Fc Glycans Research Articles

IgG anti-HA Fc glycoform modulation is predictive of influenza vaccine efficacy (IRC10P.412)

Abstract Protective anti-viral vaccines elicit high affinity, antigen-specific neutralizing antibodies through secondary diversification mechanisms involving the selection of B cells bearing somatically hypermutated B cell antigen receptors in the germinal center. This selection process involves the interaction of the B cell receptor with antigen retained in the form of immune complexes, thus dictating that Fc-Fc receptor (FcR) interactions contribute to the selection process. FcRs are diverse in function and can transduce immunomodulatory, activating, or inhibitory signaling depending on which receptor(s) are engaged; the sum of these activities contributes to the generation of protective IgGs through mechanisms including efficient antigen presentation and dendritic cell maturation as well as directing B cell activation and selection. Immune complex-FcR interactions are governed by the precise structure of Fc domains within an immune complex, which, in turn, depends on the subclass distribution and Fc glycan composition of antigen-specific serum IgG. We performed the first detailed structural analysis of IgG Fc domains following influenza virus vaccination and found complex regulation of both subclass and Fc glycoforms. The abundance of sialylated Fc glycans on HA-specific IgG was found to predict the quality of vaccine response suggesting a mechanism by which sFc glycans modulate B cell activation thresholds, thereby driving the production of neutralizing, high affinity IgGs.

A robust method for increasing Fc glycan high mannose level of recombinant antibodies.

High mannose (HM) glycoforms on antibody Fc glycan are recognized as critical quality attributes for therapeutic antibody products. Methods to control HM have been largely empirical, and it is challenging to target a desired HM level during antibody process development. A novel and robust method to increase antibody HM glycoforms is demonstrated in this study using multiple antibodies and cell lines without adversely impacting cell culture performance, including viable cell density, viability, and protein titer. This approach utilizes mannose as a carbon source and the ratio of mannose to total hexose (glucose and mannose) in feed media determines the extent of HM glycan content of an antibody expressed in cell culture. Scale-up of this strategy from 3 mL small scale plate to bioreactor (1.5 L) is also demonstrated with comparable results. Further full glycan map analysis shows that HM increase predominantly correlates with the decrease in G0F glycan, with minimum impact on other glycoforms. Possible hypotheses for the HM glycan modulation using mannose as carbon source are also discussed. Three pathways, including GDP-mannose biosynthesis, early protein glycosylation and UDP-N-acetylglucosamine biosynthesis, might be involved and contribute to this HM modulation.

Production of α2,6-sialylated IgG1 in CHO cells

The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and β1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.

Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs

The antiinflammatory activity of intravenous immunoglobulin (IVIG) is dependent on the presence of sialic acid in the core IgG fragment crystallizable domain (Fc) glycan, resulting in increased conformational flexibility of the CH2 domain with corresponding modulation of Fc receptor (FcR) binding specificity from type I to type II receptors. Sialylated IgG Fc (sFc) increases the activation threshold of innate effector cells to immune complexes by stimulating the up-regulation of the inhibitory receptor FcγRIIB. We have found that the structural alterations induced by sialylation can be mimicked by specific amino acid modifications to the CH2 domain. An IgG Fc variant with a point mutation at position 241 (F→A) exhibits antiinflammatory activity even in the absence of sialylation. F241A and sFc protect mice from arthritis in the K/BxN-induced model and, in the T cell-mediated experimental autoimmune encephalomyelitis (EAE) mouse model, suppress disease by specifically activating regulatory T cells (Treg cells). Protection by these antiinflammatory Fcs in both antibody- and T cell-mediated autoimmune diseases required type II FcRs and the induction of IL-33. These results further clarify the mechanism of action of IVIG in both antibody- and T cell-mediated inflammatory diseases and demonstrate that Fc variants that mimic the structural alterations induced by sialylation, such as F241A, can be promising therapeutic candidates for the treatment of various autoimmune disorders.

Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding

Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG1. FcγRI binds to the Fc in a similar mode as the low-affinity FcγRII and FcγRIII receptors. In addition to many conserved contacts, FcγRI forms additional hydrogen bonds and salt bridges with the lower hinge region of Fc. Unique to the high-affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG loop, which enables a charged KHR motif to interact with proximal carbohydrate units of the Fc glycans. Both the length and the charge of the FcγRI FG loop are well conserved among mammalian species. Ala and Glu mutations of the FG loop KHR residues showed significant contributions of His-174 and Arg-175 to antibody binding, and the loss of the FG loop-glycan interaction resulted in an ∼ 20- to 30-fold decrease in FcγRI affinity to all three subclasses of IgGs. Furthermore, deglycosylation of IgG1 resulted in a 40-fold loss in FcγRI binding, demonstrating involvement of the receptor FG loop in glycan recognition. These results highlight a unique glycan recognition in FcγRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan mimics to target FcγRI for certain autoimmune diseases.

