Galactosylation variations in marketed therapeutic antibodies

There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary (CHO) cells or mouse myeloma cells as the expression system. Because CHO and mouse myeloma cells are mammalian cells, rIgGs produced in these cell lines are typically N-glycosylated at the conserved asparagine (Asn) residues in the CH2 domain of the Fc, which is also the case with serum IgGs. The Fc glycans present in these rIgGs are for the most part complex biantennary oligosaccharides with heterogeneity associated with the presence or the absence of several different terminal sugars. The major Fc glycans of rIgGs contain 0 or 1 or 2 (G0, G1 and G2, respectively) terminal galactose residues as non-reducing termini and their relative proportions may vary depending on the cell culture conditions in which they were expressed. Since glycosylation is strongly associated with antibody effector functions and terminal galactosylation may affect some of those functions, a panel of commercially available therapeutic rIgGs expressed in CHO cells and mouse myeloma cells were examined for their galactosylation patterns. The results suggest that the rIgGs expressed in CHO cells are generally less galactosylated compared to the rIgGs expressed in mouse myeloma cells. Accordingly, rIgGs produced in CHO cells tend to contain higher levels of G0 glycans compared with rIgGs produced in mouse myeloma cell lines. Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies. Nevertheless, galactosylation may have an effect on the mechanisms of action of some therapeutic antibodies (e.g., effector pathways) and hence further studies to assess effects on product efficacy may be warranted for such antibodies. For antibodies that do not require effector functions for biological activity, however, setting a narrow specification range for galactose content may be unnecessary.     

Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies

ABSTRACT

Typical crystallizable fragment (Fc) glycans attached to the CH2 domain in therapeutic monoclonal antibodies (mAbs) are core-fucosylated and asialo-biantennary complex-type glycans, e.g., G2F (full galactosylation), G1aF (terminal galactosylation on the Man α1-6 arm), G1bF (terminal galactosylation on the Man α1-3 arm), and G0F (non-galactosylation). Terminal galactose (Gal) residues of Fc-glycans are known to influence effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (CDC), but the impact of the G1F isomers (G1aF and G1bF) on the effector functions has not been reported. Here, we prepared four types of glycoengineered anti-CD20 mAbs bearing homogeneous G2F, G1aF, G1bF, or G0F (G2F mAb, G1aF mAb, G1bF mAb, or G0F mAb, respectively), and evaluated their biological activities. Interestingly, G1aF mAb showed higher C1q- and FcγR-binding activities, CDC activity, and FcγR-activation property than G1bF mAb. The activities of G1aF mAb and G1bF mAb were at the same level as G2F mAb and G0F mAb, respectively. Hydrogen–deuterium exchange/mass spectrometry analysis of dynamic structures of mAbs revealed the greater involvement of the terminal Gal residue on the Man α1-6 arm in the structural stability of the CH2 domain. Considering that mAbs interact with FcγR and C1q via their hinge proximal region in the CH2 domain, the structural stabilization of the CH2 domain by the terminal Gal residue on the Man α1-6 arm of Fc-glycans may be important for the effector functions of mAbs. To our knowledge, this is the first report showing the impact of G1F isomers on the effector functions and dynamic structure of mAbs.

Abbreviations: ABC, ammonium bicarbonate solution; ACN, acetonitrile; ADCC, antibody-dependent cell-mediated cytotoxicity; C1q, complement component 1q; CDC, complement-dependent cytotoxicity; CQA, critical quality attribute; Endo, endo-β-N-acetylglucosaminidase; FA, formic acid; Fc, crystallizable fragment; FcγR, Fcγ receptors; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GST, glutathione S-transferase; HER2, human epidermal growth factor receptor 2; HDX, hydrogen–deuterium exchange; HILIC, hydrophilic interaction liquid chromatography; HLB-SPE, hydrophilic-lipophilic balance–solid-phase extraction; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; Man, mannose; MS, mass spectrometry; PBS, phosphate-buffered saline; SGP, hen egg yolk sialylglycopeptides.     

