Publications by authors named "Gestur Vidarsson"

122 Publications

Augmented antibody-based anti-cancer therapeutics boost neutrophil cytotoxicity.

J Clin Invest 2021 Feb 9. Epub 2021 Feb 9.

Department of Molecular Cell Biology and Immunology, Amsterdam University Medical Center, Amsterdam, Netherlands.

Most clinically used anti-cancer monoclonal antibodies (mAbs) are of the IgG isotype, which can eliminate tumor cells through natural killer (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 combine 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-epidermal growth factor receptor (EGFR) TrisomAb, but were less efficient in mediating killing in the presence of IgG α-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide high potential alternatives for cancer patients that do not benefit from current IgG mAb therapy.
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http://dx.doi.org/10.1172/JCI134680DOI Listing
February 2021

Biological stratification of clinical disease courses in childhood immune thrombocytopenia.

J Thromb Haemost 2021 Jan 1. Epub 2021 Jan 1.

Department of Immunohematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, The Netherlands.

Background: In childhood immune thrombocytopenia (ITP), an autoimmune bleeding disorder, there is a need for better prediction of individual disease courses and treatment outcomes.

Objective: To predict the response to intravenous Immunoglobulins (IVIg) and ITP disease course using genetic and immune markers.

Methods: Children aged below seven years with newly diagnosed ITP (N = 147) from the TIKI study were included, which randomized children to an IVIg or observation group. A total of 46 variables were available: clinical characteristics, targeted genotyping, lymphocyte immune phenotyping, and platelet autoantibodies.

Results: In the treatment arm, 48/80 children (60%) showed a complete response (platelets ≥100 x 10 /L) that lasted for at least one month (complete sustained response; CSR) and 32 exhibited no or a temporary response (absence of a sustained response; ASR). For a biological risk score, five variables were selected by regularized logistic regression that predicted ASR vs CSR: 1) hemoglobin; 2) platelet count; 3) genetic polymorphisms of FcγRIIc; 4) the presence of IgG anti-platelet antibodies; and 5) preceding vaccination. The ASR sensitivity was 0.91 (95% CI, 0.80 - 1.00) and specificity was 0.67 (95% CI, 0.53 - 0.80). In the 67 patients of the observation arm, this biological score was also associated with recovery during one-year follow-up. The addition of the biological score to a predefined clinical score further improved the discrimination of favorable ITP disease courses.

Conclusions: The prediction of disease courses and IVIg treatment responses in ITP is improved by using both clinical and biological stratification.
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http://dx.doi.org/10.1111/jth.15232DOI Listing
January 2021

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

Science 2021 02 23;371(6532). Epub 2020 Dec 23.

Department of Experimental Immunohematology, Sanquin Research, Amsterdam, Netherlands.

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.
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http://dx.doi.org/10.1126/science.abc8378DOI Listing
February 2021

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

J Immunol 2020 Dec 13;205(12):3400-3407. Epub 2020 Nov 13.

Amsterdam Rheumatology and Immunology Center, Department of Rheumatology and Clinical Immunology, Amsterdam University Medical Centers, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands;

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-1β, 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.
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http://dx.doi.org/10.4049/jimmunol.2000263DOI Listing
December 2020

Glycine 236 in the Lower Hinge Region of Human IgG1 Differentiates FcγR from Complement Effector Function.

J Immunol 2020 Dec 13;205(12):3456-3467. Epub 2020 Nov 13.

Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, 1066 CX, Amsterdam, the Netherlands;

Abs of the IgG isotype mediate effector functions like Ab-dependent cellular cytotoxicity and Ab-dependent cellular phagocytosis by Fc interactions with FcγRs and complement-dependent cytotoxicity upon IgG-Fc binding to C1q. In this study, we describe the crucial role of the highly conserved dual glycines at position 236-237 in the lower hinge region of human IgG, including the lack of one glycine as found in IgG2. We found several permutations in this region that either silence or largely abrogate FcγR binding and downstream FcγR effector functions, as demonstrated by surface plasmon resonance, Ab-dependent cellular phagocytosis, and Ab-dependent cellular cytotoxicity assays. Although the binding regions of FcγRs and C1q on the IgG-Fc largely overlap, IgG1 with a deletion of G236 only silences FcγR-mediated effector functions without affecting C1q-binding or activation. Several mutations resulted in only residual FcγRI binding with differing affinities that are either complement competent or silenced. Interestingly, we also found that IgG2, naturally only binding FcγRIIa, gains binding to FcγRI and FcγRIIIa after insertion of G236, highlighting the crucial importance of G236 in IgG for FcγR interaction. These mutants may become invaluable tools for FcγR-related research as well as for therapeutic purposes in which only complement-mediated functions are required without the involvement of FcγR.
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http://dx.doi.org/10.4049/jimmunol.2000961DOI Listing
December 2020

Development of a SARS-CoV-2 Total Antibody Assay and the Dynamics of Antibody Response over Time in Hospitalized and Nonhospitalized Patients with COVID-19.

