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

• Published in: PloS one Vol. 10; no. 7
• Main Authors: Kurogochi, Masaki, Mori, Masako, Osumi, Kenji, Tojino, Mami, Sugawara, Shu-ichi, Takashima, Shou, Hirose, Yuriko, Tsukimura, Wataru, Mizuno, Mamoru, Amano, Junko, Matsuda, Akio, Tomita, Masahiro, Takayanagi, Atsushi, Shoda, Shin-Ichiro, Shirai, Takashi
• Format: Journal Article
• Language: English
• Published: Public Library of Science 22.07.2015
• Subjects: Genetic engineering; Health aspects; Immunoglobulin G; Monoclonal antibodies; Oxazoles; Polysaccharides
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0132848

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-[beta]-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.sub.2-3 GlcNAc.sub.2 ), high-mannose type (Man.sub.4-9 GlcNAc.sub.2 ), and complex type (Man.sub.3 GlcNAc.sub.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.sub.3 GlcNAc.sub.1, G0; GlcNAc.sub.2 Man.sub.3 GlcNAc.sub.1, G2; Gal.sub.2 GlcNAc.sub.2 Man.sub.3 GlcNAc.sub.1, A2; NeuAc.sub.2 Gal.sub.2 GlcNAc.sub.2 Man.sub.3 GlcNAc.sub.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 Fc[gamma]RIIIa-V158 showed that the glycoform influenced the affinity for Fc[gamma]RIIIa-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.