Glycoengineering of Antibodies for Modulating Functions.

Antibodies are immunoglobulins that play essential roles in immune systems. All antibodies are glycoproteins that carry at least one or more conserved N-linked oligosaccharides (N-glycans) at the Fc domain. Many studies have demonstrated that both the presence and fine structures of the attached glycans can exert a profound impact on the biological functions and therapeutic efficacy of antibodies. However, antibodies usually exist as mixtures of heterogeneous glycoforms that are difficult to separate in pure glycoforms. Recent progress in glycoengineering has provided useful methods that enable production of glycan-defined and site-selectively modified antibodies for functional studies and for improved therapeutic efficacy. This review highlights major approaches in glycoengineering of antibodies with a focus on recent advances in three areas: glycoengineering through glycan biosynthetic pathway manipulation, glycoengineering through in vitro chemoenzymatic glycan remodeling, and glycoengineering of antibodies for site-specific antibody-drug conjugation.

Immunoglobulin G subclasses confer protection against Staphylococcus aureus bloodstream dissemination through distinct mechanisms in mouse models.

Antibodies bind target molecules with exquisite specificity. The removal of these targets is mediated by the effector functions of antibodies. We reported earlier that the monoclonal antibody (mAb) 3F6 promotes opsonophagocytic killing of Staphylococcus aureus in blood and reduces bacterial replication in animals. Here, we generated mouse immunoglobulin G (mIgG) subclass variants and observed a hierarchy in protective efficacy 3F6-mIgG2a > 3F6-mIgG1 ≥ 3F6-mIgG2b >> 3F6-mIgG3 following bloodstream challenge of C57BL/6J mice. This hierarchy was not observed in BALB/cJ mice: All IgG subclasses conferred similar protection. IgG subclasses differ in their ability to activate complement and interact with Fcγ receptors (FcγR) on immune cells. 3F6-mIgG2a-dependent protection was lost in FcγR-deficient, but not in complement-deficient C57BL/6J animals. Measurements of the relative ratio of FcγRIV over complement receptor 3 (CR3) on neutrophils suggest the preferential expression of FcγRIV in C57BL/6 mice and of CR3 in BALB/cJ mice. To determine the physiological significance of these differing ratios, blocking antibodies against FcγRIV or CR3 were administered to animals before challenge. Correlating with the relative abundance of each receptor, 3F6-mIgG2a-dependent protection in C57BL/6J mice showed a greater reliance for FcγRIV while protection in BALB/cJ mice was only impaired upon neutralization of CR3. Thus, 3F6-based clearance of S. aureus in mice relies on a strain-specific contribution of variable FcγR- and complement-dependent pathways. We surmise that these variabilities are the result of genetic polymorphism(s) that may be encountered in other mammals including humans and may have clinical implications in predicting the efficacy of mAb-based therapies.

Mechanism of high-mannose N-glycan breakdown and metabolism by Bifidobacterium longum.

Bifidobacteria are early colonizers of the human gut and play central roles in human health and metabolism. To thrive in this competitive niche, these bacteria evolved the capacity to use complex carbohydrates, including mammalian N-glycans. Herein, we elucidated pivotal biochemical steps involved in high-mannose N-glycan utilization by Bifidobacterium longum. After N-glycan release by an endo-ß-N-acetylglucosaminidase, the mannosyl arms are trimmed by the cooperative action of three functionally distinct glycoside hydrolase 38 (GH38) α-mannosidases and a specific GH125 α-1,6-mannosidase. High-resolution cryo-electron microscopy structures revealed that bifidobacterial GH38 α-mannosidases form homotetramers, with the N-terminal jelly roll domain contributing to substrate selectivity. Additionally, an α-glucosidase enables the processing of monoglucosylated N-glycans. Notably, the main degradation product, mannose, is isomerized into fructose before phosphorylation, an unconventional metabolic route connecting it to the bifid shunt pathway. These findings shed light on key molecular mechanisms used by bifidobacteria to use high-mannose N-glycans, a perennial carbon and energy source in the intestinal lumen.