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.     

Glycan engineering reveals interrelated effects of terminal galactose and core fucose on antibody-dependent cell-mediated cytotoxicity

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been identified as one of the potentially critical effector functions underlying the clinical efficacy of some therapeutic immunoglobin G1 (IgG1) antibodies. It has been well established that higher levels of afucosylated N-linked glycan structures on the Fc region enhance the IgG binding affinity to the FcγIIIa receptor and lead to increased ADCC activity. However, whether terminal galactosylation of an IgG1 impacts its ADCC activity is less understood. Here, we used a new strategy for glycan enrichment and remodeling to study the impact of terminal galactose on ADCC activity for therapeutic IgG1s. Our results indicate that the degree of influence of terminal galactose on in vitro ADCC activity depends on the presence or absence of the core fucose, which is typically linked to the first N-acetyl glucosamine residue of an N-linked glycosylation core structure. Specifically, terminal galactose on afucosylated IgG1 mAbs enhanced ADCC activity with impact coefficients (ADCC%/Gal%) more than 20, but had minimal influence on ADCC activity on fucosylated structures with impact coefficient in the range of 0.1–0.2. Knowledge gained here can be used to guide product and process development activities for biotherapeutic antibodies that require effector function for efficacy, and also highlight the complexity in modulating the immune response through N-linked glycosylation of antibodies.     

Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.     

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.     

High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans

Glycan structures attached to the C(H)2 domain of the Fc region of immunoglobulin G (IgG) are essential for specific effector functions but their role in modulating clearance is less clear. Clearance is of obvious importance for therapeutic monoclonal antibodies (Mabs) as it directly impacts efficacy. Here, we study the impact of Fc glycan structure on the clearance of four therapeutic human IgGs (one IgG1 and three IgG2s) in humans. The therapeutic IgGs were affinity purified from serum samples from human pharmacokinetic studies, and changes to the glycan profile over time were determined by peptide mapping employing high-resolution mass spectrometry. Relative levels of high-mannose 5 (M5) glycan decreased as a function of circulation time, whereas other glycans remained constant. These results demonstrate that therapeutic IgGs containing Fc high-mannose glycans are cleared more rapidly in humans than other glycan forms. The quantitative effect of this on pharmacokinetic area under the curve was calculated and shown to be relatively minor for three of the four molecules studied, but, depending on the dosing regimen and the relative level of the high-mannose glycan, this can also have significant impact. High-mannose content of therapeutic Mabs should be considered an important product quality attribute which may affect pharmacokinetic properties of therapeutic antibodies.     

Engineering nucleotide sugar synthesis pathways for independent and simultaneous modulation of N-glycan galactosylation and fucosylation in CHO cells

Glycosylation of recombinant therapeutics like monoclonal antibodies (mAbs) is a critical quality attribute. N-glycans in mAbs are known to affect various effector functions, and thereby therapeutic use of such glycoproteins can depend on a particular glycoform profile to achieve desired efficacy. However, there are currently limited options for modulating the glycoform profile, which depend mainly on over-expression or knock-out of glycosyltransferase enzymes that can introduce or eliminate specific glycans but do not allow predictable glycoform modulation over a range of values. In this study, we demonstrate the ability to predictably modulate the glycoform profile of recombinant IgG. Using CRISPR/Cas9, we have engineered nucleotide sugar synthesis pathways in CHO cells expressing recombinant IgG for combinatorial modulation of galactosylation and fucosylation. Knocking out the enzymes UDP-galactose 4′-epimerase (Gale) and GDP-L-fucose synthase (Fx) resulted in ablation of de novo synthesis of UDP-Gal and GDP-Fuc. With Gale knock-out, the array of N-glycans on recombinantly expressed IgG is narrowed to agalactosylated glycans, mainly A2F glycan (89%). In the Gale and Fx double knock-out cell line, agalactosylated and afucosylated A2 glycan is predominant (88%). In the double knock-out cell line, galactosylation and fucosylation was entirely dependent on the salvage pathway, which allowed for modulation of UDP-Gal and GDP-Fuc synthesis and intracellular nucleotide sugar availability by controlling the availability of extracellular galactose and fucose. We demonstrate that the glycoform profile of recombinant IgG can be modulated from containing predominantly agalactosylated and afucosylated glycans to up to 42% and 96% galactosylation and fucosylation, respectively, by extracellular feeding of sugars in a dose-dependent manner. By simply varying the availability of extracellular galactose and/or fucose, galactosylation and fucosylation levels can be simultaneously and independently modulated. In addition to achieving the production of tailored glycoforms, this engineered CHO host platform can cater to the rapid synthesis of variably glycoengineered proteins for evaluation of biological activity.     

