Fc N-glycans Research Articles

Generating and characterizing a comprehensive panel of CHO cells glycosylation mutants for advancing glycobiology and biotechnology research

This report describes the development and characterization of a comprehensive collection of CHO cell glycosylation mutants with significant potential for advancing glycobiology and biotechnology. EPO-Fc and trastuzumab, two model molecules, were produced using these mutants to assess the effects of mutated glycogenes, and LC-MS/MS analysis was employed to quantitatively analyse their N-glycans. EPO-Fc exhibited exclusively homogeneous Man9 glycans only when nearly all α-mannosidases in the genome were inactivated, except lysosomal MAN2B1. Some mutants lacking GnT-I activity produce mostly Man5 N-glycans, while their O-glycan and glycolipid profiles can differ due to other mutations in the cell. GnT-II deficiency prevents GnT-V from adding GlcNAc to the core N-glycan, resulting in branches attaching solely to the α1,3-linked mannose, leaving the α1,6-linked mannose free. The mutant-produced antibody’s single-branched glycan contains more sialic acid than the dual-branched glycans produced in CHO-K1 cells. Trastuzumab produced in these mutants provided insights into how Fc N-glycans impact the antibody’s interaction with FcγR1 and FcγR2a, FcγR3a, and their influence on antibody-dependent cellular cytotoxicity (ADCC). In the study of Fc glycans in Fc-FcγR1 and FcγR2a interactions, we observed a consistent glycan-related impact on binding to both receptors, indicating a common interaction mechanism between Fc glycans and both FcγRI and FcγRIIa. CHO mutants produced trimeric gp120 demonstrated distinct reactivity with multiple broadly neutralizing anti-HIV antibodies, confirming the involvement of gp120 glycans in interactions with specific broadly neutralizing antibodies. Finally, one of the mutants produced human β-glucocerebrosidase with uniform Man5 N-glycans, showcasing its potential for glycoengineered production and enhancement in therapeutic efficacy.

First site-specific conjugation method for native goat IgG antibodies via glycan remodeling at the conserved Fc region.

Despite their triumph in treating human diseases, antibody therapies for animals have gained momentum more slowly. However, the first approvals of animal antibodies for osteoarthritic pain in cats and dogs may herald the dawn of a new era. For example, goats are vital to economies around the world for their milk, meat, and hide products. It is therefore imperative to develop therapies to safeguard goats-with antibodies at the forefront. Goat antibodies will be crucial in the development of therapeutic antibodies, for example, as tracers to study antibody distribution in vivo, reagents to develop other therapeutic antibodies, and therapeutic agents themselves (e.g., antibody-drug conjugates). Hamstringing this effort is a still-burgeoning understanding of goat antibodies and their derivatization. Historically, goat antibody conjugates were generated through stochastic chemical modifications, producing numerous attachment sites and modification ratios, thereby deleteriously impacting antigen binding. Site-specific methods exist but often require substantial engineering and have not been demonstrated with goat antibodies. Nevertheless, we present herein a novel method to site-specifically conjugate native goat antibodies: chemo-enzymatic remodeling of the native Fc N-glycan introduces a reactive azide handle, after which click chemistry with strained alkyne partners affords homogeneous conjugates labeled only on the Fc domain. This process is robust, and resulting conjugates retain their antigen binding and specificity. To our knowledge, our report is the first for site-specific conjugation of native goat antibodies. Furthermore, our approach should be applicable to other animal antibodies-even with limited structural information-with similar success.

Comprehensive analysis and characterization of glycan pairing in therapeutic antibodies and Fc-containing biotherapeutics: Addressing current limitations and implications for N-glycan impact

N-glycosylation of the Fc part is a (critical) quality attribute of therapeutic antibodies and Fc-containing biotherapeutics, that impacts their stability, immunogenicity, pharmacokinetics, and effector functions. Current glycosylation analysis methods focus on the absolute amounts of glycans, neglecting the apparent glycan distribution over the entirety of proteins. The combination of the two Fc N-glycans, herein referred to as glyco-pair, therefore remains unknown, which is a major drawback for N-glycan impact assessment. This study presents a comprehensive workflow for the analysis and characterization of Fc N-glycan pairing in biotherapeutics, addressing the limitations of current glycosylation analysis methods. The applicability of the method across various biotherapeutic proteins including antibodies, bispecific antibody formats, and a Fc-Fusion protein is demonstrated, and the impact of method conditions on glycan pairing analysis is highlighted. Moreover, the influence of the molecular format, Fc backbone, production process, and cell line on glycan pairing pattern was investigated. The results underscore the significance of comprehensive glycan pairing analysis to accurately assess the impact of N-glycans on important product quality attributes of therapeutic antibodies and Fc-containing biotherapeutics.