Clinical Severity of Visceral Leishmaniasis Is Associated with Changes in Immunoglobulin G Fc N-Glycosylation

ABSTRACT Visceral leishmaniasis (VL) has a high fatality rate if not treated; nevertheless, the majority of human infections with the causative agent, Leishmania infantum chagasi, are asymptomatic. Although VL patients often present with increased levels of serum immunoglobulins, the contribution of antibodies to resistance or progression to disease remains unknown. Effector and regulatory functions of antibodies rely on their interactions with type I and II Fc receptors, and these interactions are tuned by the patterns of antibody Fc N-glycosylation. In view of these facts, we applied a robust method of IgG Fc N-glycopeptide profiling of serum samples from 187 patients with VL, 177 asymptomatic individuals, 116 endemic controls (individuals residing in areas where VL is endemic) and 43 nonendemic controls (individuals living in an area where VL is not endemic). We show that, in comparison to the overall IgG Fc N-glycan profiles of asymptomatic or uninfected healthy individuals, those of patients with VL are profoundly altered. These changes correlate with levels of serum cytokines and the inflammation marker C-reactive protein. We also fitted univariate and multivariate ordinal logistic regression models to demonstrate the ability of IgG Fc N-glycosylation features and immunity regulators present in serum to predict disease severity in VL patients. Importantly, we show that Fc N-glycosylation profiles change after treatment of VL. This study introduces important concepts contributing to the understanding of antibody responses in infections with Leishmania parasites and provides new insights into the pathology of human VL.

Protected hinge in the immunoglobulin G2-A2 disulfide isoform.

Human IgG2 consists of disulfide-mediated structural isoforms, classified by the number of Fab arms disulfide-linked to the heavy chain hinge. In the IgG2-B isoform, both Fab arms are linked to the hinge region, and in IgG2-A, neither Fab arm are linked to the hinge. IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide-linked to the hinge. Within each of these isoform types are subtypes, with subtle disulfide-linkage differences. Here we explored the structural basis for the A1 and A2 isoform subtypes. Whereas A1 isoform converts into the A/B and B isoforms under mild redox conditions, A2 does not. Characterization of the disulfide connectivities of A2 isoform revealed a similar structure to A1 isoform, with parallel inter heavy chain disulfide linkages in the hinge region. However, the hinge disulfides in A2 isoform were resistant to reduction under conditions where A1 isoform hinge disulfides became reduced and they required thermal treatment (>55 °C) to obtain thiol-dependent disulfide reduction. Structural analysis of the hinge region indicated that the protected disulfides were restricted to cysteines 219 and 220 of the upper hinge. Disruption of the upper hinge through insertion mutagenesis eliminated A2 isoform behavior. (1)H NMR studies showed that the A1 isoform Fc glycan was more dynamic than that on A2 isoform and showed some other conformational differences. Results point to an IgG2-A2 upper hinge region that is more akin to the interior of a globular protein than the flexible hinge region expected on an IgG.

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.

Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans was found to be limited by UDP-Gal biosynthesis, which was observed to be both cell line and cultivation condition-dependent. Extracellular glucose and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality.

Structural Characterization of Anti-Inflammatory Immunoglobulin G Fc Proteins

Immunoglobulin G (IgG) is a central mediator of host defense due to its ability to recognize and eliminate pathogens. The recognition and effector responses are encoded on distinct regions of IgGs. The diversity of the antigen recognition Fab domains accounts for IgG's ability to bind with high specificity to essentially any antigen. Recent studies have indicated that the Fc effector domain also displays considerable heterogeneity, accounting for its complex effector functions of inflammation, modulation, and immune suppression. Therapeutic anti-tumor antibodies, for example, require the pro-inflammatory properties of the IgG Fc to eliminate tumor cells, while the anti-inflammatory activity of intravenous IgG requires specific Fc glycans for activity. In particular, the anti-inflammatory activity of intravenous IgG is ascribed to a small population of IgGs in which the Asn297-linked complex N-glycans attached to each Fc CH2 domain include terminal α2,6-linked sialic acids. We used chemoenzymatic glycoengineering to prepare fully disialylated IgG Fc and solved its crystal structure. Comparison of the structures of asialylated Fc, sialylated Fc, and F241A Fc, a mutant that displays increased glycan sialylation, suggests that increased conformational flexibility of the CH2 domain is associated with the switch from pro-inflammatory to anti-inflammatory activity of the Fc.