J Immunol 2020 12 30;205(12):3491-3499. Epub 2020 Oct 30.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory Academic Medical Centre, 1066 CX Amsterdam, the Netherlands;

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infections often cause only mild disease that may evoke relatively low Ab titers compared with patients admitted to hospitals. Generally, total Ab bridging assays combine good sensitivity with high specificity. Therefore, we developed sensitive total Ab bridging assays for detection of SARS-CoV-2 Abs to the receptor-binding domain (RBD) and nucleocapsid protein in addition to conventional isotype-specific assays. Ab kinetics was assessed in PCR-confirmed, hospitalized coronavirus disease 2019 (COVID-19) patients ( = 41) and three populations of patients with COVID-19 symptoms not requiring hospital admission: PCR-confirmed convalescent plasmapheresis donors ( = 182), PCR-confirmed hospital care workers ( = 47), and a group of longitudinally sampled symptomatic individuals highly suspect of COVID-19 ( = 14). In nonhospitalized patients, the Ab response to RBD is weaker but follows similar kinetics, as has been observed in hospitalized patients. Across populations, the RBD bridging assay identified most patients correctly as seropositive. In 11/14 of the COVID-19-suspect cases, seroconversion in the RBD bridging assay could be demonstrated before day 12; nucleocapsid protein Abs emerged less consistently. Furthermore, we demonstrated the feasibility of finger-prick sampling for Ab detection against SARS-CoV-2 using these assays. In conclusion, the developed bridging assays reliably detect SARS-CoV-2 Abs in hospitalized and nonhospitalized patients and are therefore well suited to conduct seroprevalence studies.
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http://dx.doi.org/10.4049/jimmunol.2000767DOI Listing
December 2020

Divergent SARS-CoV-2-specific T- and B-cell responses in severe but not mild COVID-19 patients.

Eur J Immunol 2020 Dec 16;50(12):1998-2012. Epub 2020 Nov 16.

Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4 T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T- and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.
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http://dx.doi.org/10.1002/eji.202048908DOI Listing
December 2020

A clinical prediction score for transient versus persistent childhood immune thrombocytopenia.

J Thromb Haemost 2021 01 27;19(1):121-130. Epub 2020 Nov 27.

Department of Immunohematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, the Netherlands.

Essentials There is a need for improved tools to predict persistent and chronic immune thrombocytopenia (ITP). We developed and validated a clinical prediction model for recovery from newly diagnosed ITP. The Childhood ITP Recovery Score predicts transient vs. persistent ITP and response to intravenous immunoglobulins. The score may serve as a useful tool for clinicians to individualize patient care. ABSTRACT: Background Childhood immune thrombocytopenia (ITP) is an autoimmune bleeding disorder. The prognosis (transient, persistent, or chronic ITP) remains difficult to predict. The morbidity is most pronounced in children with persistent and chronic ITP. Clinical characteristics are associated with ITP outcomes, but there are no validated multivariate prediction models. Objective Development and external validatation of the Childhood ITP Recovery Score to predict transient versus persistent ITP in children with newly diagnosed ITP. Methods Patients with a diagnosis platelet count ≤ 20 × 10 /L and age below 16 years were included from two prospective multicenter studies (NOPHO ITP study, N = 377 [development cohort]; TIKI trial, N = 194 [external validation]). The primary outcome was transient ITP (complete recovery with platelets ≥100 × 10 /L 3 months after diagnosis) versus persistent ITP. Age, sex, mucosal bleeding, preceding infection/vaccination, insidious onset, and diagnosis platelet count were used as predictors. Results In external validation, the score predicted transient versus persistent ITP at 3 months follow-up with an area under the receiver operating characteristic curve of 0.71. In patients predicted to have a high chance of recovery, we observed 85%, 90%, and 95% recovered 3, 6, and 12 months after the diagnosis. For patients predicted to have a low chance of recovery, this was 32%, 46%, and 71%. The score also predicted cessation of bleeding symptoms and the response to intravenous immunoglobulins (IVIg). Conclusion The Childhood ITP Recovery Score predicts prognosis and may be useful to individualize clinical management. In future research, the additional predictive value of biomarkers can be compared to this score. A risk calculator is available (http://www.itprecoveryscore.org).
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http://dx.doi.org/10.1111/jth.15125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839442PMC
January 2021

MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc -Glycosylation.

