Available online at Terminal sugars of Fc glycans influence antibody effector functions of IgGsT Shantha RajuIgG molecules contain glycans in the CH2domain of the Fc fragment(N-glycosylation)which are highly heterogeneous, because of the presence of different terminal sugars.The heterogeneity of Fc glycans varies with species and expression system.Fc glycans influence the binding of IgG to Fc receptors and C1q,and are therefore important for IgG effector functions. Specifically,terminal sugars such as sialic acids,core fucose, bisecting N-acetylglucosamine,and mannose residues affect the binding of IgG to the Fc g RIIIa receptor and therebyinfluence ADCC activity.By contrast,terminal galactose residues affect antibody binding to C1q and thereby modulate CDC activity.Structural studies indicate that the presence or absence of specific terminal sugars may affect hydrophilic and hydrophobic interactions between sugar residues and amino acid residues in the Fc fragment,which in turn may impact antibody effector functions.AddressesDiscovery Research,Centocor R&D Inc.,145King of Prussia Road, Radnor,PA19087,USACorresponding author:Raju,T Shantha(traju@)Current Opinion in Immunology2008,20:471–478This review comes from a themed issue onAntibody TherapeuticsEdited by Paul Parren and Jan van de WinkelAvailable online17th July20080952-7915/$–see front matter#2008Elsevier Ltd.All rights reserved.DOI10.1016/j.coi.2008.06.007IntroductionImmunoglobulins(Igs)are soluble serum glycoproteins involved in protecting vertebrates against foreign sub-stances[1,2].IgG molecules consist of two Fab and one Fc fragment,which are linked through aflexible hinge region[1–3].The Fc fragment is involved in defining antibody effector functions and pharmacokinetic proper-ties by its interaction with Fc receptors,C1q,and FcRn [2,3].The Fab fragment of IgG seems relatively resistant to proteases,whereas the Fc and hinge regions are more sensitive to proteolysis[4 ,5 ,6 ].IgG molecules are N-glycosylated in the CH2domain of the Fc fragment and about30%of circulating human IgG is also glycosylated in the Fab region[3,7].In human IgG, the majority of the Fc glycans are complex biantennary structures with a high degree of heterogeneity,on account of the presence or absence of different terminal sugars [2,3,7].Fc glycosylation is required for the induction of antibody-mediated effector functions including ADCC and CDC[3,8].This review discusses the structural heterogeneity of Fc glycans and their role in determining antibody effector functions.Structural heterogeneity of Fc glycansThe N-glycans present in the Fc portion of IgG mol-ecules contain a common core region of two N-acetyl-glucosamine residues(GlcNAc)linked to asparagine 297(Asn297)via an amide bond and three mannose (Man)residues(Figure1)[2,3].This core structure is present in high mannose,hybrid or complex structures and may contain additional terminal-sugar residues such as Man,GlcNAc,galactose(Gal),core fucose(Fuc), bisecting GlcNAc,and sialic acid(Sia or NANA for N-acetylneuraminic acid)residues[2,3].The Fc glycans of human IgG are mainly of a core fucosylated complex biantennary type with heterogeneity because of sialyla-tion and galactosylation(Figure1).In addition,there are minor amounts of nonfucosylated glycans with or without bisecting GlcNAc residue in human IgG.Gly-cans found in the Fc of IgG molecules from other vertebrates are also mostly complex biantennary struc-tures with heterogeneity similar to human IgGs[9,11]. Additionally,small amounts of high mannose and hybrid structures(Man5structures with GlcNAc,Gal,and NANA residues)may also occur[9–11].This hetero-geneity in terminal sugars has a functional consequence as it influences binding of IgG molecules to Fc receptors and C1q and thereby impacts antibody effector func-tions[12].The described microheterogeneity in glycan structure varies between species and is also dependent on age, gender,and disease status[13].In humans,for example, galactosylation of IgGs decreases with age[13,14],and patients with autoimmune diseases such as rheumatoid arthritis contain less galactosylation than healthy individ-uals[13–15].Terminal Gal residues affect CDC activity of IgG moleculesUnlike serum glycoproteins that contain surface-exposed glycans that are60–95%sialylated,only about10%of Fc glycans of human serum IgG are sialylated and<5%of Fc glycans of recombinant IgGs(rIgGs)produced in Chi-nese hamster ovary(CHO)cells are sialylated[16].As a result,Fc