Monoclonal antibody (mAb)-based therapeutics have been the fastest growing class of human pharmaceuticals with applications in various clinical indications such as oncology, inflammatory diseases, organ transplantation, and bacteria and virus infection (1). Currently, more than 60 mAbs and derivatives are approved in USA and Europe for human use with some of them being blockbusters in the biopharmaceutical markets (2, 3). Under the vigorous engine of modern translational biotechnology, mAbs and derivatives are estimated to be >30% of the new licensed drugs (4). Most recombinant therapeutic mAbs are glycosylated immunoglobulin G (IgG) molecules with glycans attached to the amide nitrogen atom of asparagine 297 (N297) in the crystallizable fragment (Fc) region (Figure 1A) (5). It is well accepted that the N297-attached oligosaccharide is structurally integral to the IgG-Fc with multiple non-covalent interactions with the protein surface of the C_H2 domain (6). The extensive carbohydrate–polypeptide interactions as well as carbohydrate–carbohydrate interactions modulate the conformations of the IgG molecules, which would ultimately impact the biological functions of mAbs (7).

During the last several decades, substantial knowledge has been acquired regarding the impact of Fc glycosylation on mAbs efficacy, pharmacokinetics (PK), stability, aggregation, safety, and immunogenicity (8–10). Many mAbs exhibit biological functions through immune effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) mediated by Fc–FcγR and Fc–C1q interactions (11). Alterations of glycoforms impact effector functions through modulating these Fc–ligand interactions (12–14). The effector functions of aglycosylated or deglycosylated IgGs are significantly dampened or eliminated due to the much lower binding to FcγRI or no binding to FcγRII and FcγRIII (15). Fc N-glycans impact stability of therapeutic antibodies in terms of shelf storage, thermal and chemical stability (such as pH and urea), aggregation propensity, susceptibility to proteolysis, in vivo clearance rate, and PK properties. The biophysical properties of therapeutic antibodies including the size, mass, charge, solubility, and colloidal stability are affected by N-glycans. Thus, different glycoforms could endow antibodies with distinct physicochemical and storage stabilities. Structurally, the glycans hold together with Fc C_H2 domain with extensive non-covalent interactions, which not only protect the aggregation prone region (Phe241, Phe243, Pro244, Val262, Val264, Val303, and Val305) of C_H2 from solvent exposure but also contribute to reduce the dynamics of C_H2 and aid in C_H2 folding (16, 17). These structural features could explain the decreased thermal, chemical stability, and increased aggregation propensity of aglycosylated IgGs compared with the glycosylated counterparts (16, 18, 19). In addition, the fact that the large complex type N-glycans with terminal galactose support an “open” Fc conformation compared with the “closed” Fc sustained by small glycans indicates N-glycans can also influence the folding of the Fc part (20). On the other hand, N-glycans impact the PK of IgG via modulating IgG sensitivity to serum protease cleavage. Due to the glycans protection, glycosylated IgGs are more resistant to trypsin, chymotrypsin, and pepsin than the aglycosylated IgGs (21). Glycoforms with distinct length, branching, and charge of sugar residues relate to the different susceptibilities of IgGs to proteolysis. While the terminal GlcNAc and sialic acid residues improve the resistance to proteolysis and hence enhance in vivo stability of IgG, terminal galactose residue confers higher sensitivity to proteases (22–24). The other way of selective clearance of glycosylated IgGs is executed by the C-type lectins mediated endocytosis. N-glycans with high mannose or terminated with GlcNAc could bind to the mannose receptors on macrophages/dendritic cells leading to the accelerated clearance of IgGs (25, 26). IgG with terminal galactose residue could be bound and cleared by the asialoglycoprotein receptor expressed in the hepatocytes (27). Besides, mAbs glycosylation also correlates with their immunogenicity and safety in humans. Therapeutic mAbs heterologously produced in CHO and murine myeloma cells (Sp2/0 and NS0 cells) possess non-natural sugars compared with human IgG, such as N-glycolylneuraminic acid (NGNA) residues and terminal α-1,3-linked galactose, which could induce potential immunogenicity in humans (28, 29).

