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.

Integrated Genome and Protein Editing Swaps α‐2,6 Sialylation for α‐2,3 Sialic Acid on Recombinant Antibodies from CHO

“…For example, antibodies having glycans on the Fc region without the core fucosylation exhibited higher antibody‐dependent cellular cytotoxicity (ADCC) than its fucosylated counterparts . Interestingly, IgGs with enhanced α ‐2,6 sialylation have been recently observed to demonstrate superior ADCC compared to the conventional IgGs . What's more, intravenous immunoglobulin (IVIG) with sialylation on the glycan of the Fc portion has been reported to exhibit superior anti‐inflammatory efficacy than its asialylated counterparts.…”

Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands

“…However, using iv ERT with recombinant human enzyme preparations produced by mammalian cell lines to treat LSDs has several disadvantages, i.e. i) large-scale production of recombinant human lysosomal enzymes by mammalian cells is limited and costly in comparison to the large-scale production of therapeutic antibodies (5)(6)(7) and ii) the risk of pathogen infection and the risk of an immune response and adverse effects including production of neutralizing antibodies against enzyme preparations in patients with an LSD and an enzyme deficiency as a result of continuous administration (18). Alternative human glycoprotein expression systems need to be developed as more effective, safer, and cheaper lysosomal enzyme sources to overcome these problems.…”

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans

“…Recently, Endo-F3 was converted to a glycosynthase, and the mutant enzyme was capable of transferring bi-and triantennary complex type N-glycans using sugar oxazoline donor substrates to synthesize core-fucosylated complex glycopeptides (42). Endo-S and its glycosynthase, which specifically and efficiently acts on the IgG-Fc domain of N-glycans, have been used for chemoenzymatic synthesis of IgGs with structurally defined glycoforms for functional studies (43)(44)(45). More recently, Endo-S2 was shown to have a more flexible substrate specificity and high efficiency in transferring complex, hybrid, and high mannose-type N-glycans onto corefucosylated or non-fucosylated IgG molecules (46).…”