Publications by authors named "Ninotska I L Derksen"

24 Publications

  • Page 1 of 1

CD45RB Glycosylation and Ig Isotype Define Maturation of Functionally Distinct B Cell Subsets in Human Peripheral Blood.

Front Immunol 2022 28;13:891316. Epub 2022 Apr 28.

Landsteiner Laboratory, Sanquin Research, Department of Immunopathology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

Glycosylation of CD45RB (RB+) has recently been identified to mark antigen-experienced B cells, independent of their CD27 expression. By using a novel combination of markers including CD45RB glycosylation, CD27 and IgM/IgD isotype expression we segregated human peripheral blood B cell subsets and investigated their IGHV repertoire and functionality. We observed distinct maturation stages for CD27-RB+ cells, defined by differential expression of non-switched Ig isotypes. CD27-RB+ cells, which only express IgM, were more matured in terms of Ig gene mutation levels and function as compared to CD27-RB+ cells that express both IgM and IgD or cells that were CD27-RB-. Moreover, CD27-RB+IgM+ cells already showed remarkable rigidity in IgM isotype commitment, different from CD27-RB+IgMD+ and CD27-RB- cells that still demonstrated great plasticity in B cell fate decision. Thus, glycosylation of CD45RB is indicative for antigen-primed B cells, which are, dependent on the Ig isotype, functionally distinct.
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http://dx.doi.org/10.3389/fimmu.2022.891316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9095956PMC
April 2022

Human IgE does not bind to human FcRn.

Sci Rep 2022 01 7;12(1):62. Epub 2022 Jan 7.

Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands.

The neonatal Fc receptor (FcRn) is known to mediate placental transfer of IgG from mother to unborn. IgE is widely known for triggering immune responses to environmental antigens. Recent evidence suggests FcRn-mediated transplacental passage of IgE during pregnancy. However, direct interaction of FcRn and IgE was not investigated. Here, we compared binding of human IgE and IgG variants to recombinant soluble human FcRn with β2-microglobulin (sFcRn) in surface plasmon resonance (SPR) at pH 7.4 and pH 6.0. No interaction was found between human IgE and human sFcRn. These results imply that FcRn can only transport IgE indirectly, and thereby possibly transfer allergenic sensitivity from mother to fetus.
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http://dx.doi.org/10.1038/s41598-021-03852-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8741920PMC
January 2022

Fc Galactosylation Promotes Hexamerization of Human IgG1, Leading to Enhanced Classical Complement Activation.

J Immunol 2021 09 18;207(6):1545-1554. Epub 2021 Aug 18.

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

Human IgG contains one evolutionarily conserved -linked glycan in its Fc region at position 297. This glycan is crucial for Fc-mediated functions, including its induction of the classical complement cascade. This is induced after target recognition through the IgG-Fab regions, allowing neighboring IgG-Fc tails to associate through Fc:Fc interaction, ultimately leading to hexamer formation. This hexamerization seems crucial for IgG to enable efficient interaction with the globular heads of the first complement component C1q and subsequent complement activation. In this study, we show that galactose incorporated in the IgG1-Fc enhances C1q binding, C4, C3 deposition, and complement-dependent cellular cytotoxicity in human erythrocytes and Raji cells. IgG1-Fc sialylation slightly enhanced binding of C1q, but had little effect on downstream complement activation. Using various mutations that decrease or increase hexamerization capacity of IgG1, we show that IgG1-Fc galactosylation has no intrinsic effect on C1q binding to IgG1, but enhances IgG1 hexamerization potential and, thereby, complement activation. These data suggest that the therapeutic potential of Abs can be amplified without introducing immunogenic mutations, by relatively simple glycoengineering.
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http://dx.doi.org/10.4049/jimmunol.2100399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428746PMC
September 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 Rheumatology and Clinical Immunology, Amsterdam UMC, Amsterdam Rheumatology and Immunology Center, 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919849PMC
February 2021

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

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

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

Identification of the amino-terminal fragment of Ara h 1 as a major target of the IgE-binding activity in the basic peanut protein fraction.

