Publications by authors named "Arthur E H Bentlage"

26 Publications

  • Page 1 of 1

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

Front Immunol 2021 15;12:594773. Epub 2021 Mar 15.

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

Antibody-mediated blood disorders ensue after auto- or alloimmunization against blood cell antigens, resulting in cytopenia. Although the mechanisms of cell destruction are the same as in immunotherapies targeting tumor cells, many factors are still unknown. Antibody titers, for example, often do not strictly correlate with clinical outcome. Previously, we found C-reactive protein (CRP) levels to be elevated in thrombocytopenic patients, correlating with thrombocyte counts, and bleeding severity. Functionally, CRP amplified antibody-mediated phagocytosis of thrombocytes by phagocytes. To investigate whether CRP is a general enhancer of IgG-mediated target cell destruction, we extensively studied the effect of CRP on IgG-Fc receptor (FcγR)-mediated cell destruction: through respiratory burst, phagocytosis, and cellular cytotoxicity by a variety of effector cells. We now demonstrate that CRP also enhances IgG-mediated effector functions toward opsonized erythrocytes, in particular by activated neutrophils. We performed a first-of-a-kind profiling of CRP binding to all human FcγRs and IgA-Fc receptor I (FcαRI) using a surface plasmon resonance array. CRP bound these receptors with relative affinities of FcγRIa = FcγRIIa/b = FcγRIIIa > FcγRIIIb = FcαRI. Furthermore, FcγR blocking (in particular FcγRIa) abrogated CRP's ability to amplify IgG-mediated neutrophil effector functions toward opsonized erythrocytes. Finally, we observed that CRP also amplified killing of breast-cancer tumor cell line SKBR3 by neutrophils through anti-Her2 (trastuzumab). Altogether, we provide for the first time evidence for the involvement of specific CRP-FcγR interactions in the exacerbation of IgG-mediated cellular destruction; a trait that should be further evaluated as potential therapeutic target e.g., for tumor eradication.
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http://dx.doi.org/10.3389/fimmu.2021.594773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006934PMC
March 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919849PMC
February 2021

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

J Immunol 2020 12 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

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

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

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

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
March 2021

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

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

Reduced FcRn-mediated transcytosis of IgG2 due to a missing Glycine in its lower hinge.

Sci Rep 2019 05 14;9(1):7363. Epub 2019 May 14.

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

Neonatal Fc-receptor (FcRn), the major histocompatibility complex (MHC) class I-like Fc-receptor, transports immunoglobuline G (IgG) across cell layers, extending IgG half-life in circulation and providing newborns with humoral immunity. IgG1 and IgG2 have similar half-lives, yet IgG2 displays lower foetal than maternal concentration at term, despite all known FcRn binding residues being preserved between IgG1 and IgG2. We investigated FcRn mediated transcytosis of V-matched IgG1 and IgG2 and mutated variants thereof lacking Fc-gamma receptor (FcγR) binding in human cells expressing FcRn. We observed that FcγR binding was not required for transport and that FcRn transported less IgG2 than IgG1. Transport of IgG1 with a shortened lower hinge (ΔGly236, absent in germline IgG2), was reduced to levels equivalent to IgG2. Conversely, transport of IgG2 + Gly236 was increased to IgG1 levels. Gly236 is not a contact residue between IgG and FcRn, suggesting that its absence leads to an altered conformation of IgG, possibly due to a less flexible Fab, positioned closer to the Fc portion. This may sterically hinder FcRn binding and transport. We conclude that the lack of Gly236 is sufficient to explain the reduced FcRn-mediated IgG2 transcytosis and accounts for the low maternal/fetal IgG2 ratio at term.
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http://dx.doi.org/10.1038/s41598-019-40731-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6517591PMC
May 2019

Multiplex blood group typing by cellular surface plasmon resonance imaging.

Transfusion 2019 02 29;59(2):754-761. Epub 2018 Nov 29.

