Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in... Show moreAnti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in Fc gamma RIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77-81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fucosylation mediated more efficient ADCC and clearance than anti-D Ig. Galactosylation of B mAb-Ds was 57-83% but 15-58% for rodent mAb-Ds. HH mAb-Ds had non-human sugars. These data reveal high galactosylation like anti-D Ig (>60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis. Show less
Monomeric serum immunoglobulin A (IgA) can contribute to the development of various autoimmune diseases, but the regulation of serum IgA effector functions is not well defined. Here, we show that... Show moreMonomeric serum immunoglobulin A (IgA) can contribute to the development of various autoimmune diseases, but the regulation of serum IgA effector functions is not well defined. Here, we show that the two IgA subclasses (IgA1 and IgA2) differ in their effect on immune cells due to distinct binding and signaling properties. Whereas IgA2 acts pro-inflammatory on neutrophils and macrophages, IgA1 does not have pronounced effects. Moreover, IgA1 and IgA2 have different glycosylation profiles, with IgA1 possessing more sialic acid than IgA2. Removal of sialic acid increases the pro-inflammatory capacity of IgA1, making it comparable to IgA2. Of note, disease-specific autoantibodies in patients with rheumatoid arthritis display a shift toward the pro-inflammatory IgA2 subclass, which is associated with higher disease activity. Taken together, these data demonstrate that IgA effector functions depend on subclass and glycosylation, and that disturbances in subclass balance are associated with autoimmune disease. Show less
Erp, E.A. van; Lakerveld, A.J.; Graaf, E. de; Larsen, M.D.; Schepp, R.M.; Ederveen, A.L.H.; ... ; Kasteren, P.B. van 2020
Objectives Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, and there is no vaccine available. In early life, the most important... Show moreObjectives Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, and there is no vaccine available. In early life, the most important contributors to protection against infectious diseases are the innate immune response and maternal antibodies. However, antibody-mediated protection against RSV disease is incompletely understood, as both antibody levels and neutralisation capacity correlate poorly with protection. Since antibodies also mediate natural killer (NK) cell activation, we investigated whether this functionality correlates with RSV disease.Methods We performed an observational case-control study including infants hospitalised for RSV infection, hernia surgery or RSV-negative respiratory viral infections. We determined RSV antigen-specific antibody levels in plasma using a multiplex immunoassay. Subsequently, we measured the capacity of these antibodies to activate NK cells. Finally, we assessed Fc-glycosylation of the RSV-specific antibodies by mass spectrometry.Results We found that RSV-specific maternal antibodies activate NK cells in vitro. While concentrations of RSV-specific antibodies did not differ between cases and controls, antibodies from infants hospitalised for severe respiratory infections (RSV and/or other) induced significantly less NK cell interferon-gamma production than those from uninfected controls. Furthermore, NK cell activation correlated with Fc-fucosylation of RSV-specific antibodies, but their glycosylation status did not significantly differ between cases and controls.Conclusion Our results suggest that Fc-dependent antibody function and quality, exemplified by NK cell activation and glycosylation, contribute to protection against severe RSV disease and warrant further studies to evaluate the potential of using these properties to evaluate and improve the efficacy of novel vaccines. Show less
Singh, S.S.; Naber, A.; Dotz, V.; Schoep, E.; Memarian, E.; Slieker, R.C.; ... ; Hoek, M. van 2020
Introduction Recent studies revealedN-glycosylation signatures of type 2 diabetes, inflammation and cardiovascular risk factors. Most people with diabetes use medication to reduce cardiovascular... Show moreIntroduction Recent studies revealedN-glycosylation signatures of type 2 diabetes, inflammation and cardiovascular risk factors. Most people with diabetes use medication to reduce cardiovascular risk. The association of these medications with the plasmaN-glycome is largely unknown. We investigated the associations of metformin, statin, ACE inhibitor/angiotensin II receptor blocker (ARB), sulfonylurea (SU) derivatives and insulin use with the total plasmaN-glycome in type 2 diabetes.Research design and methods After enzymatic release from glycoproteins,N-glycans were measured by matrix-assisted laser desorption/ionization mass spectrometry in the DiaGene (n=1815) and Hoorn Diabetes Care System (n=1518) cohorts. Multiple linear regression was used to investigate associations with medication, adjusted for clinical characteristics. Results were meta-analyzed and corrected for multiple comparisons.Results Metformin and statins were associated with decreased fucosylation and increased galactosylation and sialylation in glycans unrelated to immunoglobulin G. Bisection was increased within diantennary fucosylated non-sialylated glycans, but decreased within diantennary fucosylated sialylated glycans. Only few glycans were associated with ACE inhibitor/ARBs, while none associated with insulin and SU derivative use.Conclusions We conclude that metformin and statins associate with a total plasmaN-glycome signature in type 2 diabetes. Further studies are needed to determine the causality of these relations, and futureN-glycomic research should consider medication a potential confounder. Show less
Singh, S.S.; Naber, A.; Dotz, V.; Schoep, E.; Memarian, E.; Slieker, R.C.; ... ; Hoek, M. van 2020