Synthetic nanobodies as tools to distinguish IgG Fc glycoforms.

Protein glycosylation is a crucial mediator of biological functions and is tightly regulated in health and disease. However, interrogating complex protein glycoforms is challenging, as current lectin tools are limited by cross-reactivity while mass spectrometry typically requires biochemical purification and isolation of the target protein. Here, we describe a method to identify and characterize a class of nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. We apply this technology to immunoglobulin G (IgG) Fc glycoforms and define nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. By adapting these tools to standard biochemical methods, we can clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their IgG glycan profile, selectively disrupt IgG-Fcγ receptor binding both in vitro and in vivo, and interrogate the B cell receptor (BCR) glycan structure on living cells. Ultimately, we provide a strategy for the development of reagents to identify and manipulate IgG Fc glycoforms.

LC-MS/MS-PRM Quantification of IgG Glycoforms Using Stable Isotope Labeled IgG1 Fc Glycopeptide Standard.

Targeted quantification of proteins is a standard methodology with broad utility, but targeted quantification of glycoproteins has not reached its full potential. The lack of optimized workflows and isotopically labeled standards limits the acceptance of glycoproteomics quantification. In this work, we introduce an efficient and streamlined chemoenzymatic synthesis of a library of isotopically labeled glycopeptides of IgG1 which we use for quantification in an energy optimized LC-MS/MS-PRM workflow. Incorporation of the stable isotope labeled N-acetylglucosamine enables an efficient monitoring of all major fragment ions of the glycopeptides generated under the soft higher-energy C-trap dissociation (HCD) conditions, which reduces the coefficients of variability (CVs) of the quantification to 0.7-2.8%. Our results document, for the first time, that the workflow using a combination of stable isotope labeled standards with intrascan normalization enables quantification of the glycopeptides by an electron transfer dissociation (ETD) workflow, as well as the HCD workflow, with the highest sensitivity compared to traditional workflows. This was exemplified by a rapid quantification (13 min) of IgG1 Fc glycoforms from COVID-19 patients.

"Ghost" Fragment Ions in Structure and Site-Specific Glycoproteomics Analysis.

Mass spectrometry (MS) can unlock crucial insights into the intricate world of glycosylation analysis. Despite its immense potential, the qualitative and quantitative analysis of isobaric glycopeptide structures remains one of the most daunting hurdles in the field of glycoproteomics. The ability to distinguish between these complex glycan structures poses a significant challenge, hindering our ability to accurately measure and understand the role of glycoproteins in biological systems. A few recent publications described the use of collision energy (CE) modulation to improve structural elucidation, especially for qualitative purposes. Different linkages of glycan units usually demonstrate different stabilities under CID/HCD fragmentation conditions. Fragmentation of the glycan moiety produces low molecular weight ions (oxonium ions) that can serve as a structure-specific signature for specific glycan moieties; however, the specificity of these fragments has never been examined closely. Here, we particularly focused on N-glycoproteomics analysis and investigated fragmentation specificity using synthetic stable isotope-labeled N-glycopeptide standards. These standards were isotopically labeled at the reducing terminal GlcNAc, which allowed us to resolve fragments produced by the oligomannose core moiety and fragments generated from outer antennary structures. Our research identified the potential for false-positive structure assignments due to the occurrence of "Ghost" fragments resulting from single glyco unit rearrangement or mannose core fragmentation within the collision cell. To mitigate this issue, we have established a minimal intensity threshold for these fragments to prevent misidentification of structure-specific fragments in glycoproteomics analysis. Our findings provide a crucial step forward in the quest for more accurate and reliable glycoproteomics measurements.

Catanionic Vesicles as a Facile Scaffold to Display Natural N-Glycan Ligands for Probing Multivalent Carbohydrate-Lectin Interactions.