IgG1 glycosylation highlights premature aging in Down syndrome.

Down syndrome (DS) is characterized by lowered immune competence and premature aging. We previously showed decreased antibody response following SARS-CoV-2 vaccination in adults with DS. IgG1 Fc glycosylation patterns are known to affect the effector function of IgG and are associated with aging. Here, we compare total and anti-spike (S) IgG1 glycosylation patterns following SARS-CoV-2 vaccination in DS and healthy controls (HC). Total and anti-Spike IgG1 Fc N-glycan glycoprofiles were measured in non-exposed adults with DS and controls before and after SARS-CoV-2 vaccination by liquid chromatography-mass spectrometry (LC-MS) of Fc glycopeptides. We recruited N = 44 patients and N = 40 controls. We confirmed IgG glycosylation patterns associated with aging in HC and showed premature aging in DS. In DS, we found decreased galactosylation (50.2% vs. 59.0%) and sialylation (6.7% vs. 8.5%) as well as increased fucosylation (97.0% vs. 94.6%) of total IgG. Both cohorts showed similar bisecting GlcNAc of total and anti-S IgG1 with age. In contrast, anti-S IgG1 of DS and HC showed highly comparable glycosylation profiles 28 days post vaccination. The IgG1 glycoprofile in DS exhibits strong premature aging. The combination of an early decrease in IgG1 Fc galactosylation and sialylation and increase in fucosylation is predicted to reduce complement activity and decrease FcγRIII binding and subsequent activation, respectively. The altered glycosylation patterns, combined with decreased antibody concentrations, help us understand the susceptibility to severe infections in DS. The effect of premature aging highlights the need for individuals with DS to receive tailored vaccines and/or vaccination schedules.

Platform-Specific Fc N-Glycan Profiles of an Antisperm Antibody.

IgG Fc N-glycosylation is necessary for effector functions and is an important component of quality control. The choice of antibody manufacturing platform has the potential to significantly influence the Fc glycans of an antibody and consequently alter their activity and clinical profile. The Human Contraception Antibody (HCA) is an IgG1 antisperm monoclonal antibody (mAb) currently in clinical development as a novel, non-hormonal contraceptive. Part of its development is selecting a suitable expression platform to manufacture HCA for use in the female reproductive tract. Here, we compared the Fc glycosylation of HCA produced in two novel mAb manufacturing platforms, namely transgenic tobacco plants (Nicotiana benthamiana; HCA-N) and mRNA-mediated expression in human vaginal cells (HCAmRNA). The Fc N-glycan profiles of the two HCA products were determined using mass spectrometry. Major differences in site occupancy, glycan types, and glycoform distributions were revealed. To address how these differences affect Fc function, antibody-dependent cellular phagocytosis (ADCP) assays were performed. The level of sperm phagocytosis was significantly lower in the presence of HCA-N than HCAmRNA. This study provides evidence that the two HCA manufacturing platforms produce functionally distinct HCAs; this information could be useful for the selection of an optimal platform for HCA clinical development and for mAbs in general.

Technical, preclinical, and clinical developments of Fc-glycan-specific antibody-drug conjugates.

Antibody-drug conjugates (ADCs) have emerged as a powerful avenue in the therapeutic treatment of cancer. Site-specific antibody-drug conjugations represent the latest trend in the development of ADCs, addressing the limitations of traditional random conjugation technologies. This article summarizes the innovative development of Fc-glycan-specific ADCs (gsADCs), which utilize the conserved Fc N-glycan as the anchor point for site-specific conjugation. This approach offers significant strengths, including improved ADC homogeneity and overall hydrophilicity, enhanced pharmacokinetics and therapeutic index, and potentially reduced Fc receptor-mediated side effects. Currently dozens of gsADCs are in different preclinical and clinical development stages. Notably, JSKN003 and IBI343 have demonstrated promising results in phase 1 trials and are advancing into phase 3 studies. This review discusses the advantages of Fc-glycan-conjugation, various glycan-specific conjugation techniques, and the preclinical and clinical development of gsADCs. While challenges such as increased manufacturing cost for large-scale production need continuous innovation to overcome and there are different opinions regarding the pros and cons of reduced/diminished affinities to Fc gamma receptors, ongoing research and clinical progress underscore the potential of gsADCs to renovate ADC cancer therapy.