Clin Exp Allergy 2020 03 7;50(3):401-405. Epub 2020 Feb 7.

Department of Biosciences, University of Salzburg, Salzburg, Austria.

Background: Small, basic peanut proteins are often poorly extracted in pH-neutral buffers that are optimal for the extraction of peanut storage proteins such as Ara h 1. As a result, such proteins are easily missed as potential allergens.

Objective: To analyse the allergenic composition of the basic peanut protein (BPP) fraction.

Methods: A peanut extract prepared at pH 4 was fractionated by physicochemical procedures. Chemical analysis was performed by SDS-PAGE and mass spectrometry. Because immunoblotting was found to be inefficient for most of these small basic proteins, IgE-binding activity was measured by coupling the fractions to CNBr-activated Sepharose, followed by incubation with sera from 55 Dutch peanut-allergic children and I-labelled anti-IgE.

Results: Most IgE reactivity of the BPP fraction was due to the 5-7 kDa amino-terminal fragment of Ara h 1. This finding was confirmed by the use of the fragment in recombinant form, to which 25/55 of the sera was IgE-positive.

Conclusion: The amino-terminal fragment of Ara h 1, a member of a family of small anti-microbial proteins, is an allergen independent of the carboxy-terminal fragment of Ara h 1.
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http://dx.doi.org/10.1111/cea.13554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047623PMC
March 2020

The effect of certolizumab drug concentration and anti-drug antibodies on TNF neutralisation.

Clin Exp Rheumatol 2020 Mar-Apr;38(2):306-313. Epub 2019 Aug 30.

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

Objectives: Tumour necrosis factor (TNF) inhibitors like certolizumab, elicit an immunogenic response leading to the formation of anti-drug antibodies (ADAs). We sought to mechanistically investigate the relationship between certolizumab concentrations, ADAs, and the effective TNF neutralising capacity in sera of rheumatoid arthritis (RA) patients. TNF neutralising capacity of certolizumab was compared to the neutralising capacity of adalimumab.

Methods: Serum samples were collected from 40 consecutive certolizumab-treated RA patients at baseline and 4, 16, 28 and 52 weeks after treatment initiation [Dutch Trial Register NTR (Nederlands Trial Register) Trial NL2824 no. 2965]. Certolizumab concentration and ADA titre were measured with a certolizumab bridging enzyme-linked immunosorbent assay (ELISA) and a drug-tolerant radioimmunoassay (RIA), respectively. TNF neutralisation by certolizumab and adalimumab, in presence or absence of ADAs, was analysed with the TNF-sensitive WEHI bioassay.

Results: Despite a high incidence of ADAs during one year of follow-up (65%; 26/40 patients), certolizumab levels of >10 μg/ml were measured in most patients. The capacity for TNF neutralisation highly correlated with certolizumab serum concentration, whereas no association with ADAs was observed. Similar results were obtained for adalimumab. The relative in vitro neutralising potency was higher for certolizumab compared to adalimumab.

Conclusions: Anti-certolizumab antibodies were detected in a large proportion of patients, but in most cases where ADAs were detected, certolizumab was also present in high concentrations, directly correlating with in vitro neutralising capacity. These results indicate that measurement of certolizumab drug levels, rather than ADAs, have direct clinical significance.
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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

Biased -Glycosylation Site Distribution and Acquisition across the Antibody V Region during B Cell Maturation.

J Immunol 2019 04 8;202(8):2220-2228. Epub 2019 Mar 8.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, 1066 CX Amsterdam, the Netherlands.