glycans may contain0,1,or2terminal Gal Current Opinion in Immunology2008,20:471–478residues (G0,G1,or G2)in their antennae (Figure 1).The terminal Gal content of IgG affects CDC,but not ADCC activity [17].For Rituxan,for example,an increase in terminal Gal content can increase CDC activity as a result of increased antibody binding to C1q (Figure 2a,b).However,ADCC activity is not affected by terminal Gal content as exemplified for Herceptin (Figure 2c).This is because terminal Gal residues do not affect antibody binding to the Fc g RIIIa receptor [17].In addition,terminal Gal residues do not appear to affect the binding of antibodies to their cognate antigen (Figure 2d).Effect of terminal GlcNAc residues on IgG functionsGlycoproteins containing terminal GlcNAc residues have been shown to bind to the mannose receptor and therebyexhibit reduced serum half-life [18 ].However,many rIgGs containing significant amounts of terminal GlcNAc residues in the form of G0and G1structures show considerably longer serum half-lives compared to glyco-proteins containing such terminal GlcNAc residues [2,3,18 ].Therefore,terminal GlcNAc residues of Fc glycans may not affect pharmacokinetic properties of IgG.IgG molecules with terminal GlcNAc residues have been shown to bind to the serum protein mannose binding protein and activate the alternative complement cascade in vitro [19].Furthermore,an increase in terminal GlcNAc content (and consequently a reduced Gal content)results in decreased binding of antibody to C1q and reduced CDC activity [17].Absence of core fucose results in enhanced ADCC activity of IgGFc glycans contain a core Fuc residue in a 1,6-position linked to the core GlcNAc residue (Figure 1)[10].Bio-synthesis of core-fucosylated glycans is the result of a transfer of a Fuc residue from GDP-Fuc-mediated by a 1,6-fucosyltransferase in the trans-Golgi [20].The absence of core Fuc residues in the Fc glycans substan-tially increases the ADCC activity of IgG as nonfucosy-lated antibodies bind to the Fc g RIIIa receptor with significantly increased affinity [21 ,22].However,>80%of the Fc glycans in serum IgG and >90%of rIgGs produced in normal CHO cells are fucosylated [9].To improve Fc g RIIIa binding and ADCC,several strategies have been developed to reduce fucosylation of rIgGs [23].These strategies include the development of production cell lines that either completely lack or have reduced levels of expression of a 1,6-fucosyltransferase [23,24].Alternative strategies to reduce fucosylation include silencing the a 1,6-fucosyltransferase gene using RNAi methods [24]or overexpression of GnTIII,an enzyme that adds bisecting GlcNAc residues to rIgG [25,26].The addition of bisecting GlcNAc has been shown to result in a reduction of core fucose content [27–29].Improved binding affinity of low fucosylated or nonfu-cosylated IgG to Fc g RIIIa is independent of IgG subclass [30 ].A number of rIgGs containing no or significantly reduced core fucose are currently in clinical trials for development as human therapeutics [22–24,30 ,31].Terminal sialylation affects IgG functionsThe N -glycans of serum glycoproteins are often termi-nated with sialic acid residues [32].Increased terminal sialylation can increase the serum half-life of many glycoproteins [2,3].Fc glycans of IgGs contain variable amounts of sialylated glycans [10].Increased sialylation of Fc glycans results in decreased ADCC activity of rIgGs as terminal sialylation negatively affects antibody binding to the Fc g RIIIa receptor [33 ,34 ].In addition,increased sialylation may result in decreased binding to472Antibody TherapeuticsFigure1Structure of major N -glycans found in human IgG.The majority of glycans found in the Fc of human IgG are shown.These glycans are complex biantennary structures with heterogeneity because of terminal galactose and/or sialic acid residues (NANA,N -acetylneuraminic acid;Gal,galactose;GlcNAc,N -acetylglucosamine;Man,mannose;Fuc,fucose).About 10%of these glycans may not contain core Fuc residue (not shown)[G0:no Gal;G1:one Gal on either a 1,6-branch or a 1,3-branch;G2:two Gal;G2S1:two Gal and one NANA on either a 1,6-branch or a 1,3-branch;G2S2:two Gal and two NANA].Current Opinion in Immunology 2008,20:471–478certain immobilized or cell surface expressed antigens [33 ].Fc sialylation with a 2,6-linked sialic acid residues has been suggested to act as a switch for the anti-inflammatory activity observed for human IVIg [33 ,34 ,35 ].Accordingly,Fc sialylation