Given its importance, glycosylation is considered a critical quality attribute for mAb therapeutics (30). The regulatory authorities require developers to keep glycoforms humanized and consistent with limited heterogeneity. Hence, developers need to strictly control the glycosylation profile during the development and production of mAbs. However, mAbs glycosylation is intrinsically heterogenous since glycans biosynthesis is not directly template driven. It is the product of sets of biochemical reactions involving a complex network of metabolic enzymes, which depends on the availability of sugar-nucleotide substrates, the enzyme distribution in the host cell, orchestrated process in endoplasmic reticulum (ER) and Golgi bodies and environmental factors (31, 32). Consequently, it is very challenging to achieve a highly homogenous glycoform independent of fermentation batches when expressing mAbs in eukaryotic cells. In the past decade, with significant advances in molecular and cell biology, protein and antibody engineering and gene editing, researchers have demonstrated individual glycoforms of antibodies could provide optimal efficacy for selected indications (Table 1) (33, 34). The pharmaceutical industry is increasingly pursuing the next-generation mAbs with tailored therapeutic effects. Herein, we review the current progress of mAbs Fc glycoengineering. We first present structures and biosynthesis of Fc N-glycans, followed by the discussion of impact of mAbs glycosylation on effector functions and the current glycoengineering strategies with emphasis on Fc defucosylation. Furthermore, we briefly discuss two novel therapeutic mAbs formats involving Fc glycans: aglycosylated mAbs and N-glycans targeted site-specific antibody–drug conjugates (ADCs).

The IgG-Fc N-glycan is usually of limited size with no more than three antennae (33). Typically, oligosaccharides of normal human IgGs are biantennary complex structures with a core heptasaccharide and an outer arm of sugar residues (6) (Figure 1B). The core oligosaccharide (GlcNAc2Man3GlcNAc2, designated as G0) is composed of two inner GlcNAc, three mannoses, and two GlcNAc β-1,2 linked to α-3 and α-6 mannose forming two antennae (α-3 arm and α-6 arm). One major feature of IgG-Fc glycans is the microheterogeneity, which not only stems from the linkage of sugar isomers and glycosylation site occupancy but also results from the outer arm sugar addition depending on the expression system and glycosylation enzymatic machinery (31, 64). Such additions include fucose (Fuc, G0F), galactose (Gal, G1, and G2), bisecting GlcNAc (linked to the core GlcNAc-associated mannose, which is catalyzed by GlcNAc transferases III), and sialic acid including N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) residues (9). In addition, structural studies have shown that the two N-glycans from every heavy chain are asymmetrically oriented (65, 66), which further diversifies IgG-Fc glycoforms. Consequently, Fc N-glycans possess more than 400 glycoforms considering random pairing of two different heavy chain glycans (67). Human serum IgG-Fc glycans typically contain ~30% G0F, ~35% G1F, ~16% G2F, and ~15% bisecting GlcNAc (68). Low levels of sialylation are observed in human IgG-Fc glycans with monosialylated and disialylated glycoforms accounting for approximately 5–10 and 1%, respectively (69). Interestingly, mAbs produced in recombinant expression systems share similar N-glycans structures with IgGs from human serum (13). MAbs produced in CHO, NS0, and Sp2/0 cell lines have predominant glycoforms of G0F, G1F, G2F, a paucity of sialylated glycans and do not contain bisecting GlcNAc (34, 70).

Like other glycoproteins, glycosylation of IgG occurs through the conserved ER and Golgi glycosylation pathway (32). N-glycosylation begins with the addition of a pyrophosphate-dolichol precursor (Dol-P, Glc3Man9GlcNAc2) to the consensus N-glycosylation sequon (Asn–X–Ser/Thr, where X is any amino acid except Pro) of a nascent polypeptide by a transmembrane oligosaccharyltransferase (Figure 2) (71). Thereafter, the N-glycans are subjected to series of sequential modifications by sets of glycosidases and glycosyltransferases. In the lumen of the ER, polypeptide associated Glc3Man9GlcNAc2 is sequentially trimmed by glucosidases I and II and endo-mannosidase to yield Man8GlcNAc2 (72). This process is under protein folding quality control mediated by calnexin–calreticulin complex. In the cis-Golgi, the Man8GlcNAc2 is sequentially processed by two class I α-mannosidases that act specifically on α-1,2-Man residues to give rise to the core Man5GlcNAc2 glycan for further diversification in the medial and trans-Golgi, which include stepwise addition of the outer arm monosaccharide residues, catalyzed by GlcNAc transferases I, II, and III (GnT I, II, and III), fucosyltransferases, galactosyltransferases (GalT), and sialyltransferases (SiaT).

