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

<div><p>Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain <i>N</i>-glycans at the constant region of the heavy chain (Fc domain), and their <i>N</i>-glycosylation patterns differ during various processes or among cell expression systems. The Fc <i>N</i>-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc <i>N</i>-glycans, we performed a rearrangement of Fc <i>N</i>-glycans from a heterogeneous <i>N</i>-glycosylation pattern to homogeneous <i>N</i>-glycans using chemoenzymatic approaches with two types of endo-β-<i>N</i>-acetyl glucosaminidases (ENG’ases), one that works as a hydrolase to cleave all heterogeneous <i>N</i>-glycans, another that is used as a glycosynthase to generate homogeneous <i>N</i>-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man_2-3GlcNAc₂), high-mannose type (Man_4-9GlcNAc₂), and complex type (Man₃GlcNAc_3-4) <i>N</i>-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 <i>N</i>-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 <i>N</i>-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.</p></div