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

Background: Blood-group typing of donors and patients is essential to avoid incompatible transfusions. Transfusion of incompatible RBCs may result in alloimmunization complicating future transfusions or in the presence of antibodies in adverse reactions. With more than 300 blood group antigens identified, it is difficult to provide fully compatible blood. Currently, standard practice is to match for the most immunogenic antigens. While the current agglutination-based RBC-typing methods are reliable for testing a selected number of antigens, they are not easily adaptable for high-throughput multiplex blood typing beyond the current standard.

Study Design And Methods: Surface plasmon resonance (SPR) is a label-free method to follow molecular-and, very recently, also cellular-interactions in real time. Demonstration of binding of RBCs to blood group antigen-specific antibodies by SPR has already been achieved. Here, we demonstrate the generation of an SPR array equipped with clinically relevant blood group antibodies (A, B, and Rh blood groups). To validate this method, we blindly compared typing of 946 blood donors with results of current diagnostic agglutination-based methods.

Results: RBC typing was achieved by monitoring RBC binding to blood group-specific antibodies on the sensor simultaneously within 5 minutes per sample. Regeneration of the chip was robust, allowing for typing of at least 100 samples. The typing results gave a 100% match with classical serology with all antibodies tested besides anti-E/e monoclonals, which gave inconsistent results due to low antibody specificity.

Conclusion: This study demonstrates that SPR-based RBC typing for multiple antigens can be realized simultaneously with high-quality antibodies, enabling reduced hands-on time and possibly improving cost efficiency.
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http://dx.doi.org/10.1111/trf.15071DOI Listing
February 2019

FcγR interaction is not required for effective anti-PD-L1 immunotherapy but can add additional benefit depending on the tumor model.

Int J Cancer 2019 01 12;144(2):345-354. Epub 2018 Nov 12.

Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands.

Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models.
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http://dx.doi.org/10.1002/ijc.31899DOI Listing
January 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

Rheumatoid factors do not preferentially bind to ACPA-IgG or IgG with altered galactosylation.

Rheumatology (Oxford) 2018 04;57(4):771

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

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http://dx.doi.org/10.1093/rheumatology/key056DOI Listing
April 2018

Conserved FcγR- glycan discriminates between fucosylated and afucosylated IgG in humans and mice.

Mol Immunol 2018 02 19;94:54-60. Epub 2017 Dec 19.

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

The binding strength between IgG and FcγR is influenced by the composition of the N-linked glycan at position N297 in the Fc-domain of IgG. Particularly, afucosylation increases the binding affinity of human IgG1 to human FcγRIIIa up to ∼20 fold, and additional galactosylation of the afucosylated IgG increases the affinity up to ∼40 fold. The increase in affinity for afucosylated IgG has previously been shown to depend on direct carbohydrate-carbohydrate interactions between the IgG-Fc glycan with an N-linked glycan at position 162 unique to hFcγRIIIa and hFcγRIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse FcγR, mFcγRIV. The N162-glycan in mFcγRIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFcγRIIIa. However, unlike hFcγRIIIa, mFcγRIV did not bind more avidly to IgG with increased galactose and reduced fucose. Overall, these results suggest the N162-glycan in the human FcγRIII family and its orthologous mouse FcγRIV to be functionally conserved.
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http://dx.doi.org/10.1016/j.molimm.2017.12.006DOI Listing
February 2018

Rheumatoid factors do not preferentially bind to ACPA-IgG or IgG with altered galactosylation.

Rheumatology (Oxford) 2017 11;56(11):2025-2030

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

Objectives: Recent reports describe interactions between the two most prominent RA-related autoantibodies, RFs and ACPAs. The main aim of the present study was to investigate whether RFs preferentially interact with ACPA-IgG over non-ACPA IgG. Additionally, interactions of RFs with IgG with altered galactose content in the Fc domain were examined, since ACPA-IgGs have been shown to have decreased Fc galactose content in RF+ patients.