Multivalent interactions are a key characteristic of protein-carbohydrate recognition. Phospholipid-based liposomes have been explored as a popular platform for multivalent presentation of glycans, but this platform has been plagued by the instability of typical liposomal formulations in biological media. We report here the exploitation of catanionic vesicles as a stable lipid-based nanoparticle scaffold for displaying large natural N-glycans as multivalent ligands. Hydrophobic insertion of lipidated N-glycans into the catanionic vesicle bilayer was optimized to allow for high-density display of structurally diverse N-glycans on the outer membrane leaflet. In an enzyme-linked competitive lectin-binding assay, the N-glycan-coated vesicles demonstrated a clear clustering glycoside effect, with significantly enhanced affinity for the corresponding lectins including Sambucus nigra agglutinin (SNA), concanavalin A (ConA), and human galectin-3, in comparison with their respective natural N-glycan ligands. Our results showed that relatively low density of high-mannose and sialylated complex type N-glycans gave the maximal clustering effect for binding to ConA and SNA, respectively, while relatively high-density display of the asialylated complex type N-glycan provided maximal clustering effects for binding to human galectin 3. Moreover, we also observed a macromolecular crowding effect on the binding of ConA to high-mannose N-glycans when catanionic vesicles bearing mixed high-mannose and complex-type N-glycans were used. The N-glycan-coated catanionic vesicles are stable and easy to formulate with varied density of ligands, which could serve as a feasible vehicle for drug delivery and as potent inhibitors for intervening protein-carbohydrate interactions implicated in disease.

Glycosylation-dependent opsonophagocytic activity of staphylococcal protein A antibodies.

Antibodies may bind to bacterial pathogens or their toxins to control infections, and their effector activity is mediated through the recruitment of complement component C1q or the engagement with Fcγ receptors (FcγRs). For bacterial pathogens that rely on a single toxin to cause disease, immunity correlates with toxin neutralization. Most other bacterial pathogens, including Staphylococcus aureus, secrete numerous toxins and evolved multiple mechanisms to escape opsonization and complement killing. Several vaccine candidates targeting defined surface antigens of S. aureus have failed to meet clinical endpoints. It is unclear that such failures can be solely attributed to the poor selection of antibody targets. Thus far, studies to delineate antibody-mediated uptake and killing of Gram-positive pathogens remain extremely limited. Here, we exploit 3F6-hIgG1, a human monoclonal antibody that binds and neutralizes the abundant surface-exposed Staphylococcal protein A (SpA). We find that galactosylation of 3F6-hIgG1 that favors C1q recruitment is indispensable for opsonophagocytic killing of staphylococci and for protection against bloodstream infection in animals. However, the simple removal of fucosyl residues, which results in reduced C1q binding and increased engagement with FcγR, maintains the opsonophagocytic killing and protective attributes of the antibody. We confirm these results by engineering 3F6-hIgG1 variants with biased binding toward C1q or FcγRs. While the therapeutic benefit of monoclonal antibodies against infectious disease agents may be debatable, the functional characterization of such antibodies represents a powerful tool for the development of correlates of protection that may guide future vaccine trials.

FcRn, but not FcγRs, drives maternal-fetal transplacental transport of human IgG antibodies.

