Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies

Therapeutic antibody IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, which gives rise to structural heterogeneity. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Recently, antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibodies such as anti-CD20 IgG1 rituximab (Rituxan^®) and anti-Her2/neu IgG1 trastuzumab (Herceptin^®). ADCC is triggered upon the binding of lymphocyte receptors (FcγRs) to the antibody Fc region. The activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an α-1,6-linkage, and is dramatically enhanced by a reduction in fucose. Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo, and are not likely to be immunogenic because their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of non-fucosylated antibodies is expected to be a powerful and elegant approach to the design of the next generation therapeutic antibodies with improved efficacy. In this review, we discuss the importance of the oligosaccharides attached to the Fc region of therapeutic antibodies, especially regarding the inhibitory effect of fucosylated therapeutic antibodies on the efficacy of non-fucosylated counterparts in one medical agent. The impact of completely non-fucosylated therapeutic antibodies on therapeutic fields will be also discussed.     

Antibody–receptor interactions mediate antibody-dependent cellular cytotoxicity

Binding of antibodies to their receptors is a core component of the innate immune system. Understanding the precise interactions between antibodies and their Fc receptors has led to the engineering of novel mAb biotherapeutics with tailored biological activities. One of the most significant findings is that afucosylated monoclonal antibodies demonstrate increased affinity toward the receptor FcγRIIIa, with a commensurate increase in antibody-dependent cellular cytotoxicity. Crystal structure analysis has led to the hypothesis that afucosylation in the Fc region results in reduced steric hindrance between antibody–receptor intermolecular glycan interactions, enhancing receptor affinity; however, solution-phase data have yet to corroborate this hypothesis. In addition, recent work has shown that the fragment antigen-binding (Fab) region may directly interact with Fc receptors; however, the biological consequences of these interactions remain unclear. By probing differences in solvent accessibility between native and afucosylated immunoglobulin G1 (IgG1) using hydroxyl radical footprinting–MS, we provide the first solution-phase evidence that an IgG1 bearing an afucosylated Fc region appears to require fewer conformational changes for FcγRIIIa binding. In addition, we performed extensive molecular dynamics (MD) simulations to understand the molecular mechanism behind the effects of afucosylation. The combination of these techniques provides molecular insight into the steric hindrance from the core Fc fucose in IgG1 and corroborates previously proposed Fab–receptor interactions. Furthermore, MD-guided rational mutagenesis enabled us to demonstrate that Fab–receptor interactions directly contribute to the modulation of antibody-dependent cellular cytotoxicity activity. This work demonstrates that in addition to Fc–polypeptide and glycan-mediated interactions, the Fab provides a third component that influences IgG–Fc receptor biology.     

Trastuzumab and pertuzumab plant biosimilars: Modification of Asn297-linked glycan of the mAbs produced in a plant with fucosyltransferase and xylosyltransferase gene knockouts

Plant biosimilars of anticancer therapeutic antibodies are of interest not only because of the prospects of their practical use, but also as an instrument and object for study of plant protein glycosylation. In this work, we first designed a pertuzumab plant biosimilar (PPB) and investigated the composition of its Asn297-linked glycan in comparison with trastuzumab plant biosimilar (TPB). Both biosimilars were produced in wild-type (WT) Nicotiana benthamiana plant (PPBWT and TPB-WT) and transgenic ΔXTFT N. benthamiana plant with XT and FT genes knockout (PPB-ΔXTFT and TPBΔXTFT). Western blot analysis with anti-α1,3-fucose and anti-xylose antibodies, as well as a test with peptide-N-glycosidase F, confirmed the absence of α1,3-fucose and xylose in the Asn297-linked glycan of PPB-ΔXTFT and TPB-ΔXTFT. Peptide analysis followed by the identification of glycomodified peptides using MALDI-TOF/TOF showed that PPB-WT and TPB-WT Asn297-linked glycans are mainly of complex type GnGnXF. The core of PPB-WT and TPB-WT Asn297linked GnGn-type glycan contains α1,3-fucose and β1,2-xylose, which, along with the absence of terminal galactose and sialic acid, distinguishes these plant biosimilars from human IgG. Analysis of TPB-ΔXTFT total carbohydrate content indicates the possibility of changing the composition of the carbohydrate profile not only of the Fc, but also of the Fab portion of an antibody produced in transgenic ΔXTFT N. benthamiana plants. Nevertheless, study of the antigen-binding capacity of the biosimilars showed that absence of xylose and fucose residues in the Asn297-linked glycans does not affect the ability of the glycomodified antibodies to interact with HER2/neu positive cancer cells.     