Effect of Environmental Parameters on Glycosylation of Recombinant Immunoglobulin G Produced from Recombinant CHO Cells

Site specific glycosylation of immunoglobulin G (IgG) occurs at Asn297 in the Fc region. The heterogeneous ensemble of glycoform occurs due to the degree of terminal galactosylation and sialylation, and these differences in glycosylation affect both the pharmacokinetic behavior and effector functions of the IgG, such as complementdependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). In this study, the differential glycosylation of IgG was compared and environmental physical and chemical parameters were evaluated in an attempt to promote glycosylation of recombinant antibodies, thereby creating more humanized glycoform antibodies and increasing their in vivo efficacy as therapeutic drugs. It was shown that cells at late stationary growth phase in batch cultures, cells with increased passage number, and the culture conditions of lowered temperature and pH promoted galactosylation and sialylation of antibodies. Galactose, fructose and mannose were found to elicit galactosylation and sialylation when they were used alone as a substitute of glucose. Mannose showed synergistic effects on glycosylation when used with other sugars, such as glucose and galactose. However when fructose was used with other sugars, the degree of galactosylation mechanism appeared to be decreased. These results support understandings of the glycosylation mechanisms in glycoprotein, particularly recombinant antibodies for therapeutics.     

Fc glycans terminated with N-acetylglucosamine residues increase antibody resistance to papain

Glycosylation in the CH2 domain of Fc is required for immunoglobulins G (IgGs) to exhibit immune effector functions including complement-dependent cytotoxicity (CDC) and antibody-dependent (Ab-dependent) cellular cytotoxicity (ADCC). We recently established that glycosylated Abs are more resistant to papain digestion than non-glycosylated IgGs (Biochem. Biophys. Res. Commun. 2006, 341, 797-803). To test whether specific Fc glycan structures affect Ab resistance to papain, we used in vitro glycoengineering methods to prepare homogeneous Ab glycoforms terminated with either sialic acid (G2S2), beta-galactose (G2), or N-acetylglucosamine (G0) and subjected them to papain digestions. Analyses of aliquots taken at different times during the digestions by matrix-assisted laser desorption-time-of-flight-mass spectroscopy (MALDI-TOF-MS) and high-performance liquid chromatography (HPLC) methods showed that the G0 glycoform was at least two times more resistant to papain digestion than the G2 and G2S2 glycoforms. The increased resistance of the G0 glycoform over the G2 and G2S2 glycoforms was independent of the specific Ab analyzed. A mouse/human chimeric version of Ab1, a fully human version of Ab2, and a humanized version of Ab3 exhibited a similar pattern of glycoform-dependent resistance. These data suggest that terminal sugars of Fc glycans may play important roles in Ab stability and affect resistance to proteases in addition to impacting Ab effector functions.     

