IgG Glycoforms Research Articles

Exploring the structural heterogeneity of IgG1s: A comprehensive analysis of glycosylated and deglycosylated forms using TIMS-TOF-MS technique

Denaturation of IgG is a complex and multistep process that involves various structural changes in the protein as it loses its native conformation. IgG denaturation is crucial in therapeutic antibody development, manufacturing, and storage. Glycosylated IgG is generally more resistant to denaturing conditions due to the stabilizing effect of its glycans. In contrast, deglycosylated IgG is more vulnerable to structural disruption. In this study, we examined the intact level and large fragments of IgG glycoforms. Our investigation focused on changes in structural heterogeneity through glycosylation under denaturing conditions. Collision cross-sections (CCS) of intact level and large fragments of IgG1 molecules have been determined using reversed phase-high performance liquid chromatography-electrospray ionization-trapped ion mobility-time-of-flight mass spectrometry (RP-HPLC-ESI-TIMS-TOF-MS) technique. At the intact level, deglycosylated bevacizumab (IgG1B) and trastuzumab (IgG1T) could be differentiated at specific charge states and CCS ranges. The deglycosylated IgG1B molecule showed a broader CCS range than the IgG1T molecule. This result demonstrated that the deglycosylated IgG1T was altered from deglycosylated IgG1B in the gas phase under denaturing conditions. The lowering in the collision cross-section distribution (CCSD) values indicates a decrease in the structural heterogeneity of the intact IgG after deglycosylation. The ion mobility-mass spectrometry (IM-MS) analyses performed in this study showed that the glycans on the Fc region of IgG1s can trigger structural changes in the molecule under denaturing conditions. Depending on whether they are glycosylated or deglycosylated, various conformations of IgG1 molecules could be distinguished in RP-HPLC-TIMS-TOF-MS analysis.

A unique serum IgG glycosylation signature predicts development of Crohn's disease and is associated with pathogenic antibodies to mannose glycan.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the gut. There is growing evidence in Crohn's disease (CD) of the existence of a preclinical period characterized by immunological changes preceding symptom onset that starts years before diagnosis. Gaining insight into this preclinical phase will allow disease prediction and prevention. Analysis of preclinical serum samples, up to 6 years before IBD diagnosis (from the PREDICTS cohort), revealed the identification of a unique glycosylation signature on circulating antibodies (IgGs) characterized by lower galactosylation levels of the IgG fragment crystallizable (Fc) domain that remained stable until disease diagnosis. This specific IgG2 Fc glycan trait correlated with increased levels of antimicrobial antibodies, specifically anti-Saccharomyces cerevisiae (ASCA), pinpointing a glycome-ASCA hub detected in serum that predates by years the development of CD. Mechanistically, we demonstrated that this agalactosylated glycoform of ASCA IgG, detected in the preclinical phase, elicits a proinflammatory immune pathway through the activation and reprogramming of innate immune cells, such as dendritic cells and natural killer cells, via an FcγR-dependent mechanism, triggering NF-κB and CARD9 signaling and leading to inflammasome activation. This proinflammatory role of ASCA was demonstrated to be dependent on mannose glycan recognition and galactosylation levels in the IgG Fc domain. The pathogenic properties of (anti-mannose) ASCA IgG were validated in vivo. Adoptive transfer of antibodies to mannan (ASCA) to recipient wild-type mice resulted in increased susceptibility to intestinal inflammation that was recovered in recipient FcγR-deficient mice. Here we identify a glycosylation signature in circulating IgGs that precedes CD onset and pinpoint a specific glycome-ASCA pathway as a central player in the initiation of inflammation many years before CD diagnosis. This pathogenic glyco-hub may constitute a promising new serum biomarker for CD prediction and a potential target for disease prevention.

Long-Term Culturing of FreeStyle 293-F Cells Affects Immunoglobulin G Glycome Composition.