B-Cell Targeting Anti-CD37 Humanized Antibodies Engineered for Potent Effector Functions and Extended Plasma Half-Life

Background: CD37 is a tetraspanin protein abundant on the surface of B-cells but absent in hematopoietic stem cells and plasma cells. Its expression pattern has attracted interest as a therapeutic target for B-cell malignancies including non-Hodgkin lymphomas (NHL), a heterogeneous disease with substantial unmet clinical need due to relapse and resistance to currently available treatments. During the last years, different approaches have been followed to exploit CD37 in targeting malignant B-cells, such as the generation of CAR-T cells and monoclonal antibodies. Of note, the anti-CD37 antibody-drug conjugates (ADC) naratuximab emtansine and the CD37-targeting radioimmunotherapy (RIT) Betalutin have shown efficacy in NHL patients during clinical testing. Here we report on the design of humanized anti-CD37 IgG1 antibodies (NNV029, NNV031 and NNV032) that are equipped with a unique Fc technology and N-glycan engineering for enhanced effector functions and extended plasma half-life. Methods: The humanized anti-CD37 candidates were characterized in vitro and in vivo and benchmarked against the anti-CD20 antibodies approved for NHL treatment, rituximab and obinutuzumab, and a recombinant version of the bi-paratopic anti-CD37 antibody DuoHexaBody-CD37, currently in Phase I clinical development. We assessed their Fcγ receptor binding properties and their ability to induce antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) against CD37-expressing NHL cell lines and patient-derived chronic lymphocytic leukemia (CLL) cells. Moreover, we evaluated their human FcRn binding and transport properties using the human endothelial recycling assay (HERA) followed by studies in human FcRn (Tg32) expressing mice. Lastly, we compared the therapeutic efficacy of the humanized antibodies versus obinutuzumab and recombinant DuoHexaBody-CD37 in a xenograft disseminated Daudi tumor model in Tg32 mice. Results: We revealed that the designed anti-CD37 IgG1 candidates have higher affinity towards FcγRIIa expressed on macrophages and that afucosylation enhanced FcγRIII engagement. Accordingly, using cell-based effector assays, potent ADCP and ADCC were measured. In NHL Burkitt's lymphoma cell lines, ADCP induction by the anti-CD37 humanized antibodies was higher than that displayed by obinutuzumab, rituximab and recombinant DuoHexaBody-CD37, while in DLBCL and MCL cell models it was in a similar range or slightly lower than that shown by the benchmarking antibodies. Moreover, the afucosylated version promoted very strong ADCC, which was superior to that of recombinant DuoHexaBody-CD37 and rituximab, but comparable to that of the afucosylated antibody obinutuzumab. In contrast, all the humanized anti-CD37 IgG1 candidates displayed very modest CDC activation. Fc-engineering for enhanced human FcRn binding was shown to increase human FcRn-mediated rescue from intracellular degradation, which translated into prolonged plasma half-life in Tg32 mice of the anti-CD37 IgG1 antibodies. Finally, all the designed anti-CD37 antibodies performed significantly better than vehicle control treatment in treating Daudi-induced disease in vivo, showing equal therapeutic effect as that of obinutuzumab and recombinant DuoHexaBody-CD37. Conclusions: Taken together, the humanized and Fc-engineered anti-CD37 antibodies show potent effector functions, favorable pharmacokinetic properties and in vivo therapeutic efficacy. As such, the designed CD37 antibodies should be attractive modalities in targeted treatment of B cell malignancies, such as NHL, including lymphomas non-responding to anti-CD20 treatments, but also potentially for B-cell driven autoimmune disorders.

Production of afucosylated antibodies in CHO cells by coexpression of an anti-FUT8 intrabody.

Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.

Altered IgG glycosylation at COVID-19 diagnosis predicts disease severity.