Front Immunol 2020 21;11:2049. Epub 2020 Aug 21.

Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands.

Current approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they also result in a loss of valuable information regarding certain subclasses, allotypes and co-occuring post-translational modifications (PTMs). Unfortunately, the high variability of polyclonal IgGs makes their intact mass spectrometry (MS) analysis extremely challenging. We propose here a middle-up strategy for the analysis of the intact fragment crystallizable (Fc) region of human plasma IgGs, with the aim of acquiring integrated information of the -glycosylation and other PTMs of subclasses and allotypes. Human plasma IgG was isolated using Fc-specific beads followed by an on-bead C 2 domain digestion with the enzyme IdeS. The obtained mixture of Fc subunits was analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) hyphenated with MS. CE-MS provided separation of different IgG-subclasses and allotypes, while HILIC-MS allowed resolution of the different glycoforms and their oxidized variants. The orthogonality of these techniques was key to reliably assign Fc allotypes. Five individual donors were analyzed using this approach. Heterozygosis was observed in all the analyzed donors resulting in a total of 12 allotypes identified. The assignments were further confirmed using recombinant monoclonal IgG allotypes as standards. While the glycosylation patterns were similar within allotypes of the same subclass, clear differences were observed between IgG subclasses and donors, highlighting the relevance of the proposed approach. In a single analysis, glycosylation levels specific for each allotype, relative abundances of subclasses and information on co-occurring modifications are obtained. This middle-up method represents an important step toward a comprehensive analysis of immunoglobulin G-Fc variants.
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http://dx.doi.org/10.3389/fimmu.2020.02049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472933PMC
August 2020

Cross-reactivity of mouse IgG subclasses to human Fc gamma receptors: Antibody deglycosylation only eliminates IgG2b binding.

Mol Immunol 2020 11 15;127:79-86. Epub 2020 Sep 15.

Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Electronic address:

Immunoglobulin G (IgG) antibodies are important for protection against pathogens and exert effector functions through binding to IgG-Fc receptors (FcγRs) on myeloid and natural killer cells, resulting in destruction of opsonized target cells. Despite interspecies differences, IgG subclasses and FcγRs show substantial similarities and functional conservation between mammals. Accordingly, binding of human IgG (hIgG) to mouse FcγRs (mFcγRs) has been utilized to study effector functions of hIgG in mice. In other applications, such as immunostaining with mouse IgG monoclonal antibodies (mAbs), these cross-reactivities are undesired and prone to misinterpretation. Despite this drawback, the binding of mouse IgG (mIgG) subclasses to human FcγR (hFcγR) classes has never been fully documented. Here, we report detailed and quantifiable characterization of binding affinities for all mIgG subclasses to hFcγRs, including functional polymorphic variants. mIgG subclasses show the strongest binding to hFcγRIa, with relative affinities mIgG2a = mIgG2c > mIgG3 > mIgG2b, and no binding by mIgG1. hFcγRIIa/b showed general low reactivities to all mIgG (mIgG1> mIgG2a/c > mIgG2b), with no reactivity to mIgG3. A particularly high affinity was observed for mIgG1 to the hFcγRIIa-R131 polymorphic variant. hFcγRIIIa showed lower binding (mIgG2a/c > mIgG3), slightly favouring binding to the hFcγRIIIa-V158 over the F158 polymorphic variant. No binding was observed of mIgG to hFcγRIIIb. Deglycosylation of mIgG1 did not abrogate binding to hFcγRIIa-R131, nor did deglycosylation of mIgG2a/c and mIgG3 prevent hFcγRIa binding. Importantly, deglycosylation of the least cross-reactive mIgG subclass, mIgG2b, abrogated reactivity to all hFcγRs. Together, these data document for the first time the full spectrum of cross-reactivities of mouse IgG to human FcγRs.
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http://dx.doi.org/10.1016/j.molimm.2020.08.015DOI Listing
November 2020

Site-Specific Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of Individual Healthy Donors.