Crystallizable fragment glycoforms affect the effector function of antibodies by modulating the fine structure of Fc and thus altering Fc–ligands interactions. In recent years, structural insights into how antibody Fc glycoforms impact effector functions have been acquired by X-ray crystallography, nuclear magnetic resonance spectroscopy, and thermodynamics studies. Due to the stabilization effect of protein–sugar interaction, the core structures of GlcNAc2Man3 on the 1,3 arm and 1,6 arm are usually visible in the crystal structures. These two arms adopt distinct orientations—the 1,6 arm hangs over the hydrophobic face of C_H2 domain while the 1,3 arm is orientated toward the internal space within the C_H2 dimer (73). The oligosaccharide is well conserved and spans over 500 Ų of the surface of each C_H2 domains (20) (Figure 3A). Oligosaccharides make multiple hydrophobic and polar non-covalent interactions with the inner face of the C_H2 domain (74). Impressive interactions include D265 hydrogen bonding to the inner GlcNAc and the α-1,6 arm forming strong CH-π packing with Phe241 and 243, which restricts the mobility of the glycans (75). Reciprocally, these intramolecular interactions restrain the C_H2 conformation by stabilizing the C′E loop where the Asn297 locates, through which N-glycans pre-organize the ligands (FcγRs and C1q) binding interface on Fc (15). Besides, carbohydrate–carbohydrate interactions also contribute to maintain Fc conformation. The reciprocal mannoses from the two heavy chains make sp–sp contacts with each other (Figure 3B), which is necessary to establish a proper Fc conformation for ligand binding (14).

Multifaced impacts of terminal sugars on the antibody effector function have been elucidated. While high mannose, low fucose, and bisecting GlcNAc increase ADCC due to enhanced FcγRIIIa binding, terminal sialic acid decrease ADCC of IgG (14). For CDC, terminal galactose increases CDC by improving C1q binding, whereas terminal GlcNAc and sialic acid decrease CDC (12). Among these effects, reduction in fucose and terminal galactose, which improves ADCC and CDC, is highly desirable in antibody glycoengineering (76). Regulating α-2,6-linked terminal sialic acid is also an attractive strategy due to the anti-inflammatory role of these terminal sialic acid (77).

Since different glycoforms have distinct impacts on antibody effector function, it is necessary to control antibody glycoforms. With advanced knowledge of glycobiology, it is feasible to produce homogeneously glycosylated antibodies with tailored effector function. Strategies include host cell based glycoengineering involving manipulations of biosynthetic pathways and in vitro chemoenzymatic glycosylation remodeling.

In recent years, aglycosylated full-length IgGs have gained substantial attentions due to their novel features (43, 118). Although the absence of N-glycans leads to the “closed” conformation of Fc and destabilization of C′E loop, the overall structures of aglycosylated IgG are similar to the glycosylated counterparts (119, 120). Thus, aglycosylated IgGs have almost identical antigen-binding affinity and pH-dependent FcRn binding and hence PK to glycosylated IgGs (121), which endows aglycosylated mAbs applications in the cases not requiring or avoiding undesired effector functions such as receptor blocking, targeted delivery, and anti-inflammation (122). Aglycosylated mAbs can be either produced in prokaryotic hosts (E. coli) or in eukaryotic hosts by introducing mutations at the Fc N297 or by the chemoenzymatic methods such as EndoS and PNGase-F enzyme treatment (120). Compared with the glycosylated IgG, aglycosylated IgG is devoid of glycan heterogeneity, hence significantly simplifies the biomanufacturing process leading to faster development timelines and lower developmental cost. Besides, aglycosylated IgG may be more susceptible to engineering due to the higher flexibility of the Fc conferred by the lack of N-glycans (118). Aglycosylated Fc could be engineered to restore or even improve its binding to FcγRs compared with glycosylated counterparts, which potentially extends the applications of aglycosylated IgGs into the cases effector functions are needed. For example, aglycosylated IgG-Fc could be engineered to bind to FcγRIIa and FcγRIIb comparably with glycosylated IgG-Fc by introducing double mutations S298G/T299A (123). More importantly, the higher flexibility renders the aglycosylated Fc being relatively easily engineered to exhibit unique FcγR specificity and novel effector functions. Jung et al. have used high-throughput library screening to develop an aglycosylated trastuzumab variant with five mutations (S298G/T299A/N390D/E382V/M428L) in Fc exhibiting >160-fold enhanced binding to FcγRIIa-R131 and 25-fold increased selectivity to FcγRIIa-R131 over FcRIIγb compared with the wide-type trastuzumab (124). Aglycosylated IgGs have established a new way for immunotherapy. Currently, several aglycosylated antibodies are under clinical investigation for efficacy and safety (120).