Quilizumab is an Afucosylated Humanized Anti-M1 Prime Therapeutic Antibody

Depletion of IgE-switched B cells may be an efficacious therapy for the treatment of allergic asthma. A humanized monoclonal IgG1 antibody against the M1 prime segment of human membrane IgE on IgE-switched B cells can reduce serum IgE by depleting IgE-producing cells. Here we report that removal of the core fucose in the Fc glycan of this anti-M1 prime antibody increases its binding affinity for human FcγRIII receptors compared to the wildtype antibody. The enhancement in binding to FcγRIII results in increased antibody potency for the in vitro depletion of IgE-positive cells in antibody-dependent cell-mediated cytotoxicity assays and for the in vivo reduction of serum IgE in mice reconstituted with human immune cells. This afucosylated anti-M1 prime antibody is known as quilizumab and is in clinical development for the treatment of allergic asthma.

Blood Plasma IgG Fc Glycans are Significantly Altered in Alzheimer's Disease and Progressive Mild Cognitive Impairment

Blood-based anti-amyloid-β (Aβ) immunoglobulins (IgGs) and peripheral inflammation are factors correlating with development of Alzheimer's disease (AD). IgG functionality can drastically change from anti- to pro-inflammatory via alterations in the IgG-Fc N-glycan structure. Herein, we tested if IgG-Fc glycosylation in plasma is indeed altered during the development of AD. Samples from age-matched subjects of 23 controls, 58 patients with stable mild cognitive impairment (SMCI), 34 patients with progressive (P)MCI, and 31 patients with AD were investigated. Label-free shotgun proteomics was applied without glycoprotein enrichment. Glycans on peptides EEQYNSTYR (IgG1) and EEQFNSTFR (IgG2) were quantified, and their abundances were normalized to total IgGn glycoform abundance. Univariate and multivariate statistics were employed to investigate the correlations between the patients groups and the abundances of the IgG glycoforms as well as those of inflammatory mediating proteins. Significant differences (p ≤ 0.05) were found, with a lower abundance of complex galactosylated and sialylated forms in AD. For females, a decline in glycoform complexity correlated with disease progress but an inverse change was found in males prior to the onset of AD. Principal component analysis (PCA; Males: R(2)X(cum) = 0.65, Q(2)(cum) = 0.34; Females: R(2)X(cum) = 0.62, Q(2)(cum) = 0.36), confirmed the gender similarities (for controls, SMCI and AD) as well as differences (for PMCI), and showed a close correlation between pro-inflammatory protein markers, AD, female PMCI, and truncated IgG-Fc glycans. The differences observed between genders prior to the onset of AD may indicate a lower ability in females to suppress peripheral inflammation, which may lead to exacerbated disease progression.

High-Throughput Biophysical Analysis and Data Visualization of Conformational Stability of an IgG1 Monoclonal Antibody After Deglycosylation

The structural integrity and conformational stability of an IgG1 monoclonal antibody (mAb), after partial or complete enzymatic removal of the N-linked Fc glycan, were compared with the untreated mAb over a wide range of temperature (10°C-90°C) and solution pH (3-8) using circular dichroism, fluorescence spectroscopy, and static light scattering combined with data visualization employing empirical phase diagrams. Subtle-to-larger stability differences between the different glycoforms were observed. Improved detection of physical stability differences was then demonstrated over narrower pH range (4.0-6.0) using smaller temperature increments, especially when combined with an alternative data visualization method (radar plots). Differential scanning calorimetry and differential scanning fluorimetry were then utilized and also showed an improved ability to detect differences in the physical stability of a mAb glycoform. On the basis of these results, a two-step methodology was used in which conformational stability of a mAb glycoform is first screened with a wide variety of instruments and environmental stresses, followed by a second evaluation with optimally sensitive experimental conditions, analytical techniques, and data visualization methods. With this approach, a high-throughput biophysical analysis to assess relatively subtle conformational stability differences in protein glycoforms is demonstrated.