has both positive and negative effects on antibody functions [33 ,34 ,35 ].Effect of high mannose structures on ADCC activity of IgGsHuman IgG molecules may contain small amounts of Man5GlcNAc2structure [9–11].Other high mannose type glycans such as Man6GlcNAc2,Man7GlcNAc2,Man8GlcNAc2,Man9GlcNAc2,and Glc3Man9GlcNAc2structures that are present in chicken IgGs were either not present or only at very low levels in human IgG [9].In rIgG,the high mannose content varies with cell lines and batches [5 ,6 ,36].Glycoproteins containing high mannose structures bind the mannose receptor and therefore exhibit a reduced serum half-life [2,18 ,36].Similarly,it has been shown that IgG-containing high mannose structures with term-inal Man residues are also cleared from the serum at a faster rate than the IgG-containing complex glycans [37 ].In contrast to this,Millward et al .[38 ]performed a pharmacokinetic study in mice and found no significant difference in the serum half-life between an IgG prep-aration which was enriched for complex-type glycans and a preparation enriched for high mannose type glycans.Therefore,it appears that the effect of high mannose type Fc glycans on serum half-life of IgGs may vary from antibody to antibody,with the mechanism not yet resolved [37 ,38 ].In addition to their conflicting influence on serum half-life,high mannose type Fc glycans may also influence antibodyTerminal sugars of Fc glycans Raju 473Figure2Increase in terminal Gal content increases CDC activity (a)and C1q binding (b)of rIgGs but does not affect ADCC activity (c)and antigen binding (d).G2,G0,and/or Gno (no glycans)glycoforms of Rituxan and/or Herceptin were prepared by in vitro glycosylation methods.These glycoforms along with control antibody samples (untreated)were subjected to CDC (Rituxan glycoforms),C1q binding (Rituxan glycoforms),ADCC (Herceptin glycoforms),and antigen binding to HER2-ECD (Herceptin glycoforms).Rituxan is a chimeric antibody against CD20and elicits CDC activity but shows very little ADCC activity.Herceptin is a humanized antibody against HER2-neu antigen and elicits ADCC activity but no CDC activity.Current Opinion in Immunology 2008,20:471–478effector functions.Zhou et al .[39 ]showed that high mannose containing rIgGs had improved ADCC activity.These high mannose containing antibodies bind to the Fc g RIIIa receptor with increased affinity,similarly to antibodies-lacking core fucose residues.It should be noted,however,that such high mannose type N -glycan structures are not fucosylated in the core region [9].It is not clear whether the presence of high mannose structures with terminal Man residue or the absence of core Fuc residues are responsible for the observed improvement in Fc g RIIIa binding and ADCC.In the same study it was also shown that the IgG molecules containing high mannose structures bind C1q with reduced affinity and had decreased CDC activity [39 ].On the basis of these observations it is suggested that high mannose type Fc glycans may impact IgGs by:first,affecting the serum half-life of some IgGs because of their affinity to bind to the mannose receptor in the liver;second,increasing Fc g RIIIa binding and ADCC,possibly owing to the absence of core Fuc on these glycans;and third,decreasing C1q binding and CDC,possibly by the absence of terminal Gal residues.Hydrophobic and hydrophilic interactions between terminal-sugar residues and amino acid residues in the FcThe impact of terminal-sugar residues on antibody effec-tor functions may be because of hydrophobic and hydro-philic interactions between amino acid and sugar residues,as observed in structural analyses of human IgG Fc fragments using X-ray crystallography,NMR spectroscopy,and differential scanning microcalorimetry [40,41 ,42 ,43,44 ].Fc fragments of G2,G0,and G-2glycoforms of IgG1(see Figure 3for glycan structures)were crystallized in complex with miniZ peptide,which represents an engineered 2-helix version (Z34C)of the B domain of protein A [45].The crystal structures of the three glyco-forms were resolved at 1.65,2.3,and 2.45A˚(Figure 3)[41 ,42 ,43].All the three Fc glycoforms contain two identical core fucosylated complex biantennary oligo-saccharides linked to Asn297of each heavy chain.In the Fc fragment,the interactions between sugar and amino acid residues,as well as among sugar residues,are both hydrophilic and hydrophobic in nature as discussed below.Interactions between sugar residues and amino acid residuesIn the crystal