Fc-glycosylation influences Fcγ receptor binding and cell-mediated anti-HIV activity of monoclonal antibody 2G12

Interactions between the Fc segment of IgG and FcγRs on a variety of cells are likely to play an important role in the anti-HIV activity of Abs. Because the nature of the glycan structure on the Fc domain is a critical determinant of Fc-FcγR binding, proper Fc glycosylation may contribute to Ab-mediated protection. We have generated five different glycoforms of the broadly HIV-1-neutralizing mAb 2G12 in wild-type and glycoengineered plants and Chinese hamster ovary cells. Plant-derived 2G12 exhibited highly homogeneous glycosylation profiles with a single dominant N-glycan species. Using flow cytometry with FcγR-expressing cell lines, all 2G12 glycoforms demonstrated similar binding to FcγRI, FcγRIIa, and FcγRIIb. In contrast, two glycoforms derived from glycoengineered plants that lack plant-specific xylose and core α1,3-fucose, and instead carry human-like glycosylation with great uniformity, showed significantly enhanced binding to FcγRIIIa compared with Chinese hamster ovary or wild-type plant-derived 2G12. Using surface plasmon resonance, we show that binding of 2G12 to FcγRIIIa is markedly affected by core fucose, irrespective of its plant-specific α1,3 or mammalian-type α1,6 linkage. Consistent with this finding, 2G12 glycoforms lacking core fucose (and xylose) mediated higher antiviral activity against HIV-1 or simian immunodeficiency virus as measured by Ab-dependent cell-mediated virus inhibition. This is, to our knowledge, the first demonstration that specific alterations of Fc glycosylation can improve antiviral activity. Such alterations may result in better immunotherapeutic reagents. Moreover, biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy.     

Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its “regular” fucosylated counterpart and a series of mixtures containing varying proportions of “regular” and afucosylated materials. Compared with the “regular” fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC₅₀ values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.     

Monomeric IgA can be produced in planta as efficient as IgG,yet receives different N‐glycans

The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1κ and IgG1κ versions of three therapeutic antibodies directed against pro‐inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF‐α, and Ustekinumab, directed against the interleukin‐12p40 subunit. We evaluated antibody yield, quality and N‐glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell‐based assay. However, IgA1κ‐based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1κ‐ and IgG1κ‐based Infliximab were enriched in oligomannose‐type N‐glycan structures. For IgG1κ‐based Ustekinumab and Adalimumab, the major N‐glycan type was the typical plant complex N‐glycan, biantennary with terminal N‐acetylglucosamine, β1,2‐xylose and core α1,3‐fucose. In contrast, the major N‐glycan on the IgA‐based antibodies was xylosylated, but lacked core α1,3‐fucose and one terminal N‐acetylglucosamine. This type of N‐glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant‐produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N‐glycan an antibody receives.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin: Characterization of the sequence Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21.90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and ¹H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N′-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (α2-6) or (α2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (α1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man(α1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(α1-6). In fraction mTf-V, which was found to be very heterogeneous by ¹H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri′-antennary glycans sialylated by Neu5Gc α-2,6- and α-2,3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(α2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (α2-6)GlcNAc sialyltransferase.     