HIV-Specific Antibody Immunity Mediated Through NK Cells and Monocytes

The partial success of the RV144 trial re-energized the field of HIV vaccine research, which had stalled after vaccines based on neutralizing antibody and cytotoxic T cells had failed to induce protection. A large post-vaccine research effort has focused attention on the role of non-neutralizing antibodies in the protection afforded by the RV144 vaccine. These binding antibodies can initiate immune responses such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) and combine elements of the adaptive and innate immune system in the form of antibodies and effector cells (including NK cells, monocytes and granulocytes). A complex interplay exists between the variable portion of the binding antibody and its HIV antigen target on one hand and the constant region of the antibody and the Fcγ-receptor of the effector cell on the other hand. Technical advances have revolutionized the abilities of scientist to detect the targets of non-neutralizing antibodies, including both envelope and non-envelope epitopes, and their role in forcing escape. Our understanding of the antibody characteristics (including IgG subclasses and Fc glycan profile) is providing valuable insights into their optimal structure and function. We expand on critical research on ADCC effector cells, particularly education of NK cells. We introduce the concept of HIV antibodydependent trogocytosis by monocytes as a potentially important aspect of HIV immunity. In summary, this review highlights recent advances in HIV-specific antibody immunity mediated through NK cells and monocytes.

The carbohydrate switch between pathogenic and immunosuppressive antigen‐specific antibodies

IgG antibodies have one conserved N-glycosylation site at Asn 297 in each of their constant heavy chain regions. These Fc glycans influence the overall structure and pro- or anti-inflammatory effector functions of IgG antibodies. The biantennary core glycan structure, consisting of four N-acetyl-glucosamine (GlcNAc) and three mannose residues, can be further decorated with fucose, a bisecting GlcNAc and terminal galactose or galactose plus sialic acid. Non-galactosylated (agalactosylated; G0) IgG antibodies have long been associated with pro-inflammatory effector functions in autoimmune patients with rheumatoid arthritis (RA). In contrast, it has been shown that sialylated IgGs are responsible for anti-inflammatory effects of intravenous immunoglobulin (IVIG; purified IgG from pooled human plasma), which is administered at high doses (2g/kg) for the systemic treatment of autoimmune patients. It has become increasingly evident that pro-inflammatory immune responses, such as autoimmune reactions, primarily induce antigen-specific G0 IgGs, whereas tolerance induces immunosuppressive galactosylated and sialylated IgGs. Under physiological conditions, differentially glycosylated IgGs mediate their pro- or anti-inflammatory effector functions obviously as immune complexes (IC) in an antigen-specific manner. Therefore, antigen-specific galactosylated and sialylated IgGs may be a promising therapeutic tool for re-establishing tolerance against defined (self-) antigens in autoimmune or allergic patients. Here, we summarize these findings and outline our viewpoint on the development and function of differentially glycosylated antigen-specific IgG antibodies.

Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.

Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein–carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan–protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man5GlcNAc2) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan–protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein–carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.

Antibody Glycosylation and Inflammation

IgG antibodies are the basis of some of the most effective therapeutics developed over the last 20 years. These antibodies are highly specific, have long serum-half lives, and can be produced relatively routinely, making them ideal drugs for immunotherapy. The degree of regulation on IgG antibody effector functions by the composition of the single, N-linked glycan attached to the Fc is increasingly appreciated. IgG antibodies with identical protein sequences can gain a 50-fold potency, in terms of initiating antibody-dependent cellular cytotoxicity (ADCC) by removal of the single fucose residue from the Fc glycan. Conversely, the addition of sialic acid to the terminus of the Fc glycan converts IgG antibodies into anti-inflammatory mediators, capable of suppressing autoantibody driven inflammation. This review will discuss the contribution of the Fc glycan to IgG antibody effector functions, the regulation of the antibody glycosylation in vivo, implications for the rational design of IgG antibody-based therapeutics, and touch upon the contribution of glycosylation to other immunoglobulin isotypes.

Chemical and structural analysis of an antibody folding intermediate trapped during glycan biosynthesis.

Human IgG Fc glycosylation modulates immunological effector functions such as antibody-dependent cellular cytotoxicity and phagocytosis. Engineering of Fc glycans therefore enables fine-tuning of the therapeutic properties of monoclonal antibodies. The N-linked glycans of Fc are typically complex-type, forming a network of noncovalent interactions along the protein surface of the Cγ2 domain. Here, we manipulate the mammalian glycan-processing pathway to trap IgG1 Fc at sequential stages of maturation, from oligomannose- to hybrid- to complex-type glycans, and show that the Fc is structurally stabilized following the transition of glycans from their hybrid- to complex-type state. X-ray crystallographic analysis of this hybrid-type intermediate reveals that N-linked glycans undergo conformational changes upon maturation, including a flip within the trimannosyl core. Our crystal structure of this intermediate reveals a molecular basis for antibody biogenesis and provides a template for the structure-guided engineering of the protein–glycan interface of therapeutic antibodies.