structures,sugar residues in the a 1,6-branch of G2,G0,and G-2glycoforms have low temperature factors and are well resolved,whereas sugar residues of the a 1!3-branch are poorly resolved and have high temperature factors.In the G2glycoform the Gal and GlcNAc residues in the a 1,3-branch appear to occur in multiple conformations.The Fuc attached to the core GlcNAc also has a high temperature factor and may alsoadopt multiple conformations.A total of six hydrogen bonds (H-bonds)between sugar residues and amino acid residues were observed in the G2glycoform (Figure 4a),whereas there were only two H-bonds in the G0and G-2glycoforms (Figure 4b,c).Owing to the absence of the Gal residue in the a 1,6-branch,the stretch from residue 244to residue 247is slightly destabilized in the G0glycoform.A molecule of water occupies the space near where the Gal resides and bridges the carbonyl oxygen of Pro244to Thr260,providing some stabilizing energy.In the absence of Gal and GlcNAc residues,the G-2glycoform has similar structural properties to the G0glycoform.The truncation in the carbohydrate structure appears to affect the tertiary structure of the Fc fragment as demonstrated by a loss of resolution in the X-ray structure (see Figure 3)and a uniform increase in B-factors of sugar residues [40,41 ].Comparative studies of G2and G0glycoforms of Fc fragments using differential scanning microcalorimetry suggest a lower enthalpy for the G0relative to the G2glycoform,possibly reflecting the loss of hydrophilic interactions (H-bonds)between sugar residues and amino acid residues in the two CH 2domains [42 ,43,44 ,45–47].These molecular interactions between galactose and amino acid residues may play a role in increasing the binding affinity of galactosylated antibodies to C1q.Although no solution or crystal structural data are avail-able for sialylated antibodies,it is tempting to speculate that the molecular interactions between sialic acid residues and amino acid residues are the reasons for474Antibody TherapeuticsFigure3X-ray crystal data and structure of G2,G0,and G-2Fc glycoforms.The G2,G0,and G-2glycoforms of human IgG Fc were prepared by using in vitro glycosylation methods [36]and treated with carboxypeptidase B to remove C-terminal Lys heterogeneity.These glycoforms werecocrystallized as complexes with miniZ peptide,an engineered 2-helix version (Z34C)of the B domain of protein A [45].In the crystal structure each Fc glycoform formed complexes with two-miniZ peptide molecules.Current Opinion in Immunology 2008,20:471–478decreased resistance of sialylated antibodies to proteases [6 ].Sialic acid is negatively charged and bulkier than most monosaccharides [48].Accordingly,sialic acid resi-dues can form ionic,hydrophobic,and hydrophilic inter-actions with amino acid residues along with imposing spatial constraints because of its bulkiness [49].The charge and bulkiness of sialic acid residues might affect Fc conformation and antibody effector function.Carbohydrate–carbohydrate interactionsThe sugar residues from both Fc monomers have very little contact with each other except for a carbohydrate–carbohydrate interaction between the two Man residues (Figure 5)in the a 1,3-branch of the two oligosaccharides.This carbohydrate–carbohydrate interaction consists of hydrophobic interactions between the two Man residues in the a 1,3-branch.The a 1,3-branch moves by as much as0.83A˚when comparing the GlcNAc residue in the G2and G0glycoforms (Figure 4a,b).The data from an NMR study support these X-ray crystallography data [43,46].Both X-ray and NMR studies show a similar hydrophobic interaction between the two core Man residues in the a 1,3-branch of two glycan chains (Figure 5)[40,41 ,42 ,43,44 ,45,46];this hydrophobic interaction seems necessary to maintain a proper Fc conformation for IgG molecules to exhibit their ADCC and CDC activities.In the absence of this hydrophobic interaction,the antibody molecule may bulge a little in the CH 2domain of the Fc,which in turn increases the hydrodyn-amic volume (as observed by measuring sedimentation coefficients of glycosylated and deglycosylated Fc frag-ments);thus altering the conformation.This structural perturbation may be responsible for the loss of ADCC and CDC activity along with the increased sensitivity of aglycosylated (or deglycosylated)antibodies to proteases [4 ,5 ,6 ].Along with the X-ray and NMR analyses,studies using differential scanning microcalorimetry revealed significant differences in the