The nature of the immune responses associated with COVID‐19 pathogenesis and disease severity, as well as the breadth of vaccine coverage and duration of immunity, is still unclear. Given the unpredictability for developing a severe/complicated disease, there is an urgent need in the field for predictive biomarkers of COVID‐19. We have analyzed IgG Fc N‐glycan traits of 82 SARS‐CoV‐2+ unvaccinated patients, at diagnosis, by nano‐LC‐ESI‐MS. We determined the impact of IgG Fc glyco‐variations in the induction of NK cells activation, further evaluating the association between IgG Fc N‐glycans and disease severity/prognosis. We found that SARS‐CoV‐2+ individuals display, at diagnosis, variations in the glycans composition of circulating IgGs. Importantly, levels of galactose and sialic acid structures on IgGs are able to predict the development of a poor COVID‐19 disease. Mechanistically, we demonstrated that a deficiency on galactose structures on IgG Fc in COVID‐19 patients appears to induce NK cells activation associated with increased release of IFN‐γ and TNF‐α, which indicates the presence of pro‐inflammatory immunoglobulins and higher immune activation, associated with a poor disease course. This study brings to light a novel blood biomarker based on IgG Fc glycome composition with capacity to stratify patients at diagnosis.

Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain.

Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc N-glycan, and eliminating the N-glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated N-glycans that are unsuitable for therapeutics. However, Fc with a specifically truncated N-glycan still engages receptors with considerable affinity. Here we describe the creation and applications of a novel Saccharomyces cerevisiae strain that specifically modifies the IgG1 Fc domain with an N-glycan consisting of a single N-acetylglucosamine residue. This strain displayed glycoengineered Fc on its surface for screening yeast surface display libraries and also served as an alternative platform to produce glycoengineered Rituximab. An IgG-specific endoglycosidase (EndoS2) truncates the IgG1 Fc N-glycan. EndoS2 was targeted to the yeast ER using the signal peptide from the yeast protein disulfide isomerase (PDI) and a yeast ER retention signal (HDEL). Furthermore, >99% of the yeast expressed Rituximab displayed the truncated glycoform as determined by SDS-PAGE and ESI-MS analyses. Lastly, the yeast expressed Rituximab engaged the FcγRIIIa with the expected affinity (KD = 2.0 ± 0.5 μM) and bound CD20 on Raji B cells.

Correlation of N-glycan dynamics and interaction network with allosteric antigen binding and Fc receptor recognition

Aim: Fragment crystallizable (Fc) glycans modulate Fc conformations and functions, and glycan may also regulate antigen recognition. In the antibody drug development, glycosylation patterns affect antibody drug characteristics and quality control. In order to provide a global feature of N-glycan interactions in response to antigen and Fc receptor bindings, the interactions among Fc N-glycans and N-glycans’ interaction with Fc CH2 and CH3 domains have been studied. Methods: Molecular dynamics simulations were used to generate conformation ensembles of free antibody, antibody-antigen complex, antibody-human Fc-gamma-receptor-I (hFcγRI) and antibody-antigen-hFcγRI, the hydrogen bonds and radial distance distribution involving N-glycans carbohydrate chains have been analyzed. Results: Two important interaction patterns have been observed. The first is the strong but non-specific interactions between two carbohydrate chains in free antibody. Secondly, it has been found that N-glycans carbohydrate chains can directly interact with CH3 domain in free antibody, and that the distance distribution between carbohydrate chains and CH3 domain clearly differentiate the free antibody, antibody-antigen complex, antibody-hFcγRI complex, and final antibody-antigen-hFcγRI complex. Conclusions: N-glycans partially acts as allosteric sensor and respond to antigen and hFcγRI binding.

Fc Glycosylation Characterization of Human Immunoglobulins G Using Immunocapture and LC-MS.

Immunoglobulins G (IgG) are proteins produced by the immune system of higher life forms that play a central role in the defense against microbial pathogens. IgG bind pathogens with the hypervariable Fab component and mediate a diversity of effector functions by binding to immune effector cells via their crystallizable (Fc) component. All IgG Fc carry a polymorphic N-glycan that regulates its binding properties and thereby its effector functions. The glycosylation profile of IgG Fc is modulated by physiological and pathological conditions, including infectious diseases and inflammatory disorders. Characterization of IgG Fc glycosylation profiles is a promising approach to understand the pathogenesis of diseases involving the immune system and to develop novel biomarkers of disease activity. Measuring the proportion of the different IgG Fc glycoforms remains an analytical challenge, that requires a sensitive and reproducible analytical approach.This chapter describes an optimized approach for the preparation and the analysis of Fc N-glycans from total serum or plasma IgG using magnetic beads, RapiFluor MS label©, and LC-MS.