Methods: (Auto)antibody interactions were studied in a surface plasmon resonance imaging assay and with ELISA. Target antibodies were isolated from RA patient plasma (polyclonal ACPA- and non-ACPA-IgG) or recombinantly produced to obtain monoclonal IgG with well-defined Fc galactose content. Interacting autoantibodies were studied using autoantibody positive patient sera and two recombinantly produced IgM-RFs.

Results: The sera from 41 RF+ RA patients showed similar RF binding to ACPA- and non-ACPA-IgG and no differences in binding to IgG with normal, high or low levels of Fc galactosylation. Two monoclonal IgM-RFs, one interacting with the CH2-CH3 interface and one binding close to the C-terminal end of the CH3 domain showed no influence of the Fc glycan on IgG binding by IgM-RF.

Conclusion: Although interactions between RF and ACPA may play a role in inflammatory processes in RA, RFs do not preferentially interact with ACPA-IgG over non-ACPA-IgG nor with agalatosylated IgG over IgG with normal or high galactosylation.
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http://dx.doi.org/10.1093/rheumatology/kex284DOI Listing
November 2017

Decoding the Human Immunoglobulin G-Glycan Repertoire Reveals a Spectrum of Fc-Receptor- and Complement-Mediated-Effector Activities.

Front Immunol 2017 2;8:877. Epub 2017 Aug 2.

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

Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.
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http://dx.doi.org/10.3389/fimmu.2017.00877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5539844PMC
August 2017

DARC extracellular domain remodeling in maturating reticulocytes explains tropism.

Blood 2017 09 28;130(12):1441-1444. Epub 2017 Jul 28.

Department Hematopoiesis, Sanquin Research, Amsterdam, The Netherlands.

is the most prevalent parasite species that causes malaria in humans and exclusively infects reticulocytes. Reticulocyte infection is facilitated by Duffy binding protein (DBP), which utilizes DARC (Duffy antigen receptor for chemokines) as an entry point. However, the selective tropism of for transferrin receptor (CD71)-positive reticulocytes remained unexplained, given the constitutive expression of DARC during reticulocyte maturation. CD71/RNA double staining of reticulocytes enriched from adult peripheral blood reveals 4 distinct reticulocyte populations: CD71/RNA (∼0.016%), CD71/RNA (∼0.059%), CD71/RNA (∼0.37%), CD71/RNA (∼0.55%), and erythrocytes CD71/RNA (∼99%). We hypothesized that selective association of DBP with a small population of immature reticulocytes could explain the preference of for reticulocytes. Binding of specific monoclonal anti-DARC antibodies and recombinant DBP to CD71/RNA reticulocytes was significantly higher compared with other reticulocyte populations and erythrocytes. Interestingly, the total DARC protein throughout reticulocyte maturation was constant. The data suggest that selective exposure of the DBP binding site within DARC is key to the preferential binding of DBP to immature reticulocytes, which is the potential mechanism underlying the preferential infection of a reticulocyte subset by .
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http://dx.doi.org/10.1182/blood-2017-03-774364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609335PMC
September 2017

Identification of sequence variants influencing immunoglobulin levels.

Nat Genet 2017 Aug 19;49(8):1182-1191. Epub 2017 Jun 19.

deCODE Genetics/Amgen, Inc., Reykjavik, Iceland.

Immunoglobulins are the effector molecules of the adaptive humoral immune system. In a genome-wide association study of 19,219 individuals, we found 38 new variants and replicated 5 known variants associating with IgA, IgG or IgM levels or with composite immunoglobulin traits, accounted for by 32 loci. Variants at these loci also affect the risk of autoimmune diseases and blood malignancies and influence blood cell development. Notable associations include a rare variant at RUNX3 decreasing IgA levels by shifting isoform proportions (rs188468174[C>T]: P = 8.3 × 10, β = -0.90 s.d.), a rare in-frame deletion in FCGR2B abolishing IgG binding to the encoded receptor (p.Asn106del: P = 4.2 × 10, β = 1.03 s.d.), four IGH locus variants influencing class switching, and ten new associations with the HLA region. Our results provide new insight into the regulation of humoral immunity.
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http://dx.doi.org/10.1038/ng.3897DOI Listing
August 2017

Enhanced Effector Functions Due to Antibody Defucosylation Depend on the Effector Cell Fcγ Receptor Profile.

