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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-β- 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 (Man 2-3 GlcNAc 2 ), high-mannose type (Man 4-9 GlcNAc 2 ), and complex type (Man 3 GlcNAc 3-4 )  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; Man 3 GlcNAc 1 , G0; GlcNAc 2 Man 3 GlcNAc 1 , G2; Gal 2 GlcNAc 2 Man 3 GlcNAc 1 , A2; NeuAc 2 Gal 2 GlcNAc 2 Man 3 GlcNAc 1 ) 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.

plos one

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