Preparation and biological activities of anti-HER2 monoclonal antibodies with multibranched complex-type N-glycans.

Immunoglobulin G (IgG) has a conserved N-glycosylation site at Asn297 in the fragment crystallizable (Fc) region. Previous studies have shown that N-glycosylation of this site is a critical mediator of the antibody's effector functions, such as antibody-dependent cellular cytotoxicity. While the N-glycan structures attached to the IgG-Fc region are generally heterogenous, IgGs engineered to be homogenously glycosylated with functional N-glycans may improve the efficacy of antibodies. The major glycoforms of the N-glycans on the IgG-Fc region are bi-antennary complex-type N-glycans, while multibranched complex-type N-glycans are not typically found. However, IgGs with tri-antennary complex-type N-glycans have been generated using the N-glycan remodeling technique, suggesting that more branched N-glycans might be artificially attached. At present, little is known about the properties of these IgGs. In this study, IgGs with multibranched N-glycans on the Fc region were prepared by using a combination of the glycosynthase/oxazoline substrate-based N-glycan remodeling technique and successive reactions with glycosyltransferases. Among the IgGs produced by these methods, the largest N-glycan attached was a bisecting N-acetylglucosamine containing a sialylated penta-antennary structure. Concerning the Fc-mediated effector functions, the majority of IgGs with tri- and tetra-antennary N-glycans on their Fc region showed properties similar to IgGs with ordinary bi-antennary N-glycans.

Novel endo-ß-N-acetylglucosaminidases from Tannerella species hydrolyze multibranched complex-type N-glycans with different specificities.

Endo-ß-N-acetylglucosaminidases are enzymes that hydrolyze the N,N'-diacetylchitobiose unit of N-glycans. Many endo-ß-N-acetylglucosaminidases also exhibit transglycosylation activity, which corresponds to the reverse of the hydrolysis reaction. Because of these activities, some of these enzymes have recently been used as powerful tools for glycan remodeling of glycoproteins. Although many endo-ß-N-acetylglucosaminidases have been identified and characterized to date, there are few enzymes that exhibit hydrolysis activity toward multibranched (tetra-antennary or more) complex-type N-glycans on glycoproteins. Therefore, we searched for novel endo-ß-N-acetylglucosaminidases that exhibit hydrolysis activity toward multibranched complex-type N-glycans in this study. From database searches, we selected three candidate enzymes from Tannerella species-Endo-Tsp1006, Endo-Tsp1263 and Endo-Tsp1457-and prepared them as recombinant proteins. We analyzed the hydrolysis activity of these enzymes toward N-glycans on glycoproteins and found that Endo-Tsp1006 and Endo-Tsp1263 exhibited hydrolysis activity toward complex-type N-glycans, including multibranched N-glycans, preferentially, whereas Endo-Tsp1457 exhibited hydrolysis activity toward high-mannose-type N-glycans exclusively. We further analyzed substrate specificities of Endo-Tsp1006 and Endo-Tsp1263 using 18 defined glycopeptides as substrates, each having a different N-glycan structure. We found that Endo-Tsp1006 preferred N-glycans with galactose or α2,6-linked sialic acid residues in their nonreducing ends as substrates, whereas Endo-Tsp1263 preferred N-glycans with N-acetylglucosamine residues in their nonreducing ends as substrates.

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

Typical crystallizable fragment (Fc) glycans attached to the CH2 domain in therapeutic monoclonal antibodies (mAbs) are core-fucosylated and asialo-biantennary complex-type glycans, e.g., G2F (full galactosylation), G1aF (terminal galactosylation on the Man α1-6 arm), G1bF (terminal galactosylation on the Man α1-3 arm), and G0F (non-galactosylation). Terminal galactose (Gal) residues of Fc-glycans are known to influence effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (CDC), but the impact of the G1F isomers (G1aF and G1bF) on the effector functions has not been reported. Here, we prepared four types of glycoengineered anti-CD20 mAbs bearing homogeneous G2F, G1aF, G1bF, or G0F (G2F mAb, G1aF mAb, G1bF mAb, or G0F mAb, respectively), and evaluated their biological activities. Interestingly, G1aF mAb showed higher C1q- and FcγR-binding activities, CDC activity, and FcγR-activation property than G1bF mAb. The activities of G1aF mAb and G1bF mAb were at the same level as G2F mAb and G0F mAb, respectively. Hydrogen-deuterium exchange/mass spectrometry analysis of dynamic structures of mAbs revealed the greater involvement of the terminal Gal residue on the Man α1-6 arm in the structural stability of the CH2 domain. Considering that mAbs interact with FcγR and C1q via their hinge proximal region in the CH2 domain, the structural stabilization of the CH2 domain by the terminal Gal residue on the Man α1-6 arm of Fc-glycans may be important for the effector functions of mAbs. To our knowledge, this is the first report showing the impact of G1F isomers on the effector functions and dynamic structure of mAbs. Abbreviations: ABC, ammonium bicarbonate solution; ACN, acetonitrile; ADCC, antibody-dependent cell-mediated cytotoxicity; C1q, complement component 1q; CDC, complement-dependent cytotoxicity; CQA, critical quality attribute; Endo, endo-ß-N-acetylglucosaminidase; FA, formic acid; Fc, crystallizable fragment; FcγR, Fcγ receptors; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GST, glutathione S-transferase; HER2, human epidermal growth factor receptor 2; HDX, hydrogen-deuterium exchange; HILIC, hydrophilic interaction liquid chromatography; HLB-SPE, hydrophilic-lipophilic balance-solid-phase extraction; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; Man, mannose; MS, mass spectrometry; PBS, phosphate-buffered saline; SGP, hen egg yolk sialylglycopeptides.