Anal Chem 2020 10 22;92(19):13172-13181. Epub 2020 Sep 22.

Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Fc gamma receptors (FcγRs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and FcγR impacts their interaction dramatically. Regarding FcγR glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map FcγRIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for FcγRIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying FcγRIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous FcγRIIIa on natural killer cells. This method will allow assessment of differences in FcγRIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions.
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http://dx.doi.org/10.1021/acs.analchem.0c02342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547861PMC
October 2020

Biological and structural characterization of murine TRALI antibody reveals increased Fc-mediated complement activation.

Blood Adv 2020 08;4(16):3875-3885

Department of Experimental Immunohematology, Sanquin Research, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Transfusion-related acute lung injury (TRALI) remains a leading cause of transfusion-related deaths. In most cases, anti-leukocyte antibodies in the transfusion product trigger TRALI, but not all anti-leukocyte antibodies cause TRALI. It has been shown that the anti-major histocompatibility complex (MHC) class I antibody 34-1-2S (anti-H-2Kd) causes TRALI in BALB/c mice (MHC class I haplotype H-2Kd), whereas SF1.1.10 (anti-H-2Kd) does not. In C57BL/6 mice (MHC class I haplotype H-2Kb), TRALI only occurs when anti-MHC class I antibody AF6-88.5.5.3 (anti-H-2Kb) is administered together with a high dose of 34-1-2S. It remains unknown which specific antibody characteristics are responsible for eliciting TRALI. We therefore investigated several biological and structural features of 34-1-2S compared with other anti-MHC class I antibodies, which on their own do not cause TRALI: SF1.1.10 and AF6-88.5.5.3. No substantial differences were observed between the TRALI-causing 34-1-2S and the TRALI-resistant SF1.1.10 regarding binding affinity to H-2Kd. Regarding binding affinity to H-2Kb, only AF6-88.5.5.3 potently bound to H-2Kb, whereas 34-1-2S exhibited weak but significant cross-reactivity. Furthermore, the binding affinity to FcγRs as well as the Fc glycan composition seemed to be similar for all antibodies. Similar Fc glycosylation profiles were also observed for human TRALI-causing donor anti-HLA antibodies compared with human anti-HLA antibodies from control donors. 34-1-2S, however, displayed superior complement activation capacity, which was fully Fc dependent and not significantly dependent on Fc glycosylation. We conclude that TRALI induction is not correlated with Fab- and Fc-binding affinities for antigen and FcγRs, respectively, nor with the composition of Fc glycans; but increased Fc-mediated complement activation is correlated with TRALI induction.
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http://dx.doi.org/10.1182/bloodadvances.2020002291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448591PMC
August 2020

IgG Subclass Determines Suppression Versus Enhancement of Humoral Alloimmunity to Kell RBC Antigens in Mice.

Front Immunol 2020 16;11:1516. Epub 2020 Jul 16.

Bloodworks Northwest Research Institute, Seattle, WA, United States.

It has long been appreciated that immunoglobulins are not just the effector endpoint of humoral immunity, but rather have a complex role in regulating antibody responses themselves. Donor derived anti-RhD IgG has been used for over 50 years as an immunoprophylactic to prevent maternal alloimmunization to RhD. Although anti-RhD has dramatically decreased rates of hemolytic disease of the fetus and newborn (for the RhD alloantigen), anti-RhD also fails in some cases, and can even paradoxically enhance immune responses in some circumstances. Attempts to generate a monoclonal anti-RhD have largely failed, with some monoclonals suppressing less than donor derived anti-RhD and others enhancing immunity. These difficulties likely result, in part, because the mechanism of anti-RhD remains unclear. However, substantial evidence exists to reject the common explanations of simple clearance of RhD + RBCs or masking of antigen. Donor derived anti-RhD is a mixture of 4 different IgG subtypes. To the best of our knowledge an analysis of the role different IgG subtypes play in immunoregulation has not been carried out; and, only IgG1 and IgG3 have been tested as monoclonals. Multiple attempts to elicit alloimmune responses to human RhD epitopes in mice have failed. To circumvent this limitation, we utilize a tractable animal model of RBC alloimmunization using the human Kell glycoprotein as an antigen to test the effect of IgG subtype on immunoregulation by antibodies to RBC alloantigens. We report that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is a function of the IgG subclass in this model system.
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http://dx.doi.org/10.3389/fimmu.2020.01516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378678PMC
July 2020

Author Correction: Human DC-SIGN and CD23 do not interact with human IgG.

