Remco Visser

A prominent lack of IgG1 Fc-fucosylation of platelet-alloantibodies in pregnancy

Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.

Low anti‐RhD IgG‐Fc‐fucosylation in pregnancy: a new variable predicting severity in haemolytic disease of the fetus and newborn

Haemolytic disease of the fetus and newborn (HDFN) may occur when maternal IgG antibodies against red blood cells (RBCs), often anti‐RhD (anti‐D) antibodies, cross the placenta and mediate the destruction of RBCs via phagocytic IgG‐Fc‐receptors (FcγR). Clinical severity is not strictly related to titre and is more accurately predicted by the diagnostically‐applied monocyte‐based antibody‐dependent cellular cytotoxicity (ADCC), a sensitive test with relatively low specificity. This suggests that other factors are involved in the pathogenesis of HDFN. Binding of IgG to FcγR requires the N‐linked glycan at position 297 in the IgG‐Fc‐region, consisting of several different glycoforms. We therefore systematically analysed IgG‐derived glycopeptides by mass spectrometry from 70 anti‐D IgG1 antibodies purified from the plasma of alloimmunized pregnant women. This revealed a variable decrease in Fc‐fucosylation in the majority of anti‐D IgG1 (even down to 12%), whereas the total IgG of these patients remained highly fucosylated, like in healthy individuals (>90%). The degree of anti‐D fucosylation correlated significantly with CD16 (FcγRIIIa)‐mediated ADCC, in agreement with increased affinity of defucosylated IgG to human FcγRIIIa. Additionally, low anti‐D fucosylation correlated significantly with low fetal‐neonatal haemoglobin levels, thus with increased haemolysis, suggesting IgG‐fucosylation to be an important pathological feature in HDFN with diagnostic potential.

Surface catalyzed formation of polychlorinated dibenzo-p-dioxins/dibenzofurans during municipal waste incineration

A series of reactions was carried out on different surfaces using carbon and CuCl2, and KCl or HCl as a Cl-source. The PCDD/PCDF congener distribution was seen to vary to a great extent but the isomer distribution remained fairly constant. It is concluded that thermodynamic properties are largely responsible for the PCDD/PCDF isomer distribution. With pentachlorobenzene Cl2 elimination, assisted by oxygen, appears to be the major pathway for PCDD/PCDF formation.

H435-containing immunoglobulin G3 allotypes are transported efficiently across the human placenta: implications for alloantibody-mediated diseases of the newborn

The neonatal receptor (FcRn) extends the half‐life of human immunoglobulin (Ig)G and transports it across the placenta, providing the newborn with humoral immunity. Of the four subclasses, IgG3 stands out with strong effector functions, short half‐life (7u2009days vs. 21 days for other subclasses), and poor placental transport. We recently described how a single‐amino‐acid polymorphism at Position 435 in IgG3 is sufficient to explain the short half‐life of R435‐containing IgG3 and demonstrated that H435‐IgG3 has a normal half‐life of 21 days. Here, we investigated whether the R435 also explains the relatively poor placental transport of IgG3.

Hinge-Region O-Glycosylation of Human Immunoglobulin G3 (IgG3)

Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out. For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes.

Monophosphoryl lipid A plus IFNγ maturation of dendritic cells induces antigen-specific CD8+ cytotoxic T cells with high cytolytic potential

Dendritic cells (DCs) are promising antigen presenting cells for cancer treatment. Previously, we showed that the combination of monophosphoryl lipid A (MPLA) with IFNγ generates mature DCs that produce IL-12 and polarize CD4+ T cells towards a Th1 phenotype. Here, we extended these observations by showing that the DCs generated with the clinical grade maturation cocktail of MPLA/IFNγ induce superior tumour antigen-specific CD8+ CTL responses compared to the cytokine cocktail matured DCs that are currently used in the clinic. MPLA/IFNγ DCs can induce CTL responses in healthy individuals as well as in melanoma patients. The CTL induction was mainly dependent on the IL-12 produced by the MPLA/IFNγ DCs. The high amounts of induced CTLs are functional as they produce IFNγ and lyse target cells and this cytolytic activity is antigen specific and HLA restricted. Furthermore, the CTLs proved to kill tumour cells expressing endogenous tumour antigen in vitro. Therefore, MPLA/IFNγ DCs are very promising for the use in future cancer immunotherapy.

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

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.

Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans.

Immunoglobulin G (IgG) mediates its immune functions through complement and cellular IgG-Fc receptors (FcγR). IgG contains an evolutionary conserved N-linked glycan at position Asn297 in the Fc-domain. This glycan consists of variable levels of fucose, galactose, sialic acid, and bisecting N-acetylglucosamine (bisection). Of these variations, the lack of fucose strongly enhances binding to the human FcγRIII, a finding which is currently used to improve the efficacy of therapeutic monoclonal antibodies. The influence of the other glycan traits is largely unknown, mostly due to lack of glyco-engineering tools. We describe general methods to produce recombinant proteins of any desired glycoform in eukaryotic cells. Decoy substrates were used to decrease the level of fucosylation or galactosylation, glycosyltransferases were transiently overexpressed to enhance bisection, galactosylation and sialylation and in vitro sialylation was applied for enhanced sialylation. Combination of these techniques enable to systematically explore the biological effect of these glycosylation traits for IgG and other glycoproteins.