Significant impact of single N-glycan residues on the biological activity of Fc-based antibody-like fragments

Recent studies have demonstrated that IgG-Fc fragments (Fcabs) can be engineered to form antigen-binding sites with antibody properties. Thus they may serve as an attractive alternative to conventional antibodies in therapeutic applications. The critical influence of Fc glycosylation on effector functions of IgGs is well documented; however, whether this applies to Fcabs is not known. Here we used human cells, wild type, and glycoengineered plants to generate four different glycoforms of H10-03-6, an Fcab with engineered HER2/neu-binding sites. Plant-derived H10-03-6 differed in the presence/absence of single oligosaccharide residues, i.e., core fucose and xylose, and terminal galactose. All of the glycoforms had similar binding to HER2/neu expressed on human tumor cells. By contrast, glycoforms that lacked core oligosaccharide modifications (i.e., core α1,3-fucose and β1,2-xylose) showed significantly enhanced binding to the Fcγ receptor IIIa, irrespective of whether plant or human expression systems were used. Consistent with this finding, plant-derived H10-03-6 glycoforms lacking core N-glycan residues mediated higher antibody-dependent cellular cytotoxicity against human tumor cells. No alteration in γ-receptor binding and antibody-dependent cellular cytotoxicity activity was observed upon decoration of N-glycans by terminal galactose. The results point to a significant impact of distinct N-glycan residues on effector functions of Fcabs. Moreover, the outcomes imply that the effector functions mediated by H10-03-6 can be optimized by altering the N-glycosylation profile. Biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy.     

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 C_H2 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 C_H2 domain is associated with the switch from pro-inflammatory to anti-inflammatory activity of the Fc.     

N‐linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG

We studied the variations in N‐linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO‐K1SV cells. The glycans detected on the Fc fragment were mainly of the core‐fucosylated complex type containing zero or one galactose and little to no sialic acid. The glycosylation was highly consistent for the same cell line when grown multiple times, indicating the robustness of the production and glycan analysis procedure. However, a twofold to threefold difference was observed in the level of galactosylation and/or non‐core‐fucosylation between the 105 different cell lines, suggesting clone‐to‐clone variation. These differences may change the Fc‐mediated effector functions by such antibodies. Large variation was also observed in the oligomannose‐5 glycan content, which, when present, may lead to undesired rapid clearance of the antibody in vivo. Statistically significant differences were noticed between the various glycan parameters for the six different antibodies, indicating that the variable domains and/or light chain isotype influence Fc glycosylation. The glycosylation altered when batch production in shaker was changed to fed‐batch production in bioreactor, but was consistent again when the process was scaled from 400 to 5,000 L. Taken together, the observed clone‐to‐clone glycosylation variation but batch‐to‐batch consistency provides a rationale for selection of optimal production cell lines for large‐scale manufacturing of biopharmaceutical human IgG. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Differences in N-glycan structures found on recombinant IgA1 and IgA2 produced in murine myeloma and CHO cell lines

The development and production of recombinant monoclonal antibodies is well established. Although most of these are IgGs, there is also great interest in producing recombinant IgAs since this isotype plays a critical role in providing immunologic protection at mucosal surfaces. The choice of expression system for production of recombinant antibodies is crucial because they are glycoproteins containing at least one N-linked carbohydrate. These glycans have been shown to contribute to the stability, pharmacokinetics and biologic function of antibodies. We have produced recombinant human IgA1 and all three allotypes of IgA2 in murine myeloma and CHO cell lines to systematically characterize and compare the N-linked glycans. Recombinant IgAs produced in murine myelomas differ significantly from IgA found in humans in that they contain the highly immunogenic Galα(1,3)Gal epitope and N-glycolylneuraminic acid residues, indicating that murine myeloma is not the optimal expression system for the production of human IgA. In contrast, IgAs produced in CHO cells contained glycans that were more similar to those found on human IgA. Expression of IgA1 and IgA2 in Lec2 and Lec8 cell lines that are defective in glycan processing resulted in a less complex pool of N-glycans. In addition, the level of sialylation of rIgAs produced in murine and CHO cells was significantly lower than that previously reported for serum IgA1. These data underscore the importance of choosing the appropriate cell line for the production of glycoproteins with therapeutic potential.     