The IgG Fc domain has the capacity to interact with diverse types of receptors, including the neonatal Fc receptor (FcRn) and Fcγ receptors (FcγRs), which confer pleiotropic biological activities. Whereas FcRn regulates IgG epithelial transport and recycling, Fc effector activities, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis, are mediated by FcγRs, which upon cross-linking transduce signals that modulate the function of effector leukocytes. Despite the well-defined and nonoverlapping functional properties of FcRn and FcγRs, recent studies have suggested that FcγRs mediate transplacental IgG transport, as certain Fc glycoforms were reported to be enriched in fetal circulation. To determine the contribution of FcγRs and FcRn to the maternal-fetal transport of IgG, we characterized the IgG Fc glycosylation in paired maternal-fetal samples from patient cohorts from Uganda and Nicaragua. No differences in IgG1 Fc glycan profiles and minimal differences in IgG2 Fc glycans were noted, whereas the presence or absence of galactose on the Fc glycan of IgG1 did not alter FcγRIIIa or FcRn binding, half-life, or their ability to deplete target cells in FcγR/FcRn humanized mice. Modeling maternal-fetal transport in FcγR/FcRn humanized mice confirmed that only FcRn contributed to transplacental transport of IgG; IgG selectively enhanced for FcRn binding resulted in enhanced accumulation of maternal antibody in the fetus. In contrast, enhancing FcγRIIIa binding did not result in enhanced maternal-fetal transport. These results argue against a role for FcγRs in IgG transplacental transport, suggesting Fc engineering of maternally administered antibody to enhance only FcRn binding as a means to improve maternal-fetal transport of IgG.

GH18 endo-ß-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans.

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-ß-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.

Synthesis and Evaluation of Three Azide-Modified Disaccharide Oxazolines as Enzyme Substrates for Single-Step Fc Glycan-Mediated Antibody-Drug Conjugation.

Antibody-drug conjugates (ADCs) hold great promise for targeted cancer cell killing. Site-specific antibody-drug conjugation is highly desirable for synthesizing homogeneous ADCs with optimal safety profiles and high efficacy. We have recently reported that azide-functionalized disaccharide oxazolines of the Manß1,4GlcNAc core were an efficient substrate of wild-type endoglycosidase Endo-S2 for Fc glycan remodeling and conjugation. In this paper, we report the synthesis and evaluation of new disaccharide oxazolines as enzyme substrates for examining the scope of the site-specific conjugation. Thus, azide-functionalized disaccharide oxazolines derived from Manß1,4GlcNAc, Glcß1,4GlcNAc, and Galß1,4GlcNAc (LacNAc) were synthesized. Enzymatic evaluation revealed that wild-type Endo-S2 demonstrated highly relaxed substrate specificity and could accommodate all the three types of disaccharide derivatives for transglycosylation to provide site-specific azide-tagged antibodies, which were readily clicked with a payload to generate homogeneous ADCs. Moreover, we also found that Endo-S2 was able to accommodate drug-preloaded minimal disaccharide oxazolines as donor substrates for efficient glycan transfer, enabling a single-step and site-specific antibody-drug conjugation without the need of an antibody click reaction. The ability of Endo-S2 to accommodate simpler and more easily synthesized disaccharide oxazoline derivatives for Fc glycan remodeling further expanded the scope of this bioconjugation method for constructing homogeneous antibody-drug conjugates in a single-step manner. Finally, cell-based assays indicated that the synthetic homogeneous ADCs demonstrated potent targeted cancer cell killing.

Synthesis and Immunological Study of N-Glycan-Bacteriophage Qß Conjugates Reveal Dominant Antibody Responses to the Conserved Chitobiose Core.

N-Glycosylation plays an important role in many biological recognition processes. However, very few N-glycan-specific antibodies are available for functional studies and potentially for therapeutic development. In this study, we sought to synthesize bacteriophage Qß conjugates with representative N-glycans and investigate their immunogenicity for raising N-glycan-specific antibodies. An array of Qß glycoconjugates bearing five different human N-glycans and two different chemical linkers were synthesized, and the immunization of the N-glycan-Qß conjugates was performed in mice. We found that the N-glycan-Qß conjugates raised significant IgG antibodies that recognize N-glycans, but, surprisingly, most of the glycan-dependent antibodies were directed to the shared chitobiose core and were nonspecific for respective N-glycan structures. The linker chemistry was found to affect antibody specificity with adipic acid-linked N-glycan-Qß immunogens raising antibodies capable of recognizing both the N-acetylglucosamine (GlcNAc) moieties of the chitobiose core. In contrast, antibodies raised by N-glycan-Qß immunogens with a triazole linker preferentially recognized the innermost N-acetylglucosamine moiety at the reducing end. We also found that sialylation of the N-glycans significantly suppressed the immune response. Furthermore, the N-glycan-Qß immunogens with an adipic acid linker elicited higher glycan-specific antibody titers than the N-glycan-triazole-Qß immunogens. These findings delineate several challenges in eliciting mammalian N-glycan-specific antibodies through the conventional glycoconjugate vaccine design and immunization.