Identification of core alpha 1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster. Potential basis of the neural anti-horseadish peroxidase epitope

For many years, polyclonal antibodies raised against the plant glycoprotein horseradish peroxidase have been used to specifically stain the neural and male reproductive tissue of Drosophila melanogaster. This epitope is considered to be of carbohydrate origin, but no glycan structure from Drosophila has yet been isolated that could account for this cross-reactivity. Here we report that N-glycan core alpha1,3-linked fucose is, as judged by preabsorption experiments, indispensable for recognition of Drosophila embryonic nervous system by anti-horseradish peroxidase antibody. Further, we describe the identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and high performance liquid chromatography of two Drosophila N-glycans that, as already detected in other insects, carry both alpha1,3- and alpha1,6-linked fucose residues on the proximal core GlcNAc. Moreover, we have isolated three cDNAs encoding alpha1,3-fucosyltransferase homologues from Drosophila. One of the cDNAs, when transformed into Pichia pastoris, was found to direct expression of core alpha1,3-fucosyltransferase activity. This recombinant enzyme preferred as substrate a biantennary core alpha1,6-fucosylated N-glycan carrying two non-reducing N-acetylglucosamine residues (GnGnF6; Km 11 microm) over the same structure lacking a core fucose residue (GnGn; Km 46 microm). The Drosophila core alpha1,3-fucosyltransferase enzyme was also shown to be able to fucosylate N-glycan structures of human transferrin in vitro, this modification correlating with the acquisition of binding to anti-horseradish peroxidase antibody.     

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.     

Molecular dynamics simulation of glycoprotein-glycans of immunoglobulin G and Immunoglobulin M

Glycoprotein-glycans have recently been implicated to play a variety of functional roles. The same glycan chain have been found complexed with proteins of diverse functions. In this article two such glycan chains found attached to Fc regions of immunoglobulin G and immunoglobulin M have been studied. An extensive simulated annealing procedure have been adopted to arrive at a low-energy minimum of the two oligosaccharides. Molecular dynamics simulations have been performed to study the flexibility of the glycosidic linkages. It was found that both glycan chains can undergo conformational transitions and adopt folded and extended conformations. The two β(1–2) linkages of complex-type glycan had been found to prefer different conformational regime and the terminal fucose linked to the GlcNAc residue drastically modifies the GlcNAc β(1–4)GlcNAc linkage conformation. In the high-mannose type glycan chain α(1–3) linkages can induce flexibility in addition to the α(1–6) linkages. The results have been compared with recent experimental nmr and fluorescence energy transfer data. © 1998 John Wiley & Sons, Inc. Biopoly 45: 177–190, 1998     

Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose

The plant glycosyltransferases, beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT), are responsible for the transfer of beta1,2-linked xylose and core alpha1,3-linked fucose residues to glycoprotein N-glycans. These glycan epitopes are not present in humans and thus may cause immunological responses, which represent a limitation for the therapeutic use of recombinant mammalian glycoproteins produced in transgenic plants. Here we report the genetic modification of the N-glycosylation pathway in Arabidopsis thaliana plants. Knockout plants were generated with complete deficiency of XylT and FucT. These plants lack antigenic protein-bound N-glycans and instead synthesise predominantly structures with two terminal betaN-acetylglucosamine residues (GlcNAc(2)Man(3)GlcNAc(2)).     

The production of human glucocerebrosidase in glyco‐engineered Nicotiana benthamiana plants

For the production of therapeutic proteins in plants, the presence of β1,2‐xylose and core α1,3‐fucose on plants’ N‐glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down‐regulate the endogenous N‐acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco‐engineered line (NbGNTI‐RNAi) showed a strong reduction of plant‐specific N‐glycans, with the result that as much as 90.9% of the total N‐glycans were of high‐mannose type. Therefore, this NbGNTI‐RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The NbGNTI‐RNAi plant was cross‐pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gaucher's disease, requires terminal mannose for its therapeutic efficacy. The N‐glycan structures that were presented on all of the four occupied N‐glycosylation sites of recombinant GC in NbGNTI‐RNAi plants (GC^gnt1) showed that the majority (ranging from 73.3% up to 85.5%) of the N‐glycans had mannose‐type structures lacking potential immunogenic β1,2‐xylose and α1,3‐fucose epitopes. Moreover, GC^gnt1 could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gaucher's disease. Notably, the NbGNTI‐RNAi line, producing GC, was stable and the NbGNTI‐RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N‐glycan structures.     