Impact of IgG1 N-glycosylation on their interaction with Fc gamma receptors

The effector functions of the IgGs are modulated by the N-glycosylation of their Fc region. Particularly, the absence of core fucosylation is known to increase the affinity of IgG1s for the Fcγ receptor IIIa expressed by immune cells, in turn translating in an improvement in the antibody-dependent cellular cytotoxicity. However, the impact of galactosylation and sialylation is still debated in the literature. In this study, we have investigated the influence of high and low levels of core fucosylation, terminal galactosylation and terminal α2,6-sialylation of the Fc N-glycans of trastuzumab on its affinity for the FcγRIIIa. A large panel of antibody glycoforms (i.e., highly α2,6-sialylated or galactosylated IgG1s, with high or low levels of core fucosylation) were generated and characterized, while their interactions with the FcγRs were analysed by a robust surface plasmon resonance-based assay as well as in a cell-based reporter bioassay. Overall, IgG1 glycoforms with reduced fucosylation display a stronger affinity for the FcγRIIIa. In addition, fucosylation, and the presence of terminal galactose and sialic acids are shown to increase the affinity for the FcγRIIIa as compared to the agalactosylated forms. These observations perfectly translate in the response observed in our reporter bioassay.

FRI0304 SERUM IGG-UNDERGALACTOSYLATION PROFILES REFLECT CUMULATIVE EXPOSURE TO SYSTEMIC INFLAMMATION IN SPONDYLOARTHRITIS PATIENTS

Background:Inflammation in spondyloarthritis (SpA) is often not reflected by elevated acute phase reactants such as C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). It has been shown that IgG glycosylation patterns are subject to specific alterations (i.e. undergalactosylation) in chronic inflammatory diseases. Since these changes only occur in persistent inflammatory processes, lasting at least one to two t1/2of IgG (24 days), it was hypothesized that IgG-glycan profiles could serve as a surrogate marker for chronic inflammation in SpA patients.Objectives:To assess the value of serum IgG-undergalactosylation in SpA patients in relation to outcome measures for disease activity, determined by patient reported outcomes, serum inflammatory markers and imaging outcomes.Methods:Serum samples were obtained from SpA patients at the baseline visit of Be-Giant: a Belgian observational cohort including SpA patients who fulfill the ASAS classification criteria for axial or peripheral SpA. IgG Fc N-glycans were released directly in whole serum by endo-β-N-acetyl-glucosaminidase fromStreptococcus pyogenes(EndoS), fluorescently labeled with ATPS and analyzed by capillary electrophoresis, rendering glycan profiles with six peaks (Figure 1). Relative peak heights were combined in the undergalactosylation score (UGS), capturing the relative upregulation of non-galactosylated glycans normalized to the total peak height (1). Baseline radiographs (X-SIJ) and magnetic resonance images (MRI) of the sacroiliac joints (SIJ) were assessed by three calibrated readers for sacroiliitis (fulfillment of the modified New York criteria; grading 0 to 4 per SIJ) and for inflammatory lesions according to the Spondyloarthritis Research Consortium of Canada (SPARCC) method (score from 0 – 72) respectively. Grades and inflammatory lesions that were seen by at least 2 readers were used for further analysis.Figure 1.Example of a serum IgG-specific glycan profile. Adapted from (1), with permission.Results:Glycan profiles were obtained from 376 SpA patients; UGS was scaled (mean = 0, SD = 1) for further analysis. UGS was independently associated with ASDAS-CRP (β1= 0.15, 95% CI 0.04 – 0.26, p = 0.006) and BASFI (β1= 0.44, 95% CI 0.16 – 0.72, p = 0.002) but not with BASDAI (β1= 0.12, 95% CI -0.13 – 0.38, p = 0.34). UGS showed a weak to moderate correlation with CRP (Rs= 0.30, p < 0.001) and ESR (Rs= 0.27, p <0.001). In axial SpA, UGS was significantly higher in patients with ankylosing spondylitis compared to non-radiographic axial SpA (OR = 2.41, 95% CI 1.60 – 3.73, p < 0.001) and showed an independent association with the total grading of the SIJ (β1= 0.44, 95% CI 0.09 – 0.80, p = 0.01, Figure 2) and SPARCC score (β1= 2.64, 95% CI 0.98 – 4.31, p = 0.002). All models were adjusted for age, gender, BMI, CRP, anti-TNF treatment and symptom duration.Conclusion:This study shows and independent association of serum IgG undergalactosylation with disease activity and functional impairment in SpA patients. Moreover, UGS was significantly higher in advanced compared to early-stage axial disease and therefore may reflect the cumulative exposure to systemic inflammation.