Synthetic Antibody-Rhamnose Cluster Conjugates Show Potent Complement-Dependent Cell Killing by Recruiting Natural Antibodies.

Monoclonal antibodies (mAbs) are one of the most rapidly growing drug classes used for the treatment of cancer, infectious and autoimmune diseases. Complement-dependent cytotoxicity (CDC) is one of the effector functions for antibodies to deplete target cells. We report here an efficient chemoenzymatic synthesis of structurally well-defined conjugates of a monoclonal antibody with a rhamnose- and an αGal trisaccharide-cluster to recruit natural anti-rhamnose and anti-αGal antibodies, respectively, to enhance the CDC-dependent targeted cell killing. The synthesis was achieved by using a modular antibody Fc-glycan remodeling method that includes site-specific chemoenzymatic Fc-glycan functionalization and subsequent click conjugation of synthetic rhamnose- and αGal trisaccharide-cluster to provide the respective homogeneous antibody conjugates. Cell-based assays indicated that the antibody-rhamnose cluster conjugates could mediate potent CDC activity for targeted cancer cell killing and showed much more potent efficacy than the antibody-αGal trisaccharide cluster conjugates for CDC effects.

The interplay of protein engineering and glycoengineering to fine-tune antibody glycosylation and its impact on effector functions.

The N-glycan pattern of an IgG antibody, attached at a conserved site within the fragment crystallizable (Fc) region, is a critical antibody quality attribute whose structural variability can also impact antibody function. For tailoring the Fc glycoprofile, glycoengineering in cell lines as well as Fc amino acid mutations have been applied. Multiple glycoengineered Chinese hamster ovary cell lines were generated, including defucosylated (FUT8KO), α-2,6-sialylated (ST6KI), and defucosylated α-2,6-sialylated (FUT8KOST6KI), expressing either a wild-type anti-CD20 IgG (WT) or phenylalanine to alanine (F241A) mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry characterization of antibody N-glycans revealed that the F241A mutation significantly increased galactosylation and sialylation content and glycan branching. Furthermore, overexpression of recombinant human α-2,6-sialyltransferase resulted in a predominance of α-2,6-sialylation rather than α-2,3-sialylation for both WT and heavily sialylated F241A antibody N-glycans. Interestingly, knocking out α-1,6-fucosyltransferase (FUT8KO), which removed core fucose, lowered the content of N-glycans with terminal Gal and increased levels of terminal GlcNAc and Man5 groups on WT antibody. Further complement-dependent cytotoxicity (CDC) analysis revealed that, regardless of the production cells, WT antibody samples have higher cytotoxic CDC activity with more exposed Gal residues compared to their individual F241A mutants. However, the FUT8KO WT antibody, with a large fraction of bi-GlcNAc structures (G0), displayed the lowest CDC activity of all WT antibody samples. Furthermore, for the F241A mutants, a higher CDC activity was observed for α-2,6- compared to α-2,3-sialylation. Antibody-dependent cellular cytotoxicity (ADCC) analysis revealed that the defucosylated WT and F241A mutants showed enhanced in vitro ADCC performance compared to their fucosylated counterparts, with the defucosylated WT antibodies displaying the highest overall ADCC activity, regardless of sialic acid substitution. Moreover, the FcγRIIIA receptor binding by antibodies did not always correspond directly with ADCC result. This study demonstrates that glycoengineering and protein engineering can both promote and inhibit antibody effector functions and represent practical approaches for varying glycan composition and functionalities during antibody development.

