title: 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.
abstract: 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 (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.
authors.affiliation: Laboratory of Glycobiology, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Kurogochi
authors.affiliation: Laboratory of Glyco-Bioengineering, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Mori
authors.affiliation: Laboratory of Glyco-organic Chemistry, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Osumi
authors.affiliation: Laboratory of Glyco-organic Chemistry, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Tojino
authors.affiliation: Laboratory of Glyco-organic Chemistry, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Shu-Ichi
authors.initials: S
authors.lastname: Sugawara
authors.affiliation: Laboratory of Glycobiology, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Shou
authors.initials: S
authors.lastname: Takashima
authors.affiliation: Laboratory of Glyco-organic Chemistry, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Hirose
authors.affiliation: Laboratory of Glyco-Bioengineering, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Tsukimura
authors.affiliation: Laboratory of Glyco-organic Chemistry, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Mizuno
authors.affiliation: Laboratory of Glycobiology, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Amano
authors.affiliation: Laboratory of Glyco-Bioengineering, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Matsuda
authors.affiliation: Immuno-Biological Laboratories Co., Ltd., 1091-1 Naka, Fujioka-shi, Gunma, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tomita
authors.affiliation: Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takayanagi
authors.affiliation: Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai, Japan.
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Shoda
authors.affiliation: Laboratory of Glycobiology, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan; Laboratory of Glyco-Bioengineering, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan; Laboratory of Glyco-organic Chemistry, The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shirai
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D000920:Antibody-Dependent Cell Cytotoxicity; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry*; D000068878:Trastuzumab / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D011134:Polysaccharides; D000118:Acetylglucosaminidase; D000068878:Trastuzumab