Fc-Linked IgG N-Glycosylation in FcγR Knock-Out Mice.

Immunoglobulin G (IgG) is the most abundant immunoglobulin isotype in the blood and is involved in the pathogenesis and progression of various diseases. Glycosylation of the IgG fragment crystallizable (Fc) region is shown to vary in different physiological and pathological states. Fc N-glycan composition can alter the effector functions of IgG by modulating its affinity for ligands, such as Fcγ receptors (FcγRs). However, it is not known whether IgG glycosylation is affected by the available repertoire of FcγRs, and if the Fc-linked N-glycome can compensate for modulation of the IgG–FcγR interaction. To explore this, we examined the subclass-specific Fc IgG glycoprofiles of healthy male and female FcγR knock-out mice on C57BL/6 and BALB/c backgrounds. We observed slight changes in IgG Fc N-glycan profiles in different knock-outs; however, it seems that the strain background and sex have a stronger effect on N-glycosylation of IgG Fc regions than the FcγR repertoire.

LPS-binding IgG arrests actively motile Salmonella Typhimurium in gastrointestinal mucus.

The gastrointestinal (GI) mucosa is coated with a continuously secreted mucus layer that serves as the first line of defense against invading enteric bacteria. We have previously shown that antigen-specific immunoglobulin G (IgG) can immobilize viruses in both human airway and genital mucus secretions through multiple low-affinity bonds between the array of virion-bound IgG and mucins, thereby facilitating their rapid elimination from mucosal surfaces and preventing mucosal transmission. Nevertheless, it remains unclear whether weak IgG-mucin crosslinks could reinforce the mucus barrier against the permeation of bacteria driven by active flagella beating, or in predominantly MUC2 mucus gel. Here, we performed high-resolution multiple particle tracking to capture the real-time motion of hundreds of individual fluorescent Salmonella Typhimurium in fresh, undiluted GI mucus from Rag1-/- mice, and analyzed the motion using a hidden Markov model framework. In contrast to control IgG, the addition of anti-lipopolysaccharide IgG to GI mucus markedly reduced the progressive motility of Salmonella by lowering the swim speed and retaining individual bacteria in an undirected motion state. Effective crosslinking of Salmonella to mucins was dependent on Fc N-glycans. Our findings implicate IgG-mucin crosslinking as a broadly conserved function that reduces mucous penetration of both bacterial and viral pathogens.

Mass spectrometric analysis of core fucosylation and sequence variation in a human-camelid monoclonal antibody.

Electrospray mass spectrometry (ESI-MS) was used to measure the masses of an intact dimeric monoclonal antibody (Mab) and assess the fucosylation level. The Mab under study was EG2-hFc, a chimeric human-camelid antibody of about 80 kDa (A. Bell et al., Cancer Lett., 2010, 289(1), 81-90). It was obtained from cell culture with and without a fucosylation inhibitor, and treated with EndoS which cleaves between the two core N-acetyl glucosamine (GlcNAc) residues. It is the first time that this combined approach with a unique mass spectrometer was used to measure 146 Da differences as part of a large intact dimeric antibody. Results showed that in the dimer, both heavy chains were fucosylated on the core GlcNAc of the Fc Asn site equivalent to Asn297. In the presence of the fucosylation inhibitor, fucosylation was lost on both subunits. Following reduction, monomers were analyzed and the masses obtained corroborated the dimer results. Dimeric EG2-hFc Mab treated with PNGase F, to deglycosylate the protein, was also measured by MS for mass comparison. In spite of the success of fucosylation level measurements, the experimental masses of deglycosylated dimers and GlcNAc-Fuc bearing dimers did not correspond to masses of our sequence of reference (A. Bell et al., Cancer Lett., 2010, 289(1), 81-90; ; ), which prompted experiments to determine the protein backbone sequence. Digest mixtures from trypsin, GluC, as well as trypsin + GluC proteolysis were analyzed by matrix-assisted laser desorption/ionization (MALDI) MS and MS/MS. A few variations were found relative to the reference sequence, which are discussed in detail herein. These measurements allowed us to build a new "experimental" sequence for the EG2-hFc samples investigated in this work, although there are still ambiguities to be resolved in this new sequence. MALDI-MS/MS also confirmed the fucosylation pattern in the Fc tryptic peptide EEQYNSTYR.