Glycosylation of IgG regulates the effector function of this antibody in the immune response. Glycosylated IgG is a potent therapeutic used for both research and clinical purposes. While there is ample research on how different cell culture conditions affect IgG glycosylation, the data are missing on the stability of IgG glycome during long cell passaging, i.e., cell "aging". To test this, we performed three independent time course experiments in FreeStyle 293-F cells, which secrete IgG with a human-like glycosylation pattern and are frequently used to generate defined IgG glycoforms. During long-term cell culturing, IgG glycome stayed fairly stable except for galactosylation, which appeared extremely variable. Cell transcriptome analysis revealed no correlation in galactosyltransferase B4GALT1 expression with galactosylation change, but with expression of EEF1A1 and SLC38A10, genes previously associated with IgG galactosylation through GWAS. The FreeStyle 293-F cell-based system for IgG production is a good model for studies of mechanisms underlying IgG glycosylation, but results from the present study point to the utmost importance of the need to control IgG galactosylation in both in vitro and in vivo systems. This is especially important for improving the production of precisely glycosylated IgG for therapeutic purposes, since IgG galactosylation affects the inflammatory potential of IgG.

Enhancing the therapeutic activity of hyperimmune IgG against chikungunya virus using FcγRIIIa affinity chromatography.

Chikungunya virus (CHIKV) is a re-emerging mosquito transmitted alphavirus of global concern. Neutralizing antibodies and antibody Fc-effector functions have been shown to reduce CHIKV disease and infection in animals. However, the ability to improve the therapeutic activity of CHIKV-specific polyclonal IgG by enhancing Fc-effector functions through modulation of IgG subclass and glycoforms remains unknown. Here, we evaluated the protective efficacy of CHIKV-immune IgG enriched for binding to Fc-gamma receptor IIIa (FcγRIIIa) to select for IgG with enhanced Fc effector functions. Total IgG was isolated from CHIKV-immune convalescent donors with and without additional purification by FcγRIIIa affinity chromatography. The enriched IgG was characterized in biophysical and biological assays and assessed for therapeutic efficacy during CHIKV infection in mice. FcγRIIIa-column purification enriched for afucosylated IgG glycoforms. In vitro characterization showed the enriched CHIKV-immune IgG had enhanced human FcγRIIIa and mouse FcγRIV affinity and FcγR-mediated effector function without reducing virus neutralization in cellular assays. When administered as post-exposure therapy in mice, CHIKV-immune IgG enriched in afucosylated glycoforms promoted reduction in viral load. Our study provides evidence that, in mice, increasing Fc engagement of FcγRs on effector cells, by leveraging FcγRIIIa-affinity chromatography, enhanced the antiviral activity of CHIKV-immune IgG and reveals a path to produce more effective therapeutics against these and potentially other emerging viruses.

Role of IgG glycans in severe COVID-19 inflammatory cytokine responses

Abstract Severe COVID-19 and multisystem inflammatory syndrome in children (MIS-C) are characterized by hyperinflammation. Severe COVID in adults is associated with development of afucosylated SARS-CoV-2 IgG that induce monocytes to secrete inflammatory cytokines. This study aimed to analyze the relationship between spike IgG Fc glycosylation and inflammatory markers during acute pediatric COVID or MIS-C or following COVID vaccination in adults. We assessed cross-sectional and longitudinal samples from 195 participants: Adult vaccinees, acute COVID patients, and healthy controls, as well as pediatric acute COVID patients, MIS-C patients, and healthy controls. We developed capillary electrophoresis (CE) methods to analyze bulk and spike IgG Fc glycans and measured ten relevant cytokines/chemokines by multiplexed assay. Analysis of healthy control bulk IgG by CE found 95.3–99.6% fucosylated, 2.7–6.9% bisected, 55.8–66.8% galactosylated, and 4.6–15.6% sialylated Fc glycans, compared to 99.8%, 6.7%, 45.4%, and 8.2% by mass spectrometry, respectively. IFN-γ, IL-10, CXCL10, and CCL3 were significantly increased in MIS-C compared to pediatric healthy controls; IL-10, CCL2, and TNF were only increased in severe adult COVID compared to healthy controls or less severe COVID. Initial results confirmed significantly more afucosylated spike IgG in adult severe COVID than afucosylated bulk IgG in controls. Our high-throughput methods for IgG Fc glycan profiling generated comparable results to mass spectrometry. Continued assessment will determine if COVID- or vaccine-induced spike IgG glycoforms correlate with inflammatory markers. Future mechanistic work will examine how modified glycosylation induces inflammatory cytokines. Supported by an IDCRC New Investigator Pilot Award to E.M.S. (NIH UM1AI148684), by the Centers for Disease Control and Prevention through a cooperative agreement with the Georgia Emerging Infection Program (grant no. U50CK000485), and by grant from NIH to P.A. (R24GM137782).