Sci Rep 2020 Jul 23;10(1):12560. Epub 2020 Jul 23.

Department Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-68760-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378241PMC
July 2020

Editorial: Roles of Fc Receptors in Disease and Therapy.

Front Immunol 2020 12;11:1232. Epub 2020 Jun 12.

Sanquin Research and Landsteiner Laboratory, Department of Experimental Immunohematology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

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http://dx.doi.org/10.3389/fimmu.2020.01232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325973PMC
June 2020

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

Science 2020 08 15;369(6504):643-650. Epub 2020 Jun 15.

Department of Medical Microbiology, Amsterdam UMC, University of Amsterdam, Amsterdam Institute for Infection and Immunity, 1105AZ Amsterdam, Netherlands.

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.
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http://dx.doi.org/10.1126/science.abc5902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299281PMC
August 2020

Propranolol Is an Effective Topical and Systemic Treatment Option for Experimental Epidermolysis Bullosa Acquisita.

J Invest Dermatol 2020 Dec 22;140(12):2408-2420. Epub 2020 May 22.

Lübeck Institute of Experimental Dermatology, University of Lübeck, Germany. Electronic address:

Propranolol is an ADRB2 blocker that regulates heart muscle contractions, smooth muscle relaxation, and glycogenolysis. In addition, an increasing number of applications in dermatology have been described, most prominently, the use as a first-line treatment for infantile hemangiomas. We here show that propranolol enhances IL-8-induced neutrophil chemotaxis and reduces the release of ROS after immune complex stimulation. To obtain further molecular insights into the modulatory effects of propranolol in activated neutrophils, we performed RNA sequencing of immune complex-stimulated neutrophils in the absence and presence of the drug. We identified the transcriptomic signature of propranolol and demonstrated an ADR2-independent immunomodulatory effect. To determine if the anti-inflammatory transcriptomic signature of propranolol also translates into clinical effects, we next evaluated the impact of propranolol in a prototypical neutrophil-dependent skin disease, specifically, antibody transfer-induced epidermolysis bullosa acquisita in mice. To validate the identified propranolol gene signature obtained in human neutrophils, we analyzed a selection of genes by RT-PCR in mouse epidermolysis bullosa acquisita skin and confirmed TNF, among others, to be differentially regulated by propranolol treatment. Our data clearly indicate that, based on its molecular impact on immune complex-activated neutrophils, propranolol is a potential treatment option for neutrophil-mediated inflammatory skin diseases.
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http://dx.doi.org/10.1016/j.jid.2020.04.025DOI Listing
December 2020

FcγR Binding and ADCC Activity of Human IgG Allotypes.

Front Immunol 2020 6;11:740. Epub 2020 May 6.

Sanquin Research and Landsteiner Laboratory, Department of Experimental Immunohematology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

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.
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http://dx.doi.org/10.3389/fimmu.2020.00740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218058PMC
May 2020

Natural killer cell activation by respiratory syncytial virus-specific antibodies is decreased in infants with severe respiratory infections and correlates with Fc-glycosylation.

Clin Transl Immunology 2020 19;9(2):e1112. Epub 2020 Feb 19.

Centre for Infectious Disease Control National Institute for Public Health and the Environment (RIVM) Bilthoven The Netherlands.

Objectives: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, and there is no vaccine available. In early life, the most important contributors to protection against infectious diseases are the innate immune response and maternal antibodies. However, antibody-mediated protection against RSV disease is incompletely understood, as both antibody levels and neutralisation capacity correlate poorly with protection. Since antibodies also mediate natural killer (NK) cell activation, we investigated whether this functionality correlates with RSV disease.

Methods: We performed an observational case-control study including infants hospitalised for RSV infection, hernia surgery or RSV-negative respiratory viral infections. We determined RSV antigen-specific antibody levels in plasma using a multiplex immunoassay. Subsequently, we measured the capacity of these antibodies to activate NK cells. Finally, we assessed Fc-glycosylation of the RSV-specific antibodies by mass spectrometry.

Results: We found that RSV-specific maternal antibodies activate NK cells . While concentrations of RSV-specific antibodies did not differ between cases and controls, antibodies from infants hospitalised for severe respiratory infections (RSV and/or other) induced significantly less NK cell interferon-γ production than those from uninfected controls. Furthermore, NK cell activation correlated with Fc-fucosylation of RSV-specific antibodies, but their glycosylation status did not significantly differ between cases and controls.

