Fushimi Pharmaceutical Co., Ltd.

Immunoglobulin G (IgG) antibodies possess a conserved N-glycosylation site in the Fc domain. In FcγRIIIa affinity column chromatography, unglycosylated, hemiglycosylated, and fully glycosylated IgG retention times differ considerably. Using retention-time differences, 66 different trastuzumab antibodies with symmetric and asymmetric homogeneous glycans were prepared systematically, substantially expanding the scope of IgGs with homogeneous glycans. Using the prepared trastuzumab with homogeneous glycans, thermal stability and antibody-dependent cellular cytotoxicity were investigated. In some glycan series, a directly proportional relationship was observed between the thermal unfolding temperature (T_m) and the calorimetric unfolding heat (ΔH_cal). Antibody function could be deduced from the combination of a pair of glycans in an intact form. Controlling glycan structure through the combination of a pair of glycans permits the precise tuning of stability and effector functions of IgG. Overall, our technology can be used to investigate the effects of glycans on antibody functions.

Endo-β-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze N-linked glycans. Many ENGases have been characterized, but few have been identified with hydrolytic activity towards multi-branched complex-type N-glycans. In this study, three candidate ENGases were identified from Barnesiella intestinihominis based on database searches and phylogenetic analysis. A domain search identified the N x E motif in all three candidates, suggesting that they were members of glycosyl hydrolase family 85 (GH85). The three candidate ENGases, named Endo-BIN1, Endo-BIN2, and Endo-BIN3, were expressed in Escherichia coli cells, and their hydrolytic activity towards N-glycans and glycoproteins was measured by high performance liquid chromatography analysis and SDS-PAGE analysis. All ENGases showed hydrolytic activity towards glycoproteins, but only Endo-BIN2 and Endo-BIN3 showed hydrolytic activity towards pyridylaminated N-glycans. The optimum pH of Endo-BIN1, Endo-BIN2, and End-BIN3 was pH 6.5, 4.0, and 7.0, respectively. We measured substrate specificities of Endo-BIN2 and Endo-BIN3 towards pyridylaminated N-glycans, and found that the two Endo-BIN enzymes showed similar substrate specificity, preferring bi-antennary complex-type N-glycans with galactose or α2,6-linked sialic acid residues at the non-reducing ends. Endo-BIN2 and Endo-BIN3 were also able to hydrolyze multi-branched complex-type N-glycans. SDS-PAGE analysis revealed that all Endo-BIN enzymes were capable of releasing complex-type N-glycans from glycoproteins such as rituximab, transferrin, and fetuin. We expect that B. intestinihominis possesses ENGases to facilitate the utilization of complex-type N-glycans from host cells. These findings will have applications in N-glycan remodeling of glycoproteins and the development of pharmaceuticals.

N-Linked glycosylation on IgG has a profound impact on antibody functions. The relationship between the N-glycan structure and the binding affinity of FcγRIIIa, relating to antibody-dependent cell-mediated cytotoxicity (ADCC) activity, is important for the efficient development of a therapeutic antibody. Here, we report an influence of the N-glycan structure of IgGs, Fc fragments, and antibody-drug conjugates (ADCs) on FcγRIIIa affinity column chromatography. We compared the retention time of several IgGs with heterogeneous and homogeneous N-glycans. IgGs with a heterogeneous N-glycan structure provided several peaks in column chromatography. On the other hand, homogeneous IgGs and ADCs gave a single peak in column chromatography. The length of glycan on IgG also affected the retention time of the FcγRIIIa column, suggesting that the length of glycan is also impacted by binding affinity to FcγRIIIa, resulting in ADCC activity. This analytic methodology provides evaluation of the binding affinity of FcγRIIIa and ADCC activity, not only full-length IgG but also Fc fragments, which are difficult to measure in a cell-based assay. Furthermore, we showed that the glycan-remodeling strategy controls the ADCC activity of IgGs, Fc fragment, and ADCs.