Anti–Tumor Necrosis Factor With a Glyco-Engineered Fc-Region Has Increased Efficacy in Mice With Colitis

BACKGROUND & AIMSnAlthough tumor necrosis factor (TNF) antagonists reduce many clinical features of inflammatory bowel disease, complete mucosal healing occurs in fewer than 50% of patients. The Fc-region of monoclonal antibodies against TNF has immunosuppressive properties via effects on macrophage polarization. We examined the interaction between the anti-TNF Fc-region and Fcγ receptors (FcγR), and whether the absence of the Fc core fucose (which increases binding to FcγRIIIa) increases the efficacy of anti-TNF in mice with colitis.nnnMETHODSnWe generated Rag1-/- mice that lackxa0all activating FcγRs (FcγRI, FcγRIII, and FcγRIV; called FcγR-/-Rag1-/- mice). We produced hypo-fucosylated antibodies against mouse and human TNF (adalimumab). Colitis was induced in mice by transfer of CD4+CD45RBhi to FcγR-/-Rag1-/- or Rag1-/- littermates; mice were given different antibodies against TNF or isotype (control) antibodies and disease activity index scores were determined. Colon tissues were collected and analyzed by histology. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors. T-cell proliferation and proportions of CD206+ (immune regulatory) macrophages were measured in mixed lymphocyte reactions. Human PBMCs were genotyped for FCGR3A158 (the FcγRIIIa-158F allotype displays a lower Fc binding affinity) using the TaqMan single nucleotide polymorphism genotype assay.nnnRESULTSnRag1-/- mice with colitis given anti-TNF had near complete mucosal healing and Rag1-/- mice given an isotype control antibody developed severe colitis. In contrast, FcγR-/-Rag1-/- mice were refractory to the effects of anti-TNF: their histological colitis scores were as severe as those from FcγR-/-Rag1-/- mice given a control antibody. Colons from Rag1-/- mice that received anti-TNF had an increased number of CD206+ macrophages compared with Rag1-/- mice given control antibody; in FcγR-/-Rag1-/- mice given anti-TNF these numbers were as low as FcγR-/-Rag1-/- given the control antibody. In human PBMCs, anti-TNF increased the number of CD206+ macrophages: this required expression of FcγRIIIa; numbers of these cells were reduced in PBMCs with the low-affinity FcγRIIIa-158F genotype. A hypo-fucosylated form of adalimumab bound human FcγRIIIa with a higher affinity than control adalimumab. When hypo-fucosylated adalimumab was added to PBMCs, a larger number of CD206+ macrophages formed and T-cell proliferation was reduced, compared with addition of a control adalimumab. Hypo-fucosylated adalimumab increased the number of CD206+ macrophages in PMBCs that expressed the low-affinity FcγRIIIa. In mice with colitis, hypo-fucosylated anti-TNF significantly increased the number of CD206+ macrophages in the colon compared with control anti-TNF and was more effective in reducing colitis severity as measured by histology.nnnCONCLUSIONSnIn a study of the inxa0vitro and inxa0vivo mechanisms of anti-TNF, we found FcγR engagement by anti-TNF to be required for reduction of colitis in mice and development of CD206+ macrophages. A hypo-fucosylated form of anti-TNF binds FcγRIIIa with higher affinity and induces development of CD206+ macrophages in human PBMCs, especially PBMCs that express low-affinity FcγRIIIa. Hypo-fucosylated anti-TNF might be more effective in patients with inflammatory bowel disease.

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

HIGHLIGHTSThe N162 glycan is conserved between the orthologous human Fc&ggr;RIIIa and mouse Fc&ggr;RIV.This glycan endows the receptors to discriminate between fucosylated and afucosylated IgG.Human afucosylated IgG/Fc&ggr;RIIIa affinity is further directed by additional galactosylation.Mouse afucosylated IgG/Fc&ggr;RIV affinity remains unaffected by additional galactosylation. ABSTRACT The binding strength between IgG and Fc&ggr;R 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 Fc&ggr;RIIIa 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 hFc&ggr;RIIIa and hFc&ggr;RIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse Fc&ggr;R, mFc&ggr;RIV. The N162‐glycan in mFc&ggr;RIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFc&ggr;RIIIa. However, unlike hFc&ggr;RIIIa, mFc&ggr;RIV did not bind more avidly to IgG with increased galactose and reduced fucose. Overall, these results suggest the N162‐glycan in the human Fc&ggr;RIII family and its orthologous mouse Fc&ggr;RIV to be functionally conserved.