An atomistic perspective on antibody-dependent cellular cytotoxicity quenching by core-fucosylation of IgG1 Fc N-glycans from enhanced sampling molecular dynamics.

The immunoglobulin type G (IgG) Fc N-glycans are known to modulate the interaction with membrane-bound Fc γ receptors (FcγRs), fine-tuning the antibody's effector function in a sequence-dependent manner. Particularly interesting in this respect are the roles of galactosylation, which levels are linked to autoimmune conditions and aging, of core fucosylation, which is known to reduce significantly the antibody-dependent cellular cytotoxicity (ADCC), and of sialylation, which also reduces antibody-dependent cellular cytotoxicity (ADCC) but only in the context of core-fucosylation. In this article, we provide an atomistic level perspective through enhanced sampling computer simulations, based on replica exchange molecular dynamics (REMD), to understand the molecular determinants linking the Fc N-glycans sequence to the observed IgG1 function. Our results indicate that the two symmetrically opposed N-glycans interact extensively through their core trimannose residues. At room temperature, the terminal galactose on the α (1-6) arm is restrained to the protein through a network of interactions that keep the arm outstretched; meanwhile, the α (1-3) arm extends toward the solvent where a terminal sialic acid remains fully accessible. We also find that the presence of core fucose interferes with the extended sialylated α (1-3) arm, altering its conformational propensity and as a consequence of steric hindrance, significantly enhancing the Fc dynamics. Furthermore, structural analysis shows that the core-fucose position within the Fc core obstructs the access of N162 glycosylated FcγRs very much like a "door-stop," potentially decreasing the IgG/FcγR binding free energy. These results provide an atomistic level-of-detail framework for the design of high potency IgG1 Fc N-glycoforms.

Hydrophobic interaction chromatography (HIC) method development and characterization of resolved drug-load variants in site-specifically conjugated pyrrolobenzodiazepine dimer-based antibody drug conjugates (PBD-ADCs)

Antibody drug conjugates (ADCs) are heterogeneous biopharmaceutical products that demand extensive characterization to ensure batch consistency, safety, and efficacy. Hydrophobic interaction chromatography (HIC) is the state-of-the-art analytical tool to monitor conjugation-related critical quality attributes (CQAs) e.g. drug-load distribution and Drug-to-Antibody Ratio (DAR). For the next generation site-specific PBD-ADCs (PBD: pyrrolobenzodiazepine dimer), denaturing RP-HPLC (reverse-phase high-performance chromatography) is the current method to determine average DAR. In this manuscript, we have utilized native HIC for the first time to understand conjugation related CQAs in PBD-ADCs. In terms of the method development, the type of stationary phase and salt, coupled with reduction of the reactive imine in the PBD drug-linker to an amine form in the sample preparation, have played a key role in achieving the best HIC resolution for the drug-load variants. The established HIC conditions resolved DAR 0, DAR 1, and two DAR 2 peaks for PBD-ADCs. Extended characterization of the DAR 2 peaks confirmed that they have retained characteristically distinct antibody Fc N-glycan distributions (Fc = Fragment crystallization region). Therefore, the results support that the HIC conditions established for PBD-ADCs is valuable in not only determining DAR values but also other important attributes including native drug-load distribution and unique DAR 2 conformations existed as a result of the N-glycan heterogeneity.

A synopsis of recent developments defining how N-glycosylation impacts immunoglobulin G structure and function.

Therapeutic monoclonal antibodies (mAbs) are the fastest growing group of drugs with 11 new antibodies or antibody-drug conjugates approved by the Food and Drug Administration in 2018. Many mAbs require effector function for efficacy, including antibody-dependent cell-mediated cytotoxicity triggered following contact of an immunoglobulin G (IgG)-coated particle with activating crystallizable fragment (Fc) γ receptors (FcγRs) expressed by leukocytes. Interactions between IgG1 and the FcγRs require post-translational modification of the Fc with an asparagine-linked carbohydrate (N-glycan). Though the structure of IgG1 Fc and the role of Fc N-glycan composition on disease were known for decades, the underlying mechanism of how the N-glycan affected FcγR binding was not defined until recently. This review will describe the current understanding of how N-glycosylation impacts the structure and function of the IgG1 Fc and describe new techniques that are poised to provide the next critical breakthroughs.