J Immunol 2017 07 31;199(1):204-211. Epub 2017 May 31.

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

Abs of the IgG isotype are glycosylated in their Fc domain at a conserved asparagine at position 297. Removal of the core fucose of this glycan greatly increases the affinity for FcγRIII, resulting in enhanced FcγRIII-mediated effector functions. Normal plasma IgG contains ∼94% fucosylated Abs, but alloantibodies against, for example, Rhesus D (RhD) and platelet Ags frequently have reduced fucosylation that enhances their pathogenicity. The increased FcγRIII-mediated effector functions have been put to use in various afucosylated therapeutic Abs in anticancer treatment. To test the functional consequences of Ab fucosylation, we produced V-gene-matched recombinant anti-RhD IgG Abs of the four different subclasses (IgG1-4) with and without core fucose (i.e., 20% fucose remaining). Binding to all human FcγR types and their functional isoforms was assessed with surface plasmon resonance. All hypofucosylated anti-RhD IgGs of all IgG subclasses indeed showed enhanced binding affinity for isolated FcγRIII isoforms, without affecting binding affinity to other FcγRs. In contrast, when testing hypofucosylated anti-RhD Abs with FcγRIIIa-expressing NK cells, a 12- and 7-fold increased erythrocyte lysis was observed with the IgG1 and IgG3, respectively, but no increase with IgG2 and IgG4 anti-RhD Abs. Notably, none of the hypofucosylated IgGs enhanced effector function of macrophages, which, in contrast to NK cells, express a complex set of FcγRs, including FcγRIIIa. Our data suggest that the beneficial effects of afucosylated biologicals for clinical use can be particularly anticipated when there is a substantial involvement of FcγRIIIa-expressing cells, such as NK cells.
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http://dx.doi.org/10.4049/jimmunol.1700116DOI Listing
July 2017

Affinity of human IgG subclasses to mouse Fc gamma receptors.

MAbs 2017 07 2;9(5):767-773. Epub 2017 May 2.

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

Human IgG is the main antibody class used in antibody therapies because of its efficacy and longer half-life, which are completely or partly due to FcγR-mediated functions of the molecules. Preclinical testing in mouse models are frequently performed using human IgG, but no detailed information on binding of human IgG to mouse FcγRs is available. The orthologous mouse and human FcγRs share roughly 60-70% identity, suggesting some incompatibility. Here, we report binding affinities of all mouse and human IgG subclasses to mouse FcγR. Human IgGs bound to mouse FcγR with remarkably similar binding strengths as we know from binding to human ortholog receptors, with relative affinities IgG3>IgG1>IgG4>IgG2 and FcγRI>FcγRIV>FcγRIII>FcγRIIb. This suggests human IgG subclasses to have similar relative FcγR-mediated biological activities in mice.
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http://dx.doi.org/10.1080/19420862.2017.1323159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524164PMC
July 2017

Anti-Hinge Antibodies Recognize IgG Subclass- and Protease-Restricted Neoepitopes.

J Immunol 2017 01 18;198(1):82-93. Epub 2016 Nov 18.