CDP-glycerol inhibits the synthesis of the functional O-mannosyl glycan of α-dystroglycan.

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface laminin receptor. Defects in the O-mannosyl glycan of an α-DG with laminin-binding activity can cause α-dystroglycanopathy, a group of congenital muscular dystrophies. In the biosynthetic pathway of functional O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP), whose mutated genes underlie α-dystroglycanopathy, sequentially transfer ribitol phosphate (RboP) from CDP-Rbo to form a tandem RboP unit (RboP-RboP) required for the synthesis of the laminin-binding epitope on O-mannosyl glycan. Both RboP- and glycerol phosphate (GroP)-substituted glycoforms have recently been detected in recombinant α-DG. However, it is unclear how GroP is transferred to the O-mannosyl glycan or whether GroP substitution affects the synthesis of the O-mannosyl glycan. Here, we report that, in addition to having RboP transfer activity, FKTN and FKRP can transfer GroP to O-mannosyl glycans by using CDP-glycerol (CDP-Gro) as a donor substrate. Kinetic experiments indicated that CDP-Gro is a less efficient donor substrate for FKTN than is CDP-Rbo. We also show that the GroP-substituted glycoform synthesized by FKTN does not serve as an acceptor substrate for FKRP and that therefore further elongation of the outer glycan chain cannot occur with this glycoform. Finally, CDP-Gro inhibited the RboP transfer activities of both FKTN and FKRP. These results suggest that CDP-Gro inhibits the synthesis of the functional O-mannosyl glycan of α-DG by preventing further elongation of the glycan chain. This is the first report of GroP transferases in mammals.

Preparation and biological activities of anti-HER2 monoclonal antibodies with fully core-fucosylated homogeneous bi-antennary complex-type glycans.

Recently, the absence of a core-fucose residue in the N-glycan has been implicated to be important for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity of immunoglobulin G monoclonal antibodies (mAbs). Here, we first prepared anti-HER2 mAbs having two core-fucosylated N-glycan chains with the single G2F, G1aF, G1bF, or G0F structure, together with those having two N-glycan chains with a single non-core-fucosylated corresponding structure for comparison, and determined their biological activities. Dissociation constants of mAbs with core-fucosylated N-glycans bound to recombinant Fcγ-receptor type IIIa variant were 10 times higher than those with the non-core-fucosylated N-glycans, regardless of core glycan structures. mAbs with the core-fucosylated N-glycans had markedly reduced ADCC activities, while those with the non-core-fucosylated N-glycans had high activities. These results indicate that the presence of a core-fucose residue in the N-glycan suppresses the binding to the Fc-receptor and the induction of ADCC of anti-HER2 mAbs.

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-ß-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.

Glycosylation of Nα-lauryl-O-(ß-D-xylopyranosyl)-L-serinamide as a saccharide primer in cells.

N(α)-Lauryl-O-(ß-D-xylopyranosyl)-L-serinamide (Xyl-Ser-C12) was synthesized as a saccharide primer to obtain oligosaccharides of glycosaminoglycan using the glycan biosynthetic potential of mouse osteosarcoma FBJ-S1 cells and Chinese hamster ovary (CHO) cells. The glycosylated products secreted into the culture medium were collected and analyzed by liquid chromatography-mass spectrometry and glycosidase digestion. The structure of the Xyl-Ser-C12 derivatives was investigated. Several glycosaminoglycan-type oligosaccharides, such as GalNAc-(GlcA-GlcNAc)(n)-GlcA-Gal-Gal-Xyl-Ser-C12, were detected, and identified as intermediates of the biosynthesis of heparan sulfate glycosaminoglycans. Xyl-Ser-C12 exhibited greater acceptor activity for the glycosylation of glycosaminoglycan-type oligosaccharides than p-nitrophenyl-ß-D-xylopyranoside.