Abs can acquire -linked glycans in their V regions during Ag-specific B cell responses. Among others, these -linked glycans can affect Ag binding and Ab stability. Elevated -linked glycosylation has furthermore been associated with several B cell-associated pathologies. Basic knowledge about patterns of V region glycosylation at different stages of B cell development is scarce. The aim of the current study is to establish patterns of -glycosylation sites in Ab V regions of naive and memory B cell subsets. We analyzed the distribution and acquisition of -glycosylation sites within Ab V regions of peripheral blood and bone marrow B cells of 12 healthy individuals, eight myasthenia gravis patients, and six systemic lupus erythematosus patients, obtained by next-generation sequencing. -glycosylation sites are clustered around CDRs and the DE loop for both H and L chains, with similar frequencies for healthy donors and patients. No evidence was found for an overall selection bias against acquiring an -glycosylation site, except for the CDR3 of the H chain. Interestingly, both IgE and IgG4 subsets have a 2-fold higher propensity to acquire Fab glycans compared with IgG1 or IgA. When expressed as rmAb, 35 out of 38 (92%) nongermline -glycosylation sites became occupied. These results point toward a differential selection pressure of -glycosylation site acquisition during affinity maturation of B cells, which depends on the location within the V region and is isotype and subclass dependent. Elevated Fab glycosylation represents an additional hallmark of T2-like IgG4/IgE responses.
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http://dx.doi.org/10.4049/jimmunol.1801622DOI Listing
April 2019

The enzymatic removal of immunoglobulin variable domain glycans by different glycosidases.

J Immunol Methods 2019 04 8;467:58-62. Epub 2019 Feb 8.

Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands; Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

About 15% of immunoglobulin G (IgG) molecules contain glycans linked to the antigen-binding fragments (Fab arms) in addition to the glycans linked to the crystallizable fragment (Fc tail) of all IgGs. Fab glycosylation appears to be an important feature of antibodies, for example by influencing antigen binding and antibody stability. The reliable generation of antibodies that either have or lack Fab glycans would be very helpful to study the role of Fab glycans in more detail. In this study, we set out to remove Fab glycans by treating polyclonal and monoclonal human IgG antibodies with two commonly used glycosidases and an improved version of one of the two (Endo F3, PNGase F, and Rapid™ PNGase F). Fc glycans can be removed using PNGase F and Rapid™ PNGase F, but not with Endo F3. For most antibody clones, Endo F3 partially cleaved off the Fab glycans. In contrast, PNGase F left the Fab glycans of most clones unaffected, but could remove glycans of some clones. Rapid™ PNGase F showed a higher glycosidase efficacy than PNGase F, and more clones could be deglycosylated using this enzyme. In summary, not all Fab glycans can be cleaved off by the tested glycosidases (under non-denaturing conditions), suggesting that Fab glycans are exposed to different degrees.
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http://dx.doi.org/10.1016/j.jim.2019.02.005DOI Listing
April 2019

Unique patterns of glycosylation in immunoglobulin subclass G4-related disease and primary sclerosing cholangitis.

J Gastroenterol Hepatol 2019 Oct 22;34(10):1878-1886. Epub 2018 Nov 22.

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

Background And Aim: Immunoglobulin subclass G4-related disease (IgG4-RD) is characterized by an abundance of IgG4 antibodies in the serum and tissue. Glycosylation status of antibodies can impact on immune effector functions and disease pathophysiology. We sought to establish glycosylation patterns in a prospective cohort of patients with IgG4-RD and the relationship with disease activity and response to treatment.

Methods: We assessed IgG Fc-tail and Fab-arm glycosylation status in patients with IgG4-RD (n = 22), disease controls with primary sclerosing cholangitis (PSC) (n = 22), and healthy controls (n = 22). Serum IgG and subclasses were quantified using ELISA. Fc and Fab glycosylation were analyzed by mass spectrometry and lectin affinity chromatography, respectively. Disease activity, organ damage, and response to treatment were assessed using the IgG4 Responder Index.