Conclusion: Our results suggest that Fc-dependent antibody function and quality, exemplified by NK cell activation and glycosylation, contribute to protection against severe RSV disease and warrant further studies to evaluate the potential of using these properties to evaluate and improve the efficacy of novel vaccines.
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http://dx.doi.org/10.1002/cti2.1112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029726PMC
February 2020

Anti-platelet antibody immunoassays in childhood immune thrombocytopenia: a systematic review.

Vox Sang 2020 May 20;115(4):323-333. Epub 2020 Feb 20.

Department of Immunohematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, The Netherlands.

Background: In adult immune thrombocytopenia (ITP), an acquired autoimmune bleeding disorder, anti-platelet autoantibody testing may be useful as a rule-in test. Childhood ITP has different disease characteristics, and the diagnostic and prognostic value of anti-platelet antibody testing remains uncertain.

Objective: To systematically review the diagnostic accuracy of anti-platelet autoantibody testing in childhood ITP.

Methods: PubMed and EMBASE were searched for studies evaluating immunoassays in childhood ITP. Study quality was assessed (QUADAS2), and evidence was synthesized descriptively.

Results: In total, 40 studies (1606 patients) were identified. Nine studies reported sufficient data to determine diagnostic accuracy measures. Anti-platelet IgG antibody testing showed a moderate sensitivity (0·36-0·80 platelet-associated IgG [direct test]; 0·19-0·39 circulating IgG [indirect test]). In studies that reported control data, including patients with non-immune thrombocytopenia, specificity was very good (0·80-1·00). Glycoprotein-specific immunoassays showed comparable sensitivity (three studies) and predominantly identified IgG anti-GP IIb/IIIa antibodies, with few IgG anti-GP Ib/IX antibodies. Anti-platelet IgM antibodies were identified in a substantial proportion of children (sensitivity 0·62-0·64 for direct and indirect tests).

Conclusion: The diagnostic evaluation of IgG and IgM anti-platelet antibodies may be useful as a rule-in test for ITP. In children with insufficient platelets for a direct test, indirect tests may be performed instead. A negative test does not rule out the diagnosis of ITP. Future studies should evaluate the value of anti-platelet antibody tests in thrombocytopenic children with suspected ITP.
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http://dx.doi.org/10.1111/vox.12894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317748PMC
May 2020

IgG-Fc glycosylation before and after rituximab treatment in immune thrombocytopenia.

Sci Rep 2020 02 20;10(1):3051. Epub 2020 Feb 20.

Sanquin Research, Department of Experimental Immunohematology, Amsterdam, The Netherlands and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

The interactions of antibodies with myeloid Fcγ receptors and the complement system are regulated by an Asn297-linked glycan in the Fc portion of IgG. Alterations of serum IgG-Fc glycosylation have been reported in various autoimmune diseases, and correlate with treatment response and disease activity. We hypothesized that IgG-Fc glycosylation is altered in immune thrombocytopenia (ITP) and associates with response to anti-CD20 monoclonal antibody treatment (rituximab). IgG-Fc glycosylation was analyzed by liquid chromatography-mass spectrometry. We found that IgG-Fc glycosylation was identical between refractory ITP patients (HOVON64 trial; N = 108) and healthy controls (N = 120). Two months after rituximab treatment, we observed a shift in Fc glycosylation, with a mean 1.7% reduction in galactosylation for IgG1 and IgG4 and a mean 1.5% increase for bisection in IgG1, IgG2/3 and IgG4 (adjusted p < 1.7 × 10 and p < 2 × 10, respectively). Neither baseline nor longitudinal changes in IgG-Fc glycosylation after rituximab were associated with clinical treatment response. We conclude that IgG-Fc glycosylation in refractory ITP is similar to healthy controls and does not predict treatment responses to rituximab. The observed changes two months after treatment suggest that rituximab may influence total serum IgG-Fc glycosylation. Overall, our study suggests that the pathophysiology of refractory ITP may differ from other autoimmune diseases.
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http://dx.doi.org/10.1038/s41598-020-59651-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033207PMC
February 2020

Anti-platelet antibodies in childhood immune thrombocytopenia: Prevalence and prognostic implications.

J Thromb Haemost 2020 05 12;18(5):1210-1220. Epub 2020 Apr 12.

Laboratory for Platelet and Leukocyte Serology, Department of Immunohematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, the Netherlands.