Strict order of (Fuc to Asn-linked GlcNAc) fucosyltransferases forming core-difucosylated structures

In insect cells fucose can be either α1,6- or α1,3-linked to the asparagine-bound GlcNAc residue of N-glycans. Difucosylated glycans have also been found. Kinetic studies and acceptor competition experiments demonstrate that two different enzymes are responsible for this α1,6- and α1,3-linkage of fucose. Using dansylated acceptor substrates a strict order of these enzymes can be established for the formation of difucosylated structures. First, the α1,6-fucosyltransferase catalyses the transfer of fucose into α1,6-linkage to the non-fucosylated acceptor and then the α1,3-fucosyltransferase completes the difucosylation. © 1998 Rapid Science Ltd     

Deletion of fucose residues in plant N‐glycans by repression of the GDP‐mannose 4,6‐dehydratase gene using virus‐induced gene silencing and RNA interference

Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant‐produced glycoproteins have N‐glycans with plant‐specific sugar residues (core β‐1,2‐xylose and α‐1,3‐fucose) and a Lewis a (Le^a) epitope, i.e., Galβ(1‐3)[Fucα(1‐4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant‐specific core α‐1,3‐fucose and α‐1,4‐fucose residues in the Le^a epitopes by repressing the Guanosine 5′‐diphosphate (GDP)‐D‐mannose 4,6‐dehydratase (GMD) gene, which is associated with GDP‐L‐fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus‐induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose‐free N‐glycans found in total soluble protein from GMD gene‐repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild‐type plants. A small amount of putative galactose substitution in N‐glycans from the NbGMD gene‐repressed plants was observed, similar to what has been previously reported GMD‐knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) with fucose‐deleted N‐glycans was successfully produced in NbGMD‐RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants.     

The effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins

The influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. In order to determine what structural features of the oligosaccharide were required for fucosylation or where in the processing pathway fucosylation occurred, influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, [5,6-3H]fucose and [1-14C]mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the [14C]mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the [14C]mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the [3H]fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the [14C]mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.     

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.     

Reduced immunogenicity of Arabidopsis hgl1 mutant N-glycans caused by altered accessibility of xylose and core fucose epitopes

Arabidopsis N-glycosylation mutants with enhanced salt sensitivity show reduced immunoreactivity of complex N-glycans. Among them, hybrid glycosylation 1 (hgl1) alleles lacking Golgi α-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry β1,2-xylose and α1,3-fucose epitopes. To dissect the contribution of xylose and core fucose residues to plant stress responses and immunogenic potential, we prepared Arabidopsis hgl1 xylT double and hgl1 fucTa fucTb triple mutants by crossing previously established T-DNA insertion lines and verified them by mass spectrometry analyses. Root growth assays revealed that hgl1 fucTa fucTb but not hgl1 xylT plants are more salt-sensitive than hgl1, hinting at the importance of core fucose modification and masking of xylose residues. Detailed immunoblot analyses with anti-β1,2-xylose and anti-α1,3-fucose rabbit immunoglobulin G antibodies as well as cross-reactive carbohydrate determinant-specific human immunoglobulin E antibodies (present in sera of allergy patients) showed that xylose-specific reactivity of hgl1 N-glycans is indeed reduced. Based on three-dimensional modeling of plant N-glycans, we propose that xylose residues are tilted by 30° because of untrimmed mannoses in hgl1 mutants. Glycosidase treatments of protein extracts restored immunoreactivity of hgl1 N-glycans supporting these models. Furthermore, among allergy patient sera, untrimmed mannoses persisting on the α1,6-arm of hgl1 N-glycans were inhibitory to immunoreaction with core fucoses to various degrees. In summary, incompletely trimmed glycoprotein N-glycans conformationally prevent xylose and, to lesser extent, core fucose accessibility. Thus, in addition to N-acetylglucosaminyltransferase I, Golgi α-mannosidase II emerges as a so far unrecognized target for lowering the immunogenic potential of plant-derived glycoproteins.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its “regular” fucosylated counterpart and a series of mixtures containing varying proportions of “regular” and afucosylated materials. Compared with the “regular” fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC₅₀ values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.