Synthesis, binding affinity, and inhibitory capacity of cyclodextrin-based multivalent glycan ligands for human galectin-3.

Human galectin 3 (Gal-3) has been implicated to play important roles in different biological recognition processes such as tumor growth and cancer metastasis. High-affinity Gal-3 ligands are desirable for functional studies and as inhibitors for potential therapeutic development. We report here a facile synthesis of ß-cyclodextrin (CD)-based Tn and TF antigen-containing multivalent ligands via a click reaction. Binding studies indicated that the synthetic multivalent glycan ligands demonstrated a clear clustering effect in binding to human Gal-3, with up to 153-fold enhanced relative affinity in comparison with the monomeric glycan ligand. The GalNAc (Tn antigen) containing heptavalent ligand showed the highest affinity for human Gal-3 among the synthetic ligands tested, with an EC50 of 1.4 µM in binding to human Gal-3. A cell-based assay revealed that the synthetic CD-based multivalent ligands could efficiently inhibit Gal-3 binding to human airway epithelial cells, with an inhibitory capacity consistent with their binding affinity measured by SPR. The synthetic cyclodextrin-based ligands described in this study should be valuable for functional studies of human Gal-3 and potentially for therapeutic applications.

Site-selective sulfation of N-glycans by human GlcNAc-6-O-sulfotransferase 1 (CHST2) and chemoenzymatic synthesis of sulfated antibody glycoforms.

Sulfation is a common modification of glycans and glycoproteins. Sulfated N-glycans have been identified in various glycoproteins and implicated for biological functions, but in vitro synthesis of structurally well-defined full length sulfated N-glycans remains to be described. We report here the first in vitro enzymatic sulfation of biantennary complex type N-glycans by recombinant human CHST2 (GlcNAc-6-O-sulfotransferase 1, GlcNAc6ST-1). We found that the sulfotransferase showed high antennary preference and could selectively sulfate the GlcNAc moiety located on the Manα1,3Man arm of the biantennary N-glycan. The glycan chain was further elongated by bacterial ß1,4 galactosyltransferase from Neiserria meningitidis and human ß1,4 galactosyltransferase IV（B4GALT4）, which led to the formation of different sulfated N-glycans. Using rituximab as a model IgG antibody, we further demonstrated that the sulfated N-glycans could be efficiently transferred to an intact antibody by using a chemoenzymatic Fc glycan remodeling method, providing homogeneous sulfated glycoforms of antibodies. Preliminary binding analysis indicated that sulfation did not affect the apparent affinity of the antibody for FcγIIIa receptor.

Appropriate aglycone modification significantly expands the glycan substrate acceptability of α1,6-fucosyltransferase (FUT8).

The α1,6-fucosyltransferase, FUT8, is the sole enzyme catalyzing the core-fucosylation of N-glycoproteins in mammalian systems. Previous studies using free N-glycans as acceptor substrates indicated that a terminal ß1,2-GlcNAc moiety on the Man-α1,3-Man arm of N-glycan substrates is required for efficient FUT8-catalyzed core-fucosylation. In contrast, we recently demonstrated that, in a proper protein context, FUT8 could also fucosylate Man5GlcNAc2 without a GlcNAc at the non-reducing end. We describe here a further study of the substrate specificity of FUT8 using a range of N-glycans containing different aglycones. We found that FUT8 could fucosylate most of high-mannose and complex-type N-glycans, including highly branched N-glycans from chicken ovalbumin, when the aglycone moiety is modified with a 9-fluorenylmethyloxycarbonyl (Fmoc) moiety or in a suitable peptide/protein context, even if they lack the terminal GlcNAc moiety on the Man-α1,3-Man arm. FUT8 could also fucosylate paucimannose structures when they are on glycoprotein substrates. Such core-fucosylated paucimannosylation is a prominent feature of lysosomal proteins of human neutrophils and several types of cancers. We also found that sialylation of N-glycans significantly reduced their activity as a substrate of FUT8. Kinetic analysis demonstrated that Fmoc aglycone modification could either improve the turnover rate or decrease the KM value depending on the nature of the substrates, thus significantly enhancing the overall efficiency of FUT8 catalyzed fucosylation. Our results indicate that an appropriate aglycone context of N-glycans could significantly broaden the acceptor substrate specificity of FUT8 beyond what has previously been thought.

Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases.

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems.

Site-Selective Chemoenzymatic Modification on the Core Fucose of an Antibody Enhances Its Fcγ Receptor Affinity and ADCC Activity.

Fc glycosylation profoundly impacts the effector functions of antibodies and often dictates an antibody's pro- or anti-inflammatory activities. It is well established that core fucosylation of the Fc domain N-glycans of an antibody significantly reduces its affinity for FcγRIIIa receptors and antibody-dependent cellular cytotoxicity (ADCC). Previous structural studies have suggested that the presence of a core fucose remarkably decreases the unique and favorable carbohydrate-carbohydrate interactions between the Fc and the receptor N-glycans, leading to reduced affinity. We report here that in contrast to natural core fucose, special site-specific modification on the core fucose could dramatically enhance the affinity of an antibody for FcγRIIIa. The site-selective modification was achieved through an enzymatic transfucosylation with a novel fucosidase mutant, which was shown to be able to use modified α-fucosyl fluoride as the donor substrate. We found that replacement of the core l-fucose with 6-azide- or 6-hydroxy-l-fucose (l-galactose) significantly enhanced the antibody's affinity for FcγRIIIa receptors and substantially increased the ADCC activity. To understand the mechanism of the modified fucose-mediated affinity enhancement, we performed molecular dynamics simulations. Our data revealed that the number of glycan contacts between the Fc and the Fc receptor was increased by the selective core-fucose modifications, showing the importance of unique carbohydrate-carbohydrate interactions in achieving high FcγRIIIa affinity and ADCC activity of antibodies. Thus, the direct site-selective modification turns the adverse effect of the core fucose into a favorable force to promote the carbohydrate-carbohydrate interactions.

One-Pot Conversion of Free Sialoglycans to Functionalized Glycan Oxazolines and Efficient Synthesis of Homogeneous Antibody-Drug Conjugates through Site-Specific Chemoenzymatic Glycan Remodeling.

Antibody-drug conjugates (ADCs) are an important class of therapeutic agents that harness the highly specific antigen targeting property of antibodies to deliver toxic drugs for targeted cell killing. Site-specific conjugation methods are highly desirable for constructing homogeneous ADCs that possess a well-defined antibody-to-drug ratio, stability, ideal pharmacological profile, and optimal therapeutic index. We report here a facile synthesis of functionalized glycan oxazolines from free sialoglycans that are key donor substrates for enzymatic Fc glycan remodeling and the application of an efficient endoglycosidase mutant (Endo-S2 D184M) for site-specific glycan transfer to construct homogeneous ADCs. We found that by a sequential use of two coupling reagents under optimized conditions, free sialoglycans could be efficiently converted to selectively functionalized glycan oxazolines carrying azide-, cyclopropene-, and norbornene-tags, respectively, in excellent yield and in a simple one-pot manner. We further demonstrated that the recently reported Endo-S2 D184 M mutant was highly efficient for Fc glycan remodeling with the selectively modified glycan oxazolines to introduce tags into an antibody, which required a significantly smaller amount of glycan oxazolines and a much shorter reaction time than that of the Endo-S D233Q-catalyzed reaction, thus minimizing the side reactions. Finally homogeneous ADCs were constructed with three different click reactions. The resulting ADCs showed excellent serum stability, and in vitro cytotoxicity assays indicated that all the three ADCs generated from the distinct click reactions possessed potent and comparable cytotoxicity for targeted cancer cell killing.