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

Anti-hinge Abs (AHAs) target neoepitopes exposed after proteolytic cleavage of IgG. In this study, we explored the diversity of protease- and IgG subclass-restricted AHAs and their potential as immunological markers in healthy donors (HDs) and patients with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). AHA reactivity against IgG-degrading enzyme of Streptococcus pyogenes (IdeS)- or pepsin-generated F(ab') fragments of all four human IgG subclasses was determined. AHA reactivity against one or more out of eight F(ab') targets was found in 68% (68 of 100) of HDs, 69% (68 of 99) of SLE patients, and 81% (79 of 97) of RA patients. Specific recognition of hinge epitopes was dependent on IgG subclass and protease used to create the F(ab') targets, as confirmed by inhibition experiments with F(ab') fragments and hinge peptides. Reactivity against IdeS-generated F(ab') targets was found most frequently, whereas reactivity against pepsin-generated F(ab') targets better discriminated between RA and HDs or SLE, with significantly higher AHA levels against IgG1/3/4. In contrast, AHA levels against pepsin-cleaved IgG2 were comparable. No reactivity against IdeS-generated IgG2-F(ab')s was detected. The most discriminatory AHA reactivity in RA was against pepsin-cleaved IgG4, with a 35% prevalence, ≥5.8-fold higher than in HDs/SLE, and significantly higher levels (p < 0.0001). Cross-reactivity for F(ab')s generated from different IgG subclasses was only observed for subclasses having homologous F(ab') C termini (IgG1/3/4). For IgG2, two pepsin cleavage sites were identified; anti-hinge reactivity was restricted to only one of these. In conclusion, AHAs specifically recognize IgG subclass- and protease-restricted hinge neoepitopes. Their protease-restricted specificity suggests that different AHA responses developed under distinct inflammatory or infectious conditions and may be markers of, and participants in, such processes.
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http://dx.doi.org/10.4049/jimmunol.1601096DOI Listing
January 2017

C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia.

Blood 2015 Mar 29;125(11):1793-802. Epub 2014 Dec 29.

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

Immune-mediated platelet destruction is most frequently caused by allo- or autoantibodies via Fcγ receptor-dependent phagocytosis. Disease severity can be predicted neither by antibody isotype nor by titer, indicating that other factors play a role. Here we show that the acute phase protein C-reactive protein (CRP), a ligand for Fc receptors on phagocytes, enhances antibody-mediated platelet destruction by human phagocytes in vitro and in vivo in mice. Without antiplatelet antibodies, CRP was found to be inert toward platelets, but it bound to phosphorylcholine exposed after oxidation triggered by antiplatelet antibodies, thereby enhancing platelet phagocytosis. CRP levels were significantly elevated in patients with allo- and autoantibody-mediated thrombocytopenias compared with healthy controls. Within a week, intravenous immunoglobulin treatment in children with newly diagnosed immune thrombocytopenia led to significant decrease of CRP levels, increased platelet numbers, and clinically decreased bleeding severity. Furthermore, the higher the level of CRP at diagnosis, the longer it took before stable platelet counts were reached. These data suggest that CRP amplifies antibody-mediated platelet destruction and may in part explain the aggravation of thrombocytopenia on infections. Hence, targeting CRP could offer new therapeutic opportunities for these patients.
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http://dx.doi.org/10.1182/blood-2014-05-579110DOI Listing
March 2015

Label-free cell profiling.

Anal Biochem 2013 Aug 11;439(1):4-6. Epub 2013 Apr 11.

Medical Cell Biophysics Group, MIRA Institute, University of Twente, Enschede, The Netherlands.

A surface plasmon resonance (SPR) array imaging method is outlined for label-free cell profiling. Red blood cells (RBCs) were injected into a flow chamber on top of a spotted sensor surface. Spots contained antibodies to various RBC membrane antigens. A typical sensorgram showed an initial response corresponding to cell sedimentation (S) followed by a specific upward response (T) corresponding to specific binding of cells during a critical wash step. The full analysis cycle for RBC profiling was less than 6 min. The sensor surface could be regenerated at least 100 times, allowing the determination of a cell surface antigen profile of RBCs.
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http://dx.doi.org/10.1016/j.ab.2013.04.001DOI Listing
August 2013