Enhanced Immunomodulatory Effect of Intravenous Immunoglobulin by Fc Galactosylation and Nonfucosylation.

Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in the treatment of various autoimmune/inflammatory diseases although its mechanism of action remains elusive. Recently, nonfucosylated IgG has been shown to be preferentially bound to Fcγ receptor IIIa (FcγRIIIa) on circulating natural killer cells; therefore, we hypothesized that nonfucosylated IVIG may modulate immune responses through FcγRIIIa blockade. Here, homogeneous fucosylated or nonfucosylated glycoforms of normal polyclonal IgG bearing sialylated, galactosylated or nongalactosylated Fc oligosaccharides were generated by chemoenzymatic glycoengineering to investigate whether the IgG glycoforms can inhibit antibody-dependent cellular cytotoxicity (ADCC). Among the six IgG glycoforms, galactosylated, nonfucosylated IgG [(G2)2] had the highest affinity to FcγRIIIa and 20 times higher potency to inhibit ADCC than native IgG. A pilot study of IVIG treatment in mice with collagen antibody-induced arthritis highlighted the low-dose (G2)2 glycoform of IVIG (0.1 g/kg) as an effective immunomodulatory agent as the 10-fold higher dose of native IVIG. These preliminary results suggest that the anti-inflammatory activity of IVIG is in part mediated via activating FcγR blockade by galactosylated, nonfucosylated IgG and that such nonfucosylated IgG glycoforms bound to FcγRs on immune cells play immunomodulatory roles in health and disease. This study provides insights into improved therapeutic strategies for autoimmune/inflammatory diseases using glycoengineered IVIG and recombinant Fc.

Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

We have used hydrogen exchange–mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments.

Crystallizable Fragment Glycoengineering for Therapeutic Antibodies Development.

Monoclonal antibody (mAb)-based therapeutics are the fastest growing class of human pharmaceuticals. They are typically IgG1 molecules with N-glycans attached to the N297 residue on crystallizable fragment (Fc). Different Fc glycoforms impact their effector function, pharmacokinetics, stability, aggregation, safety, and immunogenicity. Fc glycoforms affect mAbs effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by modulating the Fc–FcγRs and Fc–C1q interactions. While the terminal galactose enhances CDC activity, the fucose significantly decreases ADCC. Defucosylated immunoglobulin Gs (IgGs) are thus highly pursued as next-generation therapeutic mAbs with potent ADCC at reduced doses. A plethora of cell glycoengineering and chemoenzymatic glycoengineering strategies is emerging to produce IgGs with homogenous glycoforms especially without core fucose. The chemoenzymatic glycosylation remodeling also offers useful avenues for site-specific conjugations of small molecule drugs onto mAbs. Herein, we review the current progress of IgG-Fc glycoengineering. We begin with the discussion of the structures of IgG N-glycans and biosynthesis followed by reviewing the impact of IgG glycoforms on antibody effector functions and the current Fc glycoengineering strategies with emphasis on Fc defucosylation. Furthermore, we briefly discuss two novel therapeutic mAbs formats: aglycosylated mAbs and Fc glycan specific antibody–drug conjugates (ADCs). The advances in the understanding of Fc glycobiology and development of novel glycoengineering technologies have facilitated the generation of therapeutic mAbs with homogenous glycoforms and improved therapeutic efficacy.

155 - A Survey of the Current use of Flow Cytometry for the Diagnosis of Myelodysplastic Syndromes in Latin America

We have used hydrogen exchange–mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments.

