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.
Highlights
Monoclonal antibodies of IgG have become important therapeutic agents for numerous diseases such as cancer, autoimmune, and infectious diseases [1, 2, 3]
IgG antibodies are composed of two heavy chains and two light chains, which can be divided into two regions based on amino acid sequence variability: the fragment antigen binding (Fab) region can recognize specific antigens, while the fragment crystallizable (Fc) region plays a role in modulating immune cell activity, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) [4, 5]
Hatched larvae were allowed to develop to moths, and embryos obtained by mating the moths were screened for MGFP expression to obtain transgenic silkworms carrying anti-human epidermal growth factor receptor 2 (Her2) Monoclonal antibodies (mAbs) cDNAs
Summary
Monoclonal antibodies (mAbs) of IgG have become important therapeutic agents for numerous diseases such as cancer, autoimmune, and infectious diseases [1, 2, 3]. Several cell lines (yeast, CHO cells, etc.) can produce more authentic glycoproteins with non-fucosylated complex type N-glycans using several genetic manipulations containing glyco-related enzymes genes, glycoproteins produced from silkworm cocoon do not contain a core fucosylated glycan without gene knockdown or knockout, unlike other tissues (fat body) [25]. Their Fc N-glycans doi:10.1371/journal.pone.0132848.g001 consist of the non-fucosylated pauci-mannose (Man2-3GlcNAc2), high-mannose (Man49GlcNAc2), and complex types (Man3GlcNAc3-4). We performed an ADCC-reporter gene assay for the glycoengineered anti-Her mAbs using SKBR-3 and BT-474 cells with high Her expression (~1 × 106 molecules per cell; [31]) and Jurkat/ FcγRIIIa/NFAT-Luc cells [32]
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