Introduction Recent studies revealed N-glycosylation signatures of type 2 diabetes, inflammation and cardiovascular risk factors. Most people with diabetes use medication to reduce cardiovascular... Show moreIntroduction Recent studies revealed N-glycosylation signatures of type 2 diabetes, inflammation and cardiovascular risk factors. Most people with diabetes use medication to reduce cardiovascular risk. The association of these medications with the plasma N-glycome is largely unknown. We investigated the associations of metformin, statin, ACE inhibitor/angiotensin II receptor blocker (ARB), sulfonylurea (SU) derivatives and insulin use with the total plasma N-glycome in type 2 diabetes.Research design and methods After enzymatic release from glycoproteins, N-glycans were measured by matrix-assisted laser desorption/ionization mass spectrometry in the DiaGene (n=1815) and Hoorn Diabetes Care System (n=1518) cohorts. Multiple linear regression was used to investigate associations with medication, adjusted for clinical characteristics.Results were meta-analyzed and corrected for multiple comparisons. Results Metformin and statins were associated with decreased fucosylation and increased galactosylation and sialylation in glycans unrelated to immunoglobulin G. Bisection was increased within diantennary fucosylated non-sialylated glycans, but decreased within diantennary fucosylated sialylated glycans. Only few glycans were associated with ACE inhibitor/ARBs, while none associated with insulin and SU derivative use.Conclusions We conclude that metformin and statins associate with a total plasma N-glycome signature in type 2 diabetes. Further studies are needed to determine the causality of these relations, and future N-glycomic research should consider medication a potential confounder. Show less
Gstottner, C.; Reusch, D.; Haberger, M.; Dragan, I.; Veelen, P. van; Kilgour, D.P.A.; ... ; Nicolardi, S. 2020
Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for... Show moreBispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or in vivo after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd' regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS. Show less
A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories.Glycosylation is a topic of intense current... Show moreA broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories.Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Show less
Changes in the abundance of antennary fucosylated glycans in human total plasma N-glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However... Show moreChanges in the abundance of antennary fucosylated glycans in human total plasma N-glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However, it is challenging to address this important part of the human glycome. Most commonly, time-consuming chromatographic separations are performed to differentially quantify core and antenna fucosylation. Obtaining sufficient resolution for larger, more complex glycans can be challenging. We introduce a matrix-assisted laser desorption/ionization—mass spectrometry (MALDI-MS) assay for the relative quantitation of antennary fucosylation in TPNG. N-linked glycans are released from plasma by PNGase F and further treated with a core fucosidase before performing a linkage-informative sialic acid derivatization. The core fucosylated glycans are thus depleted while the remaining antennary fucosylated glycans are quantitated. Simultaneous quantitation of α2,3-linked sialic acids and antennary fucosylation allows an estimation of the sialyl-Lewis x motif. The approach is feasible using either ultrahigh-resolution Fourier-transform ion cyclotron resonance mass spectrometry or time-of-flight mass spectrometry. The assay was used to investigate changes of antennary fucosylation as clinically relevant marker in 14 colorectal cancer patients. In accordance with a previous report, we found elevated levels of antennary fucosylation pre-surgery which decreased after tumor resection. The assay has the potential for revealing antennary fucosylation signatures in various conditions including diabetes and different types of cancer. Show less
Kissel, T.; Schie, K.A. van; Hafkenscheid, L.; Lundquist, A.; Kokkonen, H.; Wuhrer, M.; ... ; Rantapaa-Dahlqvist, S. 2019
Objective Anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) patients display a unique feature defined by the abundant presence of N-linked glycans within the variable... Show moreObjective Anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) patients display a unique feature defined by the abundant presence of N-linked glycans within the variable domains (V-domains). Recently, we showed that N-glycosylation sites, which are required for the incorporation of V-domain glycans, are introduced following somatic hypermutation. However, it is currently unclear when V-domain glycosylation occurs. Further, it is unknown which factors might trigger the generation of V-domain glycans and whether such glycans are relevant for the transition towards RA. Here, we determined the presence of ACPA-IgG V-domain glycans in paired samples of pre-symptomatic individuals and RA patients.Methods ACPA-IgG V-domain glycosylation was analysed using ultra-high performance liquid chromatography (UHPLC) in paired samples of pre-symptomatic individuals (median interquartile range (IQR) pre-dating time: 5.8 (5.9) years; n=201; 139 ACPA-positive and 62 ACPA-negative) and RA patients (n=99; 94 ACPA-positive and 5 ACPA-negative).Results V-domain glycans on ACPA-IgG were already present up to 15 years before disease in pre-symptomatic individuals and their abundance increased closer to symptom onset. Noteworthy, human leucocyte antigen class II shared epitope (HLA-SE) alleles associated with the presence of V-domain glycans on ACPA-IgG.Conclusion Our observations indicate that somatic hypermutation of ACPA, which results in the incorporation of N-linked glycosylation sites and consequently V-domain glycans, occurs already years before symptom onset in individuals that will develop RA later in life. Moreover, our findings provide first evidence that HLA-SE alleles associate with ACPA-IgG V-domain glycosylation in the pre-disease phase and thereby further refine the connection between HLA-SE and the development of ACPA-positive RA. Show less
Vreeker, G.C.M.; Bladergroen, M.R.; Nicolardi, S.; Mesker, W.E.; Tollenaar, R.A.E.M.; Burgt, Y.E.M. van der; Wuhrer, M. 2019