Patients with Autoimmune Anti-Red Blood Cell IgG1 Have Different Total- and Autoantibody -IgG1 Glycosylation Compared to Healthy Controls

IntroductionAutoimmune hemolytic anemia (AIHA) is a potentially severe disease in which red blood cells (RBC) are destroyed by autoantibodies. IgG anti-RBC autoantibodies bind at 37°C and can lead to extravascular hemolysis by binding to phagocytes in the spleen.The composition of the N-linked sugar at position 297 in the IgG-Fc tail influences the binding affinity to IgG Fc receptors (FcγR) on effector cells. By mass spectrometric analysis we recently extensively studied the contribution of this sugar moiety to the clinical effect of RBC and platelet alloantibodies in pregnant women. Compared to the IgG glycoforms detected in plasma of these women, anti-D, anti-K and anti-Human Platelet Antigen 1a were found to be skewed towards IgG1 isoforms with low fucosylation, increasing binding capacity to IgG-Fc receptors type IIIa and IIIb (Kapur et al Blood 2014;123:471-80 & BHJ 2015;166:936-45; Sonneveld et al BJH 2016; 174:310-320 and oral presentation ASH Blood 2015;126:660). This feature has only been described for anti-HIV antibodies, but never for any other immune response. Furthermore, we observed an additional role for the level of bisected GlcNAc (bisection), galactosylation and sialylation of the RBC alloantibody-bound sugar moiety in predicting clinical outcome of HDFN. It is known that in autoimmune diseases the galactosylation and sialylation of plasma IgG1 is lowered, the glycoforms of specific autoantibodies have not yet been studied.AimThe aim of this study was to investigate the type of glycoforms of RBC autoantibodies in relation to the plasma IgG1 glycoforms in these patients and occurrence of hemolysis.Methods109 patients with a positive direct antiglobulin test (DAT) with anti-IgG were included, of whom blood was sent to our laboratory for diagnostic purposes. From 90 patients clinical data were collected. Based on laboratory markers (hemoglobin (Hb), haptoglobin, lactate dehydrogenase, reticulocyte count, bilirubin level), these patients were divided in three groups: 1. with hemolysis (n=36), 2. no hemolysis (n=33) or 3. possible hemolysis (n=21). RBC-bound autoantibodies were purified by acid elution. This RBC-eluted IgG and total IgG purified from paired plasma samples were subjected to tryptic digestion followed by mass spectrometry for glycoprofiling. Total IgG1 glycosylation patterns from healthy donors were used as controls.ResultsIn agreement with the previously described low IgG1-galactosylation and sialylation in patients with autoimmune diseases, we observed that IgG1 purified from plasma of patients with RBC-bound antibodies (a positive DAT) showed decreased galactosylation and sialylation levels compared to healthy controls: average 36% (95% CI, 34-37%) versus 53% (95%CI, 50-56%) (p<0.0001) and 2.4% (95% CI, 1.8-2%) versus 4% (95% CI, 3.4-4.5%), respectively. We now observed that total bisection levels were higher in patients with RBC autoantibodies (19%) versus healthy controls (12%) (p<0.001).Compared to plasma IgG1 glycosylation, the RBC-bound autoantibodies showed a profile with lower galactosylation, higher sialylation and lower bisecting GlcNAc levels (p=0.0029, p=0.0128 and p<0.0001 respectively). Fc-fucosylation was not different between healthy controls and patients for plasma IgG1 and not for RBC-bound IgG1 fractions (p=0.4986 and p=0.29 resp.).Analysis of Fc glycoprofiles in relation to Hb levels showed that higher bisection was associated with higher Hb levels for the total group of patients (p<0.001). This correlation was not found analyzing the three patients groups separately, probably due to low sample number.In conclusion, we found a low total IgG1 Fc-galactosylation in patients with RBC-bound IgG antibodies irrespective of occurrence of hemolysis. This supports the hypothesis that galactosylation is a marker for activation of the immune system in autoimmune diseases. In addition, we found RBC-bound IgG1 to be differently skewed in the composition of the N-linked sugar at position 297 compared to plasma-derived IgG1 in these patients. DisclosuresNo relevant conflicts of interest to declare.

Data Independent Analysis of IgG Glycoforms in Samples of Unfractionated Human Plasma.