Engineering host cell lines to reduce terminal sialylation of secreted antibodies

Covalently-linked glycans on proteins have many functional roles, some of which are still not completely understood. Antibodies have a very specific glycan modification in the Fc region that is required for mediating immune effector functions. These Fc glycans are typically highly heterogeneous in structure, and this heterogeneity is influenced by many factors, such as type of cellular host and rate of Ab secretion. Glycan heterogeneity can affect the Fc-dependent activities of antibodies. It has been shown recently that increased Fc sialylation can result in decreased binding to immobilized antigens and some Fcγ receptors, as well as decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In contrast, increased Fc sialylation enhances the anti-inflammatory activity of antibodies. To produce antibodies with increased effector functions, we developed host cell lines that would limit the degree of sialylation of recombinantly-expressed antibodies. Towards this end, the catalytic domain of the Arthrobacter ureafaciens sialidase (sialidase A) was engineered for secreted expression in mammalian cell lines. Expression of this sialidase A gene in mammalian cells resulted in secreted expression of soluble enzyme that was capable of removing sialic acid from antibodies secreted into the medium. Purified antibodies secreted from these cells were found to possess very low levels of sialylation compared with the same antibodies purified from unmodified host cells. The low sialylated antibodies exhibited similar binding affinity to soluble antigens, improved ADCC activity, and they possessed pharmacokinetic properties comparable to their more sialylated counterparts. Further, it was observed that the amount of sialidase A expressed was sufficient to thoroughly remove sialic acid from Abs made in high-producing cell lines. Thus, engineering host cells to express sialidase A enzyme can be used to produce recombinant antibodies with very low levels of sialylation.Key words: antibodies, IgGs, glycans, oligosaccharides, sialic acid, sialidase, ADCC, CDC, effector functions, cells, Fc receptors, proteases     

Changes in antigen-specific IgG1 Fc N-glycosylation upon influenza and tetanus vaccination

Antibody effector functions have been shown to be influenced by the structure of the Fc N-glycans. Here we studied the changes in plasma or serum IgG Fc N-glycosylation upon vaccination of 10 Caucasian adults and 10 African children. Serum/plasma IgG was purified by affinity chromatography prior to and at two time points after vaccination. Fc N-glycosylation profiles of individual IgG subclasses were determined for both total IgG and affinity-purified anti-vaccine IgG using a recently developed fast nanoliquid chromatography-electrospray ionization MS (LC-ESI-MS) method. While vaccination had no effect on the glycosylation of total IgG, anti-vaccine IgG showed increased levels of galactosylation and sialylation upon active immunization. Interestingly, the number of sialic acids per galactose increased during the vaccination time course, suggesting a distinct regulation of galactosylation and sialylation. In addition we observed a decrease in the level of IgG1 bisecting N-acetylglucosamine whereas no significant changes were observed for the level of fucosylation. Our data indicate that dependent on the vaccination time point the infectious agent will encounter IgGs with different glycosylation profiles, which are expected to influence the antibody effector functions relevant in immunity.     

Avidity confers FcγR binding and immune effector function to aglycosylated immunoglobulin G1

Immunoglobulin G (IgG) antibodies are an integral part of the adaptive immune response that provide a direct link between humoral and cellular components of the immune system. Insights into relationships between the structure and function of human IgGs have prompted molecular engineering efforts to enhance or eliminate specific properties, such as Fc-mediated immune effector functions. Human IgGs have an N-glycosylation site at Asn297, located in the second heavy chain constant region (CH2). The composition of the Fc glycan can have substantial impacts on Fc gamma receptor(FcγR) binding. The removal of the glycan through enzymatic deglycosylation or mutagenesis of the N-linked glycosylation site has been reported to "silence" FcγR-binding and effector functions, particularly with assays that measure monomeric binding. However, interactions between IgGs and FcγRs are not limited to monomeric interactions but can be influenced by avidity, which takes into account the sum of multimeric interactions between antigen-engaged IgGs and FcγRs. We show here that under in vitro conditions, which allowed avidity binding, aglycosylated IgGs can bind to one of the FcγRs, FcγRI, and mediate effector functions. These studies highlight how the valency of a molecular interaction (monomeric binding versus avidity binding) can influence antibody/FcγR interactions such that avidity effects can translate very low intrinsic affinities into significant functional outcomes.     