Results: Immunoglobulin G Fab sialylation was increased in IgG4-RD compared with PSC and healthy control (P = 0.01), with a preferential increase in IgG4-specific Fab sialylation, which was independent of IgG4 Fab-arm exchange. There was a reduction in IgG1-specific Fc bisection and hybrid structures in IgG4-RD (P < 0.01), which recovered upon steroid treatment and correlated with disease activity. Overall, IgG Fc galactosylation was reduced in both IgG4-RD and PSC (P < 0.01), with a preferential reduction in IgG1-specific sialylation and enhancement of IgG4-specific bisection in PSC. IgG4 fucosylation and IgG1/2/3 hybrid structures negatively correlated with complement C3 and C4 levels in IgG4-RD (P < 0.01), but not PSC.

Conclusion: We report the first study showing unique antibody glycosylation status in a prospective cohort of IgG4-RD and PSC patients, which may determine modulation of the immune system and contribute to disease pathophysiology.
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http://dx.doi.org/10.1111/jgh.14512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899843PMC
October 2019

Restricted immune activation and internalisation of anti-idiotype complexes between drug and antidrug antibodies.

Ann Rheum Dis 2018 10 26;77(10):1471-1479. Epub 2018 Jun 26.

Sanquin Research and Landsteiner Laboratory, Department of Immunopathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Objectives: Therapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these 'anti-idiotype' complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies.

Methods: Size distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA.

Results: Size and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation.No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions.

Conclusions: Anti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events.
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http://dx.doi.org/10.1136/annrheumdis-2018-213299DOI Listing
October 2018

Variable Domain -Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability.

Front Immunol 2018 12;9:740. Epub 2018 Apr 12.

Sanquin Research, Department of Immunopathology, Amsterdam, Netherlands.

Immunoglobulin G (IgG) can contain -linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the C2 domains. These Fab glycans are acquired following introduction of -glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may-in addition to affecting antigen binding-contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting selection of stable, glycosylated antibodies. Collectively, our data show that variable domain -linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.
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http://dx.doi.org/10.3389/fimmu.2018.00740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906590PMC
June 2019

Dimeric IgG complexes from IVIg are incapable of inducing in vitro neutrophil degranulation or complement activation.

PLoS One 2018 10;13(4):e0195729. Epub 2018 Apr 10.

Department of Immunopathology, Sanquin Blood Supply and Landsteiner laboratory Amsterdam Medical Centre, University of Amsterdam, CX, Amsterdam, the Netherlands.

Purpose: Intravenous immunoglobulin (IVIg) products contain various amounts of dimeric IgG complexes. Current insights into the possible biological activities of these dimers remain controversial, and both immunemodulating and immune-activating effects have been reported. Here, we analyzed the putative immune-activating effects of dimers isolated from IVIg.

Methods: Dimers isolated from IVIg were purified by high-performance size-exclusion chromatography (HP-SEC) and tested for the ability to induce neutrophil degranulation in vitro.

Results: Dimers isolated from IVIg were found to be incapable of inducing in vitro neutrophil degranulation or complement activation, even at concentrations exceeding those expected to be reached upon administration in patients. These results depend on the removal of artefactual activation by using 0.1 micron filtration and the use of poloxamer to prevent adsorption of IgG onto the solid phase.

Conclusions: The data suggest dimeric IgG found in IVIg may bind to Fc-receptors without causing activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195729PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892932PMC
July 2018

Adaptive antibody diversification through -linked glycosylation of the immunoglobulin variable region.

Proc Natl Acad Sci U S A 2018 02 5;115(8):1901-1906. Epub 2018 Feb 5.

Sanquin Research, Department of Immunopathology, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, 1066 CX, Amsterdam, The Netherlands.

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire -linked glycans, a process conditional on the introduction of consensus amino acid motifs (-glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that -glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.
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http://dx.doi.org/10.1073/pnas.1711720115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828577PMC
February 2018

Antibodies to IgG4 hinge can be found in rheumatoid arthritis patients during all stages of disease and may exacerbate chronic antibody-mediated inflammation.