Glycosylation regulates functional responses mediated by the interaction of IgG with their receptors. Multiple analytical methods have been designed for the determination of the IgG N-glycan microheterogeneity, including MS methods for the analysis of site specific glycoforms of IgG. However, measurement of low abundant glycoforms remains challenging in complex samples like serum without enrichment of the IgG. We present a workflow for quantitative analysis of site specific glycoforms of IgG based on data independent acquisition (DIA) of Y-ions generated under "minimal" fragmentation conditions. The adjusted collision induced dissociation (CID) conditions generate specific Y-ions in the yield of up to 60% precursor ion intensity. These selective fragments, measured in high resolution, improve specificity of detection compared to the typically quantified B-ions which have higher overall intensity but lower signal-to-noise ratios. Under optimized conditions, we achieve label-free quantification of the majority of previously reported glycoforms of IgG (26 glycoforms of IgG1, 22 glycoforms of IgG 2/3, and 19 glycoforms of IgG4) directly in unfractionated samples of human plasma and we detect traces of previously unreported glycoforms of IgG1, including doubly fucosylated glycoforms. The SWATH data independent quantification of IgG glycoforms in pooled plasma samples of patients with liver cirrhosis detects reliably the expected changes in the quantity of major glycoforms compared to healthy controls. Our results show that optimized CID fragmentation enables DIA of IgG glycoforms and suggest that such workflow may enable quantitative analyses of the glycoproteome in complex matrixes.

Combination of IgG N-glycomics and corresponding transcriptomics data to identify anti-TNF-α treatment responders in inflammatory diseases.

Prediction of responsiveness in biological therapies is an important and challenging issue in different diseases. Analyzing glycosylation pattern changes of key serum glycoproteins is one of the possible avenues to follow disease remission. The aim of this study was to investigate the changes of serum IgG glycoforms in Crohn's disease (CD) and rheumatoid arthritis patients in response to antitumor necrosis factor alpha (anti-TNF-α) treatment. IgG was isolated from patient serum samples using Protein A affinity pull-down, followed by the release of N-glycans with peptide-N-glycosidase F. The released glycans were fluorescently tagged with 8-aminopyrene-1,3,6-trisulfonate and analyzed by CGE with laser-induced fluorescent detection. Significant alterations were detected between responders and nonresponders in both disease groups. In CD patients, disease-specific alteration was found in response to anti-TNF-α therapy, which was also confirmed by transcriptomics data analysis of the corresponding glycosyltransferases and glycosidases.

Chemoenzymatic Glyco-engineering of Monoclonal Antibodies.

Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications.

Immunoglobulin G Fc N-glycosylation in Guillain-Barré syndrome treated with intravenous immunoglobulin.

Immunoglobulin G Fc N-glycosylation in Guillain-Barré syndrome treated with intravenous immunoglobulin.

Fc gamma receptor glycosylation modulates the binding of IgG glycoforms: a requirement for stable antibody interactions.

FcγRs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of FcγR glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant FcγRs (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa(Phe158/Val158), and FcγRIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for FcγRIa and FcγRIIIa. The IgG-FcγR interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated FcγRIa and FcγRIIIa(Phe158/Val158) receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated FcγRIIIa(Phe158) was now consistent with a 1:1 binding mechanism, while binding of rituximab to FcγRIIIa(Val158) remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-FcγR interaction and the influence of FcγR glycosylation.

Fragments of truth: T-cell targets of polyclonal immunoglobulins in autoimmune diseases.

The expanding therapeutic use of high-dose intravenous immunoglobulin (IVIg) in autoimmune diseases has raised important practical and conceptual issues over the last few years. These have prompted a number of research efforts aimed at characterizing aspects of the mechanism of action of current IVIg preparations, which might lead to the development of standardized, more cost-effective agents. Although polyclonal IgG in these preparations are mostly thought to act via direct interference with disease-specific, pathogenic autoantibodies, evidence from clinical and experimental work points to the involvement of crucial checkpoints upstream of self-reactive B-cell activation and autoantibody production. Reviewed herein are the results of the most recent studies documenting the crucial role of regulatory T cells (Treg) in the immunomodulatory activity of IVIg, and the molecular mechanisms mediating the effect of specific IgG fragments and glycoforms on Treg activity and the ensuing downregulation of T-cell effector responses of different sign and magnitude. Further progress in this area of translational research may lead to the development of innovative strategies aimed at restoring tolerance in autoimmune diseases.

