pharmacological class. This first-in-human study evaluated the safety, tolerability, pharmacokinetics and pharmacodynamics of STR-324 in healthy male participants.

Data are scarce on immunogenicity of COVID-19 vaccines in patients with autoimmune diseases, who are often treated with immunosuppressive drugs. We aimed to investigate the effect of different immunosuppressive drugs on antibody development after COVID-19 vaccination in patients with autoimmune diseases.

IgG4 is the least abundant subclass of IgG in human serum and has unique functional features. IgG4 is largely unable to activate antibody-dependent immune effector responses and, furthermore, undergoes Fab (fragment antigen binding)-arm exchange, rendering it bispecific for antigen binding and functionally monovalent. These properties of IgG4 have a blocking effect, either on the immune response or on the target protein of IgG4. In this Review, we discuss the unique structural characteristics of IgG4 and how these contribute to its roles in health and disease. We highlight how, depending on the setting, IgG4 responses can be beneficial (for example, in responses to allergens or parasites) or detrimental (for example, in autoimmune diseases, in antitumour responses and in anti-biologic responses). The development of novel models for studying IgG4 (patho)physiology and understanding how IgG4 responses are regulated could offer insights into novel treatment strategies for these IgG4-associated disease settings.

Natalizumab is a monoclonal IgG4 antibody used for treatment of relapsing remitting MS. Natalizumab interferes with lymphocyte migration by blocking alpha-4 integrin (CD49d). Saturation levels of alpha-4 integrin on circulating T cells by natalizumab have been associated with clinical effectiveness of therapy. However, in most cases, measurements have been carried out using freshly isolated PBMCs. The aim of this study was to set up and evaluate a method to measure relative levels of cell-bound natalizumab using frozen PBMC samples. A new method was set up to measure cell-bound natalizumab by flow cytometry on T cell subsets using fully saturated cells as a 100% reference. A comparison was made between spike samples and samples of natalizumab-treated MS patients freshly isolated and stored in liquid nitrogen. Cell-bound natalizumab could be measured (using an anti-IgG4 antibody) on cells stored in liquid nitrogen. Natalizumab was found to slowly dissociate from the cells during isolation and subsequent sample work-up. This dissociation was more pronounced for monovalent natalizumab resulting from Fab arm exchange (the predominant isoform in patients) than bivalent natalizumab straight from the vial. We established a correction factor to account for this phenomenon. The resulting method has good accuracy compared to assessing fresh cells. The inter-assay precision (%CV) is ca. 12% using frozen cells. In conclusion, we established a method to assess relative levels of cell-bound natalizumab on cells obtained from frozen PBMC samples.

Therapeutic drug monitoring (TDM) can optimize the efficacy of infliximab (IFX) in patients with inflammatory bowel disease (IBD). Because of the delay between blood samples taken at trough and availability of results, dose adjustments can only be carried out at the next infusion, typically 8 weeks later. Dried blood samples (DBS) performed at home to measure IFX concentrations can reduce the time to adapt dose/dosing interval. Here, we aimed to validate the clinical application of DBS for IFX in IBD patients and to evaluate the feasibility of home sampling.

Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3(Cre/+) , devoid of mast cells but with intact Kit signaling.

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.

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.

To assess viral clearance, pharmacokinetics, tolerability and symptom evolution following ensovibep administration in symptomatic COVID-19 outpatients.

Binding to the neonatal Fc receptor (FcRn) extends serum half-life of IgG, and antagonizing this interaction is a promising therapeutic approach in IgG-mediated autoimmune diseases. Fc-MST-HN, designed for enhanced FcRn binding capacity, has not been evaluated in the context of a full-length antibody, and the structural properties of the attached Fab regions might affect the FcRn-mediated intracellular trafficking pathway. Here we present a comprehensive comparative analysis of the IgG salvage pathway between two full-size IgG1 variants, containing wild type and MST-HN Fc fragments, and their Fc-only counterparts. We find no evidence of Fab-regions affecting FcRn binding in cell-free assays, however, cellular assays show impaired binding of full-size IgG to FcRn, which translates into improved intracellular FcRn occupancy and intracellular accumulation of Fc-MST-HN compared to full size IgG1-MST-HN. The crystal structure of Fc-MST-HN in complex with FcRn provides a plausible explanation why the Fab disrupts the interaction only in the context of membrane-associated FcRn. Importantly, we find that Fc-MST-HN outperforms full-size IgG1-MST-HN in reducing IgG levels in cynomolgus monkeys. Collectively, our findings identify the cellular membrane context as a critical factor in FcRn biology and therapeutic targeting.

Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms.

High-affinity antibody-secreting cells (ASC) arise from terminal differentiation of B-cells after coordinated interactions with T follicular helper (Tfh) cells in germinal centers (GC). Elucidation of cues promoting human naive B-cells to progress into ASCs is challenging, as this process is notoriously difficult to induce in vitro while maintaining enough cell numbers to investigate the differentiation route(s). Here, we describe a minimalistic in vitro culture system that supports efficient differentiation of human naive B-cells into antibody-secreting cells. Upon initial stimulations, the interplay between level of CD40 costimulation and the Tfh cell-associated cytokines IL-21 and IL-4 determined the magnitude of B-cell expansion, immunoglobulin class-switching and expression of ASC regulator PRDM1. In contrast, the B-cell-specific transcriptional program was maintained, and efficient ASC formation was hampered. Renewed CD40 costimulation and Tfh cytokines exposure induced rapid secondary STAT3 signaling and extensive ASC differentiation, accompanied by repression of B-cell identity factors PAX5, BACH2 and IRF8 and further induction of PRDM1. Our work shows that, like in vivo, renewed CD40L costimulation also induces efficient terminal ASC differentiation after initial B-cell expansion in vitro. This culture system for efficient differentiation of human naive B-cells into ASCs, while also maintaining high cell numbers, may form an important tool in dissecting human naive B-cell differentiation, thereby enabling identification of novel transcriptional regulators and biomarkers for desired and detrimental antibody formation in humans.

Extended interval dosing (EID) of natalizumab treatment is increasingly used in multiple sclerosis. Besides the clear anti-inflammatory effect, natalizumab is considered to have neuroprotective properties as well.