stability of both CH 2and CH 3domains between the glycosylated and aglycosylated forms [42 ].The free energy of stabilization of the aglycosylated CH 2domain decreased,suggesting that deglycosylation makes it less structured,consistent with increased susceptibility to proteases [5 ].Structural studies of fucosylated and nonfucosylated Fc fragments reveal that the overall conformations of these glycoforms are similar,except for the hydration modeTerminal sugars of Fc glycans Raju 475Figure4Hydrophobic and hydrophilic interactions between sugar residues and amino acid residues determined in the crystal structures of G2(a),G0(b),and G-2(c)glycoforms.Interactions between sugar residues and amino acid residues in the CH 2domain of Fc fragment are shown.A total of six H-bonds between sugar residues and amino acid residues (shown in dotted lines)were observed in G2glycoform,whereas there were only two H-bonds observed for G0and G-2glycoforms.In G2glycoform the sugar residues in the a 1,6-branch also form hydrophobic interactions with amino acid residues.The flexibility of sugar residues increases (B-factors)as the carbohydrate content decreases from G2to G0to G-2glycoforms.The terminal Gal in the a 1,6-branch is highly rigid whereas the terminal Gal in the a 1,3-branch is highly flexible.The core Fucresidue is also highly flexible,as it forms no hydrophobic and hydrophilic interactions with amino acid residues or other sugar residues in the Fc.Current Opinion in Immunology 2008,20:471–478around Tyr296,as well as the observed differences in electron density maps around Asp280and Asn297resi-dues [50 ].These minor differences in the Fc confor-mation between fucosylated and nonfucosylated antibodies may not completely explain the increased affinity of nonfucosylated antibody for the Fc g RIIIa re-ceptor.An explanation may come from recent studies on Fc g RIIIa,whose glycosylation at Asn162impacts anti-body binding [51 ].The high affinity between nonfu-cosylated antibodies and Fc g RIII receptors appears to result from productive interactions between receptor carbohydrate attached at Asn162and regions of the Fc that are only accessible in the absence of a core Fuc residue [50 ,51 ].Since the core Fuc residue does not make any hydrophobic and hydrophilic interactions and is highly flexible,its presence appears to sterically hinder Fc receptor binding.Crystal and solution structural studies show that minor variations on account of the heterogeneity of terminal sugars of glycans affect antibody conformation in the Fc.These changes in Fc conformation influence antibody functions and stability.Since heterogeneity varies across species,cell lines,and batches,it is necessary to carefully monitor variations in glycosylation during manufacturing [33 ,34 ,52].ConclusionsThe presence or absence of various terminal sugars of Fc glycans increases microheterogeneity,which affects not only antibody effector functions but also antibody stability and binding to certain cell surface antigens.Structuralstudies of intact antibodies and Fc fragments suggest that hydrophobic and hydrophilic interactions between sugar residues and amino acid residues in the CH 2domain of the Fc fragment affect the fine structural conformation of IgG molecules.Terminal-sugar residues mainly dictate these molecular interactions and therefore determine the impact of Fc glycosylation on antibody functions and stability.Terminal Gal,GlcNAc,and Man residues affect C1q binding and CDC activity,whereas terminal NANA,Man,core Fuc,and bisecting GlcNAc residues affect Fc g RIIIa binding and ADCC.Furthermore,terminal Man residues may affect serum half-life of some IgGs and terminal NANA may affect antibody binding to some cell surface antigens.Owing to the potential impact of Fc glycan microheterogeneity,it is necessary to be vigilant in quality control of antibody glycosylation and limit the impact of Fc glycans on product quality and efficacy of antibody-based human therapeutics.References and recommended readingPapers of particular interest,published within the period of the review,have been highlighted as: of special interestof outstanding interest 1.Beale D,Feinstein A:Structure and function of the constant regions of immunoglobulins .Q Rev Biophys 1976,9:135-180.2.Raju TS:Glycosylation variations with expression systems and their impact on biological activity of therapeutic immunoglobulins .Bioprocess Int 2003,1:44-53.3.Wright 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