Impact on N-Glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties

Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for suppressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet. The goals of this study were comparison of structural and functional properties of human anti-D polyclonal and monoclonal Abs and assessment of the possibility to manipulate the effector properties of the mAb. N-Glycosylation and particularly the content of nonfucosylated glycans are crucial for affinity of mAb to FcγRIIIA, which plays the key role in the clearance of sensitized cells. We studied and compared glycoprofiles and FcγRIIIA-mediated hemolytic ability of human polyclonal antibodies and anti-D mAbs produced by human B-cell lines, human-rodent heterohybridomas, and a human non-lymphoid cell line PER.C6. Replacement of producing cell line and use of glycosylation modulators can convert an inert mAb into an active one. Nevertheless, rodent cell lines, as well as human non-lymphoid cells, distort natural glycosylation of human IgG and could lead to the loss of immunosuppressive properties. All of the anti-D mAbs secreted by human B-cell lines have a glycoprofile close to human serum IgG. Hence, the constant ratio of IgG glycoforms in human serum is predetermined by glycosylation at the level of the individual antibody-producing cell. The anti-D fraction of polyclonal anti-D immunoglobulin compared to the total human IgG contains more nonfucosylated glycans. Thus, only human transformed B-cells are an appropriate source for efficient anti-D mAbs that can imitate the action of polyclonal anti-D IgG.

Immunoglobulin G Fc N-glycan profiling in patients with gastric cancer by LC-ESI-MS: relation to tumor progression and survival

The IgG Fc glycans strongly influence the Fcγ receptor interactions and Fc-mediated effector mechanisms. Changes in the structure of IgG glycans are associated with various diseases, such as infections and autoimmunity. However, the possible role of Fc glycans in tumor immunity is not yet fully understood. The aim of this study was to profile the Fc N-glycans of IgG samples from patients with gastric cancer (n = 80) and controls (n = 51) using LC-ESI-MS method to correlate the findings with stage of cancer and patients survival. Analysis of 32 different IgG N-glycans revealed significant increase of agalactosylated (GnGnF, GnGn(bi)F), and decrease of galactosylated (AGn(bi), AGn(bi)F, AA(bi), AAF) and monosialylated IgG glycoforms (NaAF, NaA(bi)) in cancer patients. A statistically significant increase of Fc fucosylation was observed in tumor stage II and III whereas reverse changes were found for the presence of bisecting GlcNAc. Higher level of fully sialylated glycans and elevated expression of glycans with bisecting GlcNAc were associated with better survival rate. Our findings provide the first evidence that the changes in Fc glycan profile may predict the survival of patients with gastric cancer. Cancer stage-dependent changes in Fc fucosylation and the bisecting N-acteylglucosamine expression as well as an association of several IgG glycoforms with the survival suggest that IgG glycosylation is related to pathogenesis of cancer and progression of the disease.

Structural basis for Fc gammaRIIa recognition of human IgG and formation of inflammatory signaling complexes.

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.

Glycoforms of Human IgG in Health and Disease

今や 20 種類以上のリコンビナント抗体が，様々ながんや慢性疾患の治療用に認可されている。さらに，文字通り数百もの抗体が開発あるいは臨床の早期フェーズのさなかにある。当初，抗体の魅力は標的抗原への特異性にあった。しかし今や，免疫複合体の形成が臨床上の成否を決する下流の生物活性の引き金となることが広く認められている。そのような生物学的メカニズム（エフェクター機能）の活性化には，抗体の質量のわずか 2-3％にすぎない糖鎖による IgG-Fc の修飾が不可欠であることが示されている。さらに，Fc に結合している糖鎖の構造はタンパク質のコンフォメーションや安定性を決定し，それにより下流の生物学的な効力にも影響を及ぼし得るのである。ある疾患に対して最適となるような均一なグライコフォームの抗体を選択的に生産する細胞株を作り出すために細胞工学的な手法が取り入れてられてきている。治療抗体を高濃度に調製することには利点があるが，IgG-Fab 領域に新たな糖鎖修飾を行うことが，それを支援する技術となるかもしれない。また，生産コストを下げることによって低廉化をもたらし得る生産系も開発されつつある。