Arthritis Rheumatol 2014 May;66(5):1133-40

Sanquin Research, Academic Medical Centre, University of Amsterdam, and Jan van Breemen Research Institute | Reade, Amsterdam, The Netherlands.

Objective: In rheumatoid arthritis (RA), autoantibodies such as anti-citrullinated protein antibodies (ACPAs) develop in response to neoepitopes that are formed under conditions of chronic inflammation. These autoantibodies may subsequently be fragmented by inflammation-associated proteases, leading to the formation of F(ab')2 fragments. The hinge of F(ab')2 fragments can serve as a neoepitope, and so-called antihinge antibodies (AHAs) can be found in RA patients, which might modulate the function of (fragmented) autoantibodies. We undertook this study to investigate the presence and specificities of AHAs in different stages of RA and to study their function.

Methods: The presence of AHAs was assessed by radioimmunoassay in healthy controls, blood donors who later developed RA, patients with arthralgia, patients with early RA, and patients with established RA. Specificity of the AHAs was analyzed with inhibition assays, and complement-activating ability was studied with a C4b deposition assay.

Results: Antibodies to IgG1 hinge, IgG2 hinge, and IgG4 hinge were detected in patients with established RA, with anti-IgG4 hinge antibodies being most specific (appearing in 1% of healthy controls, 3.8% of blood donors who later developed RA, 13% of arthralgia patients, 19% of early RA patients, and 16% of established RA patients). Anti-IgG4 hinge antibodies were subclass specific and were able to restore C4b deposition by IgG4 F(ab')2 fragments. In patients with arthralgia and patients with early RA, anti-IgG4 hinge antibodies were associated with rheumatoid factors and ACPAs.

Conclusion: Anti-IgG4 hinge antibodies are present in RA patients and have low sensitivity but high specificity for RA. Since a significant proportion of ACPAs can be of the IgG4 subclass, the formation of anti-IgG4 hinge antibodies may represent one mechanism of ACPA-mediated inflammation.
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http://dx.doi.org/10.1002/art.38335DOI Listing
May 2014

Preventing adsorption of immunoglobulin G to solid surfaces using poloxamer 407 eliminates artifactual stimulation of neutrophils.

J Immunol Methods 2013 Jun 29;392(1-2):49-56. Epub 2013 Mar 29.

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

To study the effect of polyclonal intravenous immunoglobulin G (IVIG) on neutrophils in vitro, adsorption of immunoglobulin G (IgG) to solid surfaces has to be prevented, because IgG bound to a solid surface can activate neutrophils through activating FcγRs. In this study we demonstrate that poloxamer 407, a non ionic surfactant, at low concentration (0.05%) prevented the adsorption of high concentrations of IgG (5 mg/ml) better than other blocking agents without interfering with the interaction of IgG with the neutrophils. Poloxamer 407 is therefore a suitable blocking agent to prevent the interaction of immunoglobulin with solid surfaces in cell-based in vitro experiments.
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http://dx.doi.org/10.1016/j.jim.2013.03.009DOI Listing
June 2013

Nanomolar to sub-picomolar affinity measurements of antibody-antigen interactions and protein multimerizations: fluorescence-assisted high-performance liquid chromatography.

Anal Biochem 2013 Jun 13;437(2):118-22. Epub 2013 Mar 13.

Sanquin Research, 1066 CX Amsterdam, The Netherlands.

Although several techniques exist for the measurement of high-affinity interactions, it is still challenging to determine dissociation constants around or even below 1pM. During the analysis of several human-derived monoclonal antibodies to adalimumab, we found a clone with a very high affinity that could not be measured using conventional surface plasmon resonance assays. We developed a straightforward and robust method to measure affinities in the nanomolar to sub-picomolar range. The assay is based on separation of bound and free fluorescently labeled antigen using size exclusion chromatography and quantification by in-line fluorescence detection. We describe optimal conditions and procedures that result in a very sensitive assay that can be used to reliably determine ultra-high affinities. Using the method described in this article, a dissociation constant of 0.78pM could be determined for the anti-adalimumab antibody.
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http://dx.doi.org/10.1016/j.ab.2013.02.027DOI Listing
June 2013

IgE production to α-gal is accompanied by elevated levels of specific IgG1 antibodies and low amounts of IgE to blood group B.