Comparison of the Fc glycosylation of fetal and maternal immunoglobulin G

Human immunoglobulin G (IgG) molecules are composed of two Fab portions and one Fc portion. The glycans attached to the Fc portions of IgG are known to modulate its biological activity as they influence interaction with both complement and various cellular Fc receptors. IgG glycosylation changes significantly with pregnancy, showing a vast increase in galactosylation and sialylation and a concomitant decrease in the incidence of bisecting GlcNAc. Maternal IgGs are actively transported to the fetus by the neonatal Fc receptor (FcRn) expressed in syncytiotrophoblasts in the placenta, providing the fetus and newborn with immunological protection. Two earlier reports described significant differences in total glycosylation between fetal and maternal IgG, suggesting a possible glycosylation-selective transport via the placenta. These results might suggest an alternative maternal transport pathway, since FcRn binding to IgG does not depend on Fc-glycosylation. These early studies were performed by releasing N-glycans from total IgG. Here, we chose for an alternative approach analyzing IgG Fc glycosylation at the glycopeptide level in an Fc-specific manner, providing glycosylation profiles for IgG1 and IgG4 as well as combined Fc glycosylation profiles of IgG2 and 3. The analysis of ten pairs of fetal and maternal IgG samples revealed largely comparable Fc glycosylation for all the analyzed subclasses. Average levels of galactosylation, sialylation, bisecting GlcNAc and fucosylation were very similar for the fetal and maternal IgGs. Our data suggest that the placental IgG transport is not Fc glycosylation selective.     

With or Without Sugar? (A)glycosylation of Therapeutic Antibodies

Antibodies and antibody-based drugs are currently the fastest-growing class of therapeutics. Over the last three decades, more than 30 therapeutic monoclonal antibodies and derivatives thereof have been approved for and successfully applied in diverse indication areas including cancer, organ transplants, autoimmune/inflammatory disorders, and cardiovascular disease. The isotype of choice for antibody therapeutics is human IgG, whose Fc region contains a ubiquitous asparagine residue (N₂₉₇) that acts as an acceptor site for N-linked glycans. The nature of these glycans can decisively influence the therapeutic performance of a recombinant antibody, and their absence or modification can lead to the loss of Fc effector functions, greater immunogenicity, and unfavorable pharmacokinetic profiles. However, recent studies have shown that aglycosylated antibodies can be genetically engineered to display novel or enhanced effector functions and that favorable pharmacokinetic properties can be preserved. Furthermore, the ability to produce aglycosylated antibodies in lower eukaryotes and bacteria offers the potential to broaden and simplify the production platforms and avoid the problem of antibody heterogeneity, which occurs when mammalian cells are used for production. In this review, we discuss the importance of Fc glycosylation focusing on the use of aglycosylated and glyco-engineered antibodies as therapeutic proteins.     

Structural study of the N-glycans of intercellular adhesion molecule-5 (telencephalin)

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized membrane glycoprotein expressed in tissues distinct from those expressing other ICAMs. Here, we determined the N-glycan structure of ICAM-5 purified from adult rat brain and compared it with that of other ICAMs. N-glycans were released by N-glycosidase F digestion and labeled with p-amino benzoic octylester (ABOE). ABOE-labeled glycans were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. The N-glycans obtained from rat brain ICAM-5 consisted of approximately 85% neutral, 10.2% sialylated-only, 2.8% sulfated-only, and 1.2% sialylated and sulfated glycans. Compared with the N-glycan structures of human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells, rat brain ICAM-5 had less highly branched glycans, sialylated glycans, and N-acetyllactosamine structures. In contrast, high-mannose-type N-glycans and Lewis X were more commonly found in rat brain ICAM-5 than in human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells. In addition, sulfated glycans contained GlcNAc 6-O-sulfate on the non-reducing terminal side. Our data will be important for the elucidation of the roles of the N-glycans expressed in neural cells, including those present on ICAM-5.