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.

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.

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.

Serum protein adsorption and platelet adhesion on pluronic-adsorbed polysulfone membranes.

We examined plasma protein adsorption and platelet adhesion to polysulfone (PSf) flat membranes coated with Pluronic with varying polyethylene oxide (PEO) block length. Adsorption of albumin, globulin and fibrinogen to Pluronic-coated PSf membranes was independent of plasma dilution when concentrations of human blood plasma above 20% were applied. Increasing coating concentrations of aqueous Pluronic solution resulted in decreased protein adsorption by the PSf membranes. Pluronic F68, which was more hydrophilic than Pluronic L62 or L64 and had 80% of PEO content, was the most effective at suppressing the adsorption of plasma proteins and platelet adhesion to PSf membranes. We developed a mixed protein solution containing human albumin, gamma-globulin and fibrinogen to attempt to mimic the competitive and cooperative binding effects found in plasma. Fibrinogen adsorption from plasma could be recapitulated by the mixed protein solution. The number of platelets adhering to the PSf membranes decreased as the coating concentration of Pluronic solution was increased, and platelet adhesion decreased in parallel with fibrinogen adsorption. These results suggest that the bioinert property of PEO segments in the Pluronic, which is ascribed to their high flexibility in aqueous media, suppresses the adsorption of plasma proteins and platelets to the Pluronic-coated PSf membranes.