PLoS One 2013 4;8(2):e55566. Epub 2013 Feb 4.

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

IgE antibodies to gal-α-1,3-gal-β-1,4-GlcNAc (α-gal) can mediate a novel form of delayed anaphylaxis to red meat. Although IgG antibodies to α-gal (anti-α-gal or anti-Gal) are widely expressed in humans, IgE anti-α-gal is not. We explored the relationship between the IgG and IgE responses to both α-gal and the related blood group B antigen. Contradicting previous reports, antibodies to α-gal were found to be significantly less abundant in individuals with blood group B or AB. Importantly, we established a connection between IgE and IgG responses to α-gal: elevated titers of IgG anti-α-gal were found in IgE-positive subjects. In particular, proportionally more IgG1 anti-α-gal was found in IgE-positive subjects against a background of IgG2 production specific for α-gal. Thus, two types of immune response to α-gal epitopes can be distinguished: a 'typical' IgG2 response, presumably in response to gut bacteria, and an 'atypical', Th2-like response leading to IgG1 and IgE in addition to IgG2. These results suggest that IgE to a carbohydrate antigen can be formed (probably as part of a glycoprotein or glycolipid) even against a background of bacterial immune stimulation with essentially the same antigen.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055566PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563531PMC
July 2013

Enrichment of sialylated IgG by lectin fractionation does not enhance the efficacy of immunoglobulin G in a murine model of immune thrombocytopenia.

PLoS One 2011 23;6(6):e21246. Epub 2011 Jun 23.

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

Intravenous immunoglobulin G (IVIg) is widely used against a range of clinical symptoms. For its use in immune modulating therapies such as treatment of immune thrombocytopenic purpura high doses of IVIg are required. It has been suggested that only a fraction of IVIg causes this anti immune modulating effect. Recent studies indicated that this fraction is the Fc-sialylated IgG fraction. The aim of our study was to determine the efficacy of IVIg enriched for sialylated IgG (IVIg-SA⁺) in a murine model of passive immune thrombocytopenia (PIT). We enriched IVIg for sialylated IgG by Sambucus nigra agglutinin (SNA) lectin fractionation and determined the degree of sialylation. Analysis of IVIg-SA⁺ using a lectin-based ELISA revealed that we enriched predominantly for Fab-sialylated IgG, whereas we did not find an increase in Fc-sialylated IgG. Mass spectrometric analysis confirmed that Fc sialylation did not change after SNA lectin fractionation. The efficacy of sialylated IgG was measured by administering IVIg or IVIg-SA⁺ 24 hours prior to an injection of a rat anti-mouse platelet mAb. We found an 85% decrease in platelet count after injection of an anti-platelet mAb, which was reduced to a 70% decrease by injecting IVIg (p<0.01). In contrast, IVIg-SA⁺ had no effect on the platelet count. Serum levels of IVIg and IVIg-SA⁺ were similar, ruling out enhanced IgG clearance as a possible explanation. Our results indicate that SNA lectin fractionation is not a suitable method to enrich IVIg for Fc-sialylated IgG. The use of IVIg enriched for Fab-sialylated IgG abolishes the efficacy of IVIg in the murine PIT model.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021246PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3121734PMC
November 2011

Detection of conformational changes in immunoglobulin G using isothermal titration calorimetry with low-molecular-weight probes.

Anal Biochem 2008 Sep 5;380(2):303-9. Epub 2008 Jun 5.

Sanquin Research, 1066 CX Amsterdam, The Netherlands.