Antibody–drug conjugates are IgGs conjugated with cytotoxic small molecules through chemical linkers. By specifically targeting cancer cells and selective delivery of highly cytotoxic drugs, ADCs fundamentally revolutionized the way of cancer immunotherapy and chemotherapy (125). Currently, four ADCs (Adcetris^®, Kadcyla^®, Besponsa^®, and Mylotarg^®) have been approved by FDA with more than 80 ADCs under clinical evaluations (126). The mode of action of ADCs involves antigen-mediated endocytosis, followed by the release of drugs by either lysosomal degradation or hydrolytic/proteolytic cleavage (127). The methods for conjugating the small molecule drugs onto IgG represent one of the key technologies in ADC development. The conventional conjugation approaches involve random addition of drugs onto Lys or reduced Cys residues by amide coupling and maleimide alkylation chemistry, which leads to highly heterogeneous mixtures with different drug–antibody ratios and inconsistent yield (128). This heterogeneity negatively impacts the in vivo efficacy, stability, PK of ADCs (129). Thus, site-specific conjugation methods are highly pursued (130). Conjugation through IgG-Fc N-glycans represents one of the most widely used site-specific conjugation methods (131). Glycosite-specific conjugation proceeds with the introduction of a chemically active moiety onto the Fc N-glycans followed by reacting with payloads carrying another chemically active group. In this method, native IgG with heterogeneous N-glycans is deglycosylated by a wild-type endoglycosidase followed by the transglycosylation of a chemical group capped homogenous N-glycan substrate. The transglycosylation is catalyzed by an endoglycosynthase (a mutant of endoglycosidase) that lacks hydrolytic activity but possesses transglycosylation activity (132). Subsequently, the drug payload can be conjugated by biocompatible chemical reactions such as click chemistry and oximation. Boons et al. have reported the utilization of a sialyltransferase to attach an azido-tagged sialic acid moiety onto the galactosylated IgG N-glycan and conjugate the payload, doxorubicin on the azido group via the “click chemistry” (133). van Geel et al. reported a different method to produce ADC, which involved the endoglycosidase-mediated deglycosylation to obtain the Fuc-α-1,6-GlcNAc disaccharide glycoform of IgG, followed by the addition of azido-capped UDP-galactose catalyzed by a mutant galactosyltransferase (134). Our group has exploited a galactosyltransferase mutant (β-1,4-Gal-T1-Y289L) to achieve glycosite-specific conjugation by transferring the keto-tagged or azido-tagged galactose onto the degalactosylated G0F glycoform of IgG. The final ADC products, m860-AF (Auristatin F) ADC and m276-PBD (pyrrolobenzodiazepine) ADC, were obtained through the keto oximation-mediated addition of AF and the “click chemistry”-mediated addition of PBD dimer onto m860 IgG1 and m276 IgG1, respectively (110, 135). The glycosite-specific conjugation strategies provide novel routes for the preparations of ADCs with better homogeneity and drug to antibody ratios.

Unlike DNA and protein synthesis, antibody glycosylation synthesis is not directly template driven but is rather a result of networks of enzymatic reactions. Both host cells and the culture environment impact antibody glycosylation. Recombinant mAbs produced in host cells carry heterogenous Fc glycosylation, presumably with more than 400 possible glycoforms. Different glycoforms affect the in vivo efficacy, effector function, PK, stability, aggregation, safety, and immunogenicity of IgG. Among these, the impacts of Fc N-glycans on antibody effector function including ADCC and CDC are widely studied. IgG N-glycans affect their ADCC and CDC activity by altering Fc conformations and modulating the non-covalent interactions between oligosaccharides and C_H2 domains. While the terminal Gal enhances CDC activity, the core fucose significantly inhibits ADCC by sterically hindering the interactions between IgG-Fc and FcγRIIIa. Thus, the regulatory authorities require developers to keep glycoforms of mAbs consistent with limited heterogeneity, which has driven the development of multiple cell glycoengineering strategies to produce mAbs with desired glycoforms, especially without fucose. Although progress has been made, it is still challenging to consistently produce fully homogenously glycosylated antibodies by glycoengineered cell lines. The chemoenzymatic glycosylation remodeling offers revolutionized avenues to IgG with homogenous glycoforms. However, most current chemoenzymatic glycoengineering is still under lab-scale explorations, which is very challenging to scale up for industrial development. The chemoenzymatic glycoengineering approaches also provide novel routes for the productions of ADCs. On the other hand, the glyco-heterogeneity of mAbs could be bypassed by aglycosylated full-length IgGs. However, it remains to be seen for the outcomes of the clinical trials of aglycosylated antibodies in terms of the in vivo stability and immunogenicity. In the future, “omics” technologies and systems biology modeling hold promises to aid the glycoengineering for developing next-generation mAbs with homogenous glycoforms and improved therapeutic efficacy.

DD conceived the topic; WL wrote the manuscript; ZZ, YF, and WC revised the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.