Glycoside hydrolase family 89 alpha-N-acetylglucosaminidase from Clostridium perfringens specifically acts on GlcNAc alpha1,4Gal beta1R at the non-reducing terminus of O-glycans in gastric mucin.

In mammals, α-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. α-N-acetylglucosaminidases (αGNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher eukaryotes. Human lysosomal αGNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity of αGNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human αGNase, by chemically synthesizing a series of disaccharide substrates containing α-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAcα1,4Galß1pMP and GlcNAcα1pNP substrates (where pMP and pNP represent p-methoxyphenyl and p-nitrophenyl, respectively). AgnC also released GlcNAc from porcine gastric mucin and cell surface mucin. Because AgnC showed no activity against any of the GlcNAcα1,2Galß1pMP, GlcNAcα1,3Galß1pMP, GlcNAcα1,6Galß1pMP, and GlcNAcα1,4GlcAß1pMP substrates, this enzyme may represent a specific glycosidase required for degrading α-GlcNAc-capped O-glycans of the class III mucin secreted from the stomach and duodenum. Deletion of the C-terminal region containing several carbohydrate-binding module 32 (CBM32) domains significantly reduced the activity for porcine gastric mucin; however, activity against GlcNAcα1,4Galß1pMP was markedly enhanced. Dot blot and ELISA analyses revealed that the deletion construct containing the C-terminal CBM-C2 to CBM-C6 domains binds strongly to porcine gastric mucin. Consequently, tandem CBM32 domains located near the C terminus of AgnC should function by increasing the affinity for branched or clustered α-GlcNAc-containing glycans. The agnC gene-disrupted strain showed significantly reduced growth on the class III mucin-containing medium compared with the wild type strain, suggesting that AgnC might have an important role in dominant growth in intestines.

Derivatization with 1-pyrenyldiazomethane enhances ionization of glycopeptides but not peptides in matrix-assisted laser desorption/ionization mass spectrometry.

Glycoproteomics holds the promise of new advances in medical technology. However, mass spectrometry has limitations for the structural determination of glycosylated peptides because the hydrophilic nature of the oligosaccharide moiety in glycopeptides is disadvantageous for ionization, and glycopeptides ionize much less readily than nonglycosylated peptides. Therefore, conventional proteomics tools cannot detect altered glycosylation on proteins. Here, we describe an on-plate pyrene derivatization method using 1-pyrenyldiazomethane for highly sensitive matrix-assisted laser/desorption ionization-tandem mass spectrometry (MALDI-MS(n)) of glycopeptides in amounts of less than 100 fmol. This derivatization is unique, as the pyrene groups are easily released from glycopeptides during ionization when 2,5-dihydroxybenzoic acid is used as a matrix. As a result, most ions are observed as the underivatized form on the spectra. At the same time, pyrene derivatization dramatically reduces the ionization of peptides. Thus, for glycopeptides in a mixture of abundant peptides, we could obtain MS spectra in which the signals of glycopeptides were intense enough for subjection to MS(n) in order to determine the structures of both glycan and peptide. Finally, we show that the glycopeptides derived from as little as 1 ng of prostate specific antigen can be detected by this method.

Negative-ion MALDI-QIT-TOFMSn for structural determination of fucosylated and sialylated oligosaccharides labeled with a pyrene derivative.

Oligosaccharides have many isomers and MALDI-QIT-TOFMS(n) analysis is effective for determining their structures. However, it is difficult to elucidate in detail the structures of fucosylated and/or sialylated oligosaccharides that are known to be disease markers because fucose and sialic acid residues are easily released. We have introduced a technique of labeling oligosaccharides with a pyrene derivative prior to negative-ion MALDI-QIT-TOFMS(n), and we have established a reliable method using this technique for the analysis of neutral oligosaccharides, such as fucosylated oligosaccharides containing blood group antigens H, Le(a), and Le(x). Intense and stable ionization in both positive and negative modes was achieved by derivatization with pyrene. As little as 10 fmol of pyrene-labeled oligosaccharides gave sufficient signals for analysis. Specific A-, D- or Y-type ions that depend on the structures of branching antennae could be detected by MS(n) and were useful for rapid and easy structural determination. These specific fragmentations resulting from collision-induced dissociation can be used to elucidate the structures of unknown oligosaccharides even if authentic oligosaccharides are not available as standards. By using this method, we identified and quantitated isomeric oligosaccharides with different fucosyl linkages from their mixtures. Moreover, sialylated oligosaccharide was converted to the corresponding neutral oligosaccharide by amidation, and the negative-ion spectrum was shown to be more informative than that of the original acidic oligosaccharide. Structural determination of both fucosylated and sialylated isomers, such as sialylfucosyllacto-N-hexaose I and monosialyl monofucosyllacto-N-neohexaose, was successful because fragment ions bearing fucose or amidated sialic acid were obtained on negative-MS(n).