Proteins for therapeutic use may contain small amounts of partially misfolded monomeric precursors to postproduction aggregation. To detect these misfolded proteins in the presence of an excess of properly folded protein, fluorescent probes such as 8-anilino-1-naphthalene sulfonate (ANS) are commonly used. We investigated the possibility of using isothermal titration calorimetry (ITC) to improve the detection of this type of conformational change using hydrophobic probes. As a case study, conformational changes in human polyclonal immunoglobulin G (IgG) were monitored by measuring the enthalpies of binding of ANS using ITC. Results were compared with those using fluorescence spectroscopy. IgG heated at 63 degrees C was used as a model system for "damaged" IgG. Heat-treated IgG can be detected already at levels below 5% with both ITC and fluorescence. However, ITC allows a much wider molar probe-to-protein ratio to be sampled. In particular, using reverse titration experiments (allowing high probe-to-protein ratios not available to fluorescence spectroscopy), an increase in the number of binding sites with a K(d)>10 mM was observed for heat-treated IgG, reflecting subtle changes in structure. Both ITC and fluorescence spectroscopy showed low background signals for native IgG. The nature of the background signals was not clear from the fluorescence measurements. However, further analysis of the ITC background signals shows that a fraction (8%) binds ANS with a dissociation constant of approximately 0.2 mM. Measurements were also carried out at pH 4.5. Precipitation of IgG was induced by ANS at concentrations above 0.5 mM, interfering with the ITC measurements. Instead, with the nonfluorescent probes 4-amino-1-naphthalene sulfonate and 1-naphthalene sulfonate, no precipitation is observed. These probes yield differences in the enthalpies of binding to heated and nonheated IgG similar to ANS. The data illustrate that ITC with low-molecular-weight probes is a versatile tool to monitor conformational changes in proteins with a wider application potential than fluorescence measurements.
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http://dx.doi.org/10.1016/j.ab.2008.06.001DOI Listing
September 2008

Maturation of Pichia pastoris-derived recombinant pro-Der p 1 induced by deglycosylation and by the natural cysteine protease Der p 1 from house dust mite.

Eur J Biochem 2002 Jan;269(2):671-9

CLB Department of Immunopathology and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, the Netherlands.

The mature cysteine protease from Dermatophgoides pteronyssinus, Der p 1, is a major house dust mite allergen. Its enzymatic activity has been shown to have pro-inflammatory effects that could also negatively influence efficacy of allergen-specific immunotherapy. The aim of this study was to express recombinant pro-Der p 1 (rpro-Der p 1) in the yeast Pichia pastoris and to study its maturation. Expression was achieved at a concentration ranging from 45 mg.L-1 (methanol-induced expression) to 168 mg.L-1 (constitutive expression). No significant spontaneous maturation of the secreted proenzyme was observed. rpro-Der p 1 with a sequence-based molecular mass of 34 kDa was hyperglycosylated by the yeast, migrating at 50-60 kDa on SDS/PAGE. Compared with its natural counterpart (nDer p 1), the recombinant proenzyme demonstrated decreased IgE reactivity, resulting in a 30-fold lower capacity to induce histamine release from human basophils. Decreased immunoreactivity was also shown by competitive RIA and sandwich ELISA with Der p 1-specific antibody reagents. CD spectra of rpro-Der p 1 and nDer p 1 revealed significant structural differences. Deglycosylation of rpro-Der p 1 with endoglycosidase H resulted in a decrease in apparent molecular mass from 50 kDa to 34 kDa, but did not affect nDer p 1. On removal of N-glycans from rpro-Der p 1, which harbours two putative N-glycosylation sites in both propeptide and mature sequence, the mature rDer p 1 appeared. This suggests that hyperglycosylation hampers spontaneous maturation. Maturation of the recombinant pro-enzyme was also achieved by addition of the active natural cysteine protease, nDer p 1. In conclusion, high-level expression of rpro-Der p 1 in P. pastoris results in a stable hypoallergenic proenzyme with potential for use in allergen-specific immunotherapy.
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http://dx.doi.org/10.1046/j.0014-2956.2001.02700.xDOI Listing
January 2002
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