Background: Anti-platelet antibody testing may be useful for the diagnosis and management of childhood immune thrombocytopenia (ITP).

Objectives: Here we aimed to assess the prevalence and prognostic significance of anti-platelet glycoprotein-specific IgM and IgG antibodies.

Methods: Children with newly diagnosed ITP were included at diagnosis and randomized to an intravenous immunoglobulins (IVIg) or careful observation group (TIKI trial). In this well-defined and longitudinally followed cohort (N = 179), anti-platelet glycoprotein-specific IgM and IgG antibodies were determined by monoclonal antibody-immobilization of platelet antigens.

Results: The dominant circulating anti-platelet antibody class in childhood ITP was IgM (62% of patients); but IgG antibodies were also found (10%). Children without IgM platelet antibodies were older and more often female. There was weak evidence for an association between IgM anti-GP IIb/IIIa antibodies and an increased bleeding severity (P = .03). The IgM and IgG anti-platelet responses partially overlapped, and reactivity was frequently directed against multiple glycoproteins. During 1-year follow-up, children with IgM antibodies in the observation group displayed a faster platelet recovery compared to children without, also after adjustment for age and preceding infections (P = 7.1 × 10 ). The small group of patients with detectable IgG anti-platelet antibodies exhibited an almost complete response to IVIg treatment (N = 12; P = .02), suggesting that IVIg was particularly efficacious in these children.

Conclusions: Testing for circulating anti-platelet antibodies may be helpful for the clinical prognostication and the guidance of treatment decisions in newly diagnosed childhood ITP. Our data suggest that the development of even more sensitive tests may further improve the clinical value of antibody testing.
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http://dx.doi.org/10.1111/jth.14762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318215PMC
May 2020

Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy.

Sci Rep 2020 01 30;10(1):1464. Epub 2020 Jan 30.

Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands.

Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77-81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fucosylation mediated more efficient ADCC and clearance than anti-D Ig. Galactosylation of B mAb-Ds was 57-83% but 15-58% for rodent mAb-Ds. HH mAb-Ds had non-human sugars. These data reveal high galactosylation like anti-D Ig (>60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis.
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http://dx.doi.org/10.1038/s41598-019-57393-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992666PMC
January 2020

Strategies to develop a prophylaxis for the prevention of HPA-1a immunization and fetal and neonatal alloimmune thrombocytopenia.

Transfus Apher Sci 2020 Feb 31;59(1):102712. Epub 2019 Dec 31.

Department of Medical Biology, UiT- The Artic University of Norway, Tromsø, Norway.

Anti-HPA-1a-antibodies are the main cause of fetal and neonatal alloimmune thrombocytopenia (FNAIT) which may result in intracranial hemorrhage (ICH) and death among fetuses and newborns. Advances in understanding the pathogenesis of FNAIT and proof of concept for prophylaxis to prevent immunization suggest that development of hyperimmune anti-HPA-1a IgG aimed at preventing immunization against HPA-1a and FNAIT is feasible. Anti-HPA-1a IgG can be obtained either by isolating immunoglobulin from already-immunized women or by development of monoclonal anti-HPA-1a antibodies. Here we discuss recent advances that may lead to the development of a prenatal and postnatal prophylactic treatment for the prevention of HPA-1a-associated FNAIT and life-threatening FNAIT-induced complications.
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http://dx.doi.org/10.1016/j.transci.2019.102712DOI Listing
February 2020

Functional Attributes of Antibodies, Effector Cells, and Target Cells Affecting NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity.

J Immunol 2019 12 20;203(12):3126-3135. Epub 2019 Nov 20.

Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, 1066 CX Amsterdam, the Netherlands;