Structural determination by negative-ion MALDI-QIT-TOFMSn after pyrene derivatization of variously fucosylated oligosaccharides with branched decaose cores from human milk.

We prepared neutral oligosaccharide fraction from milk of a woman (blood type A, Le(b+)) by anion-exchange column chromatography after the removal of lipids and proteins. Further fractionation was performed by means of Aleuria aurantia lectin-Sepharose column chromatography and reverse-phase HPLC after labeling with a pyrene derivative. This pyrene labeling allowed identification by negative-MALDI-TOFMS(n) analysis of 22 oligosaccharides with decaose cores, among which 21 had novel structures. Negative ions could not be produced from neutral oligosaccharides without labeling on MALDI. Mono-, di-, tri-, and tetrafucosylated decaose fractions contained three, nine, six, and four isomers, respectively. Our method enables easy determination of fucosylated structures on the N-acetyllactosamine branches of these isomers. On negative-MS(n) the fragment ions included several A and D ions, from which fucosylation on the branches could be elucidated. Other characteristic ions were also detected. Y-type cleavage at the reducing side of -3GlcNAc indicated the occurrence of type 1 chain. Specific fragment ions were produced from H, Le(a), and Le(x) antigens. Linkage-specific exoglycosidase digestion confirmed the structures. The results indicate that the diversity of the oligosaccharides is due to combinations of type 1 H, Le(a), Le(x), and Le(b)/Le(y) on branched decaose cores. In typical oligosaccharides, 6-branches always consist of type 2 chain, while 3-branches, such as beta and gamma chains, are fucosylated type 1 chains. From the viewpoint of biosynthesis, the presence of fucosylation and type 1 chain may halt elongation of the N-acetyllactosamine and promote formation of branched structures.

Synthesis of mono-glucose-branched cyclodextrins with a high inclusion ability for doxorubicin and their efficient glycosylation using Mucor hiemalis endo-beta-N-acetylglucosaminidase.

The mono-glucose-branched cyclodextrins having an appropriate spacer between the beta-cyclodextrin and a glucose moiety were synthesized from beta-cyclodextrin and arbutin. They had the significantly high association constants for doxorubicin, the anticancer agent, in the range of 10(5)-10(6)M(-1), and worked as highly reactive glycosyl acceptors for the transglycosylation reaction by endo-beta-N-acetylglucosaminidase of Mucor hiemalis to produce sialo-complex type oligosaccharide-branched cyclodextrins in the high yields of 65-67%.

Mucor hiemalis endo-beta-N-acetylglucosaminidase can transglycosylate a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide.

We found that the recombinant endo-beta-N-acetylglucosaminidase of Mucor hiemalis (Endo-M) expressed in Candida boidinii had the transglycosylation activity of transferring a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide to the acceptor (p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside) in a good yield of 43%.

Transglycosylation reaction of Mucor hiemalis endo-beta-N-acetylglucosaminidase using sugar derivatives modified at C-1 or C-2 as oligosaccharide acceptors.

We investigated the transglycosylation reaction of the recombinant endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) expressed in Candida boidinii using such sugar derivatives as N-acylated d-glucosamines, C-glucosyl derivatives, and a 2-O-glycosylated disaccharide as acceptors. We found that a variety of sugar derivatives modified at C-1 or C-2 could be used as acceptors for transglycosylation by Endo-M to create novel oligosaccharides.

High efficiency of transferring a native sugar chain from a glycopeptide by a microbial endoglycosidase in organic solvents.

We examined the transglycosylation reaction by the recombinant endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) expressed in Candida boidinii in media containing organic solvents. The recombinant Endo-M could transglycosylate a disialo biantennary complex-type oligosaccharide from hen egg yolk glycopeptide to p-nitrophenyl N-acetyl-beta-D-glucosaminide even in the presence of 30% acetone, dimethyl sulfoxide, or methanol. The yield of the transglycosylation product reached 21-34% of the total amount of acceptor, while the yield was only about 14% in aqueous solution.