Lanthanide binding and IgG affinity construct: Potential applications in solution NMR, MRI, and luminescence microscopy

Paramagnetic lanthanide ions when bound to proteins offer great potential for structural investigations that utilize solution nuclear magnetic resonance spectroscopy, magnetic resonance imaging, or optical microscopy. However, many proteins do not have native metal ion binding sites and engineering a chimeric protein to bind an ion while retaining affinity for a protein of interest represents a significant challenge. Here we report the characterization of an immunoglobulin G-binding protein redesigned to include a lanthanide binding motif in place of a loop between two helices (Z-L2LBT). It was shown to bind Tb³⁺ with 130 nM affinity. Ions such as Dy³⁺, Yb³⁺, and Ce³⁺ produce paramagnetic effects on NMR spectra and the utility of these effects is illustrated by their use in determining a structural model of the metal-complexed Z-L2LBT protein and a preliminary characterization of the dynamic distribution of IgG Fc glycan positions. Furthermore, this designed protein is demonstrated to be a novel IgG-binding reagent for magnetic resonance imaging (Z-L2LBT:Gd³⁺ complex) and luminescence microscopy (Z-L2LBT: Tb³⁺ complex).

Chemoenzymatic Glycoengineering of Intact IgG Antibodies for Gain of Functions

The fine structures of Fc N-glycans can modulate the effector functions of IgG antibodies. It has been demonstrated that lack of the core fucose on the Fc N-glycans leads to drastic enhancement of antibody-dependent cellular cytotoxicity (ADCC), while terminal α2,6-sialylation of Fc glycan plays a critical role for the anti-inflammatory activity of human intravenous immunoglobulin (IVIG). We describe in this paper a highly efficient chemoenzymatic method for site-selective Fc glycoengineering of intact monoclonal antibody and IVIG. Two new glycosynthase mutants (EndoS-D233A and D233Q) were generated by site-directed mutagenesis of EndoS (an endoglycosidase from Streptococcus pyogenes ) and were found to be capable of efficiently transferring predefined N-glycans from corresponding glycan oxazolines to the Fc-deglycosylated intact IgGs without product hydrolysis. As a model study, rituximab (a therapeutic monoclonal antibody) was successfully transformed from mixtures of G0F, G1F, and G2F glycoforms to well-defined homogeneous glycoforms, including a fully sialylated (S2G2F) glycoform that may gain anti-inflammatory activity, a nonfucosylated G2 glycoform that showed significantly enhanced FcγIIIa receptor-binding activity, and an azido-tagged glycoform that can be further transformed into other glycoforms. We also found that EndoS could selectively remove the Fc N-glycans in the presence of FAB glycosylation. This finding, coupled with the remarkable transglycosylation activity of the EndoS glycosynthase mutants, permitted a highly selective glycoengineering of the IVIG's Fc glycans into a fully sialylated Fc glycoform, which may possess significantly enhanced anti-inflammatory activity. The glycoengineering approach described here provides a general platform to modulate the effector functions of IgG antibodies, enabling the optimization of therapeutic efficacy and gain of new functions of monoclonal antibodies and IVIG.

Increased serum clearance of oligomannose species present on a human IgG1 molecule

The role of Fc glycans on clearance of IgG molecule has been examined by various groups in experiments where specific glycans have been enriched or the entire spectrum of glycans was studied after administration in pre-clinical or clinical pharmacokinetic (PK) studies. The overall conclusions from these studies are inconsistent, which may result from differences in antibody structure or experimental design. In the present study a well-characterized recombinant monoclonal IgG1 molecule (mAb-1) was analyzed from serum samples obtained from a human PK study. mAb-1 was recovered from serum using its ligand cross-linked to Sepharose beads. The overall purity and recovery of all isoforms were carefully evaluated using a variety of methods. Glycans were then enzymatically cleaved, labeled using 2-aminobenzamide and analyzed by normal phase high performance liquid chromatography. The assays for recovering mAb-1 from serum and subsequent glycan analysis were rigorously qualified at a lower limit of quantitation of 15 μg/mL, thus permitting analysis to day 14 of the clinical PK study. Eight glycans were monitored and classified into two groups: (1) the oligomannose type structures (M5, M6 and M7) and (2) fucosylated biantennary oligosaccharides (FBO) structures (NGA2F, NA1F, NA2F, NA1F-GlcNAc and NGA2F-GlcNAc). We observed that the oligomannose species were cleared at a much faster rate (40%) than FBOs and conclude that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute. These observations were only possible through the application of rigorous analytical methods that we believe will need to be employed when comparing innovator and biosimilar molecules.