Ab-dependent cellular cytotoxicity (ADCC) is one of the most important effector mechanisms of tumor-targeting Abs in current immunotherapies. In ADCC and other Ab-dependent activation of myeloid effector cells, close cell-cell contact (between effector and target cell) and formation of immunological synapses are required. However, we still lack basic knowledge on the principal factors influencing ADCC potential by therapeutic Abs. In this study we investigated the combined roles of five factors affecting human NK cell-mediated ADCC, namely: 1) Ag density, 2) target cell membrane composition, 3) IgG FcγR polymorphism, 4) FcγR-blocking cytophilic Abs, and 5) Ab fucosylation. We demonstrate that the magnitude of NK cell-mediated ADCC responses is predominantly influenced by Ag density and Ab fucosylation. Afucosylation consistently induced efficient ADCC, even at very low Ag density, where fucosylated target Abs did not elicit ADCC. On the side of the effector cell, the FcγRIIIa-Val/Phe158 polymorphism influenced ADCC potency, with NK cells expressing the Val158 variant showing more potent ADCC. In addition, we identified the sialic acid content of the target cell membrane as an important inhibitory factor for ADCC. Furthermore, we found that the presence and glycosylation status of aspecific endogenous Abs bound to NK cell FcγRIIIa (cytophilic Abs) determine the blocking effect on ADCC. These five parameters affect the potency of Abs in vitro and should be further tested as predictors of in vivo capacity.
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http://dx.doi.org/10.4049/jimmunol.1900985DOI Listing
December 2019

The Ligands for Human IgG and Their Effector Functions.

Antibodies (Basel) 2019 Apr 25;8(2). Epub 2019 Apr 25.

Sanquin Research, Dept Experimental Immunohematology and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, 1066 CX Amsterdam, The Netherlands.

Activation of the humoral immune system is initiated when antibodies recognize an antigen and trigger effector functions through the interaction with Fc engaging molecules. The most abundant immunoglobulin isotype in serum is Immunoglobulin G (IgG), which is involved in many humoral immune responses, strongly interacting with effector molecules. The IgG subclass, allotype, and glycosylation pattern, among other factors, determine the interaction strength of the IgG-Fc domain with these Fc engaging molecules, and thereby the potential strength of their effector potential. The molecules responsible for the effector phase include the classical IgG-Fc receptors (FcγR), the neonatal Fc-receptor (FcRn), the Tripartite motif-containing protein 21 (TRIM21), the first component of the classical complement cascade (C1), and possibly, the Fc-receptor-like receptors (FcRL4/5). Here we provide an overview of the interactions of IgG with effector molecules and discuss how natural variation on the antibody and effector molecule side shapes the biological activities of antibodies. The increasing knowledge on the Fc-mediated effector functions of antibodies drives the development of better therapeutic antibodies for cancer immunotherapy or treatment of autoimmune diseases.
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http://dx.doi.org/10.3390/antib8020030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640714PMC
April 2019

Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering.

Sci Adv 2019 08 28;5(8):eaaw1822. Epub 2019 Aug 28.

Department of Medical Oncology, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, Netherlands.

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.
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http://dx.doi.org/10.1126/sciadv.aaw1822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713500PMC
August 2019

Anti-glycoprotein Ibα autoantibodies do not impair circulating thrombopoietin levels in immune thrombocytopenia patients.

Haematologica 2020 04 11;105(4):e172-e174. Epub 2019 Jul 11.

Sanquin Research, Department of Experimental Immunohematology and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam

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http://dx.doi.org/10.3324/haematol.2019.228908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109722PMC
April 2020

Human DC-SIGN and CD23 do not interact with human IgG.

Sci Rep 2019 07 10;9(1):9995. Epub 2019 Jul 10.

Department Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.

The precise mechanisms underlying anti-inflammatory effects of intravenous immunoglobulin (IVIg) therapies remain elusive. The sialylated IgG fraction within IVIg has been shown to be therapeutically more active in mouse models. Functionally, it has been suggested that IgG undergoes conformational changes upon Fc-sialylation which sterically impede binding to conventional FcγRs, but simultaneously allow binding to human DC-SIGN (SIGN-R1 in mice) and also CD23. These latter C-type lectins have been proposed responsible for the immunomodulatory effects in mouse models. However, there is conflicting evidence supporting direct interactions between sialylated human IgG and CD23/DC-SIGN. While cells expressing human CD23 and DC-SIGN in their native configuration bound their natural ligands IgE and ICAM-3, respectively, no IgG binding was observed, regardless of Fc-glycan sialylation in any context (with or without bisection and/or fucosylation) or presence of sialylated Fab-glycans. This was tested by both by FACS and a novel cellular Surface Plasmon Resonance imaging (cSPRi) approach allowing for monitoring low-affinity but high-avidity interactions. In summary, we find no evidence for human CD23 or DC-SIGN being bona fide receptors to human IgG, regardless of IgG Fc- or Fab-glycosylation status. However, these results do not exclude the possibility that either IgG glycosylation or C-type lectins affect IVIg therapies.
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http://dx.doi.org/10.1038/s41598-019-46484-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620288PMC
July 2019