Development of glycosynthases with broad glycan specificity for the efficient glyco-remodeling of antibodies

Shivatare, Sachin S.; Huang, Li-Hsin; Zeng, Yi‐Fang; Liao, Jung-Yu; You, Tsai-Hong; Wang, Shiyun; Cheng, Ting-Jen Rachel; Chiu, Chih‐Wei; Chao, Ping; Chen, Li-Tzu; Tsai, Tsung-I; Huang, Chien‐Hao; Wu, Chung‐Yi; Lin, Nan‐Horng; Wong, Chi‐Huey

doi:10.1039/c8cc03384f

Cited by 33 publications

(41 citation statements)

References 38 publications

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“…Since our results demonstrated significant statistical interaction between core fucosylation and terminal galactosylation, and are in strong agreement with previous studies utilizing mAbs with highly homogeneous glycoforms, [37][38][39]42 we hypothesize that the interpretation of results could be compromised when evaluating mixtures of different types of glycoform. Our evaluations of the effects of terminal sialylation in FcγRIIIa binding analysis by SPR and ADCC reporter bioassay using N-glycosylated ligand agree with previous reports that have described reduction of FcγRIIIa affinity, 13,38,40,46,49,50 but found no evidence of an unchanged affinity as previously reported. 30,34,51,52 For terminally sialylated glycoforms and terminally mannosylated mAb-HM, results regarding FcγRIIIa shown in SPR and ADCC reporter bioassay were significantly different from affinity chromatography analysis.…”

Section: Effector Functions and Affinity Characteristics To Fc Receptorssupporting

confidence: 92%

Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms

Wada

Matsui

Kawasaki

2018

mAbs

128

115

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Glycosylation of the conserved asparagine residue in each heavy chain of IgG in the CH2 domain is known as N-glycosylation. It is one of the most common post-translational modifications and important critical quality attributes of monoclonal antibody (mAb) therapeutics. Various studies have demonstrated the effects of the Fc N-glycosylation on safety, Fc effector functions, and pharmacokinetics, both dependent and independent of neonatal Fc receptor (FcRn) pathway. However, separation of various glycoforms to investigate the biological and functional relevance of glycosylation is a major challenge, and existing studies often discuss the overall impact of N-glycans, without considering the individual contributions of each glycoform when evaluating mAbs with highly heterogeneous distributions. In this study, chemoenzymatic glycoengineering incorporating an endo-β-N-acetylglucosaminidase (ENGase) EndoS2 and its mutant with transglycosylation activity was used to generate mAb glycoforms with highly homogeneous and well-defined N-glycans to better understand and precisely evaluate the effect of each N-glycan structure on Fc effector functions and protein stability. We demonstrated that the core fucosylation, non-reducing terminal galactosylation, sialylation, and mannosylation of IgG1 mAb N-glycans impact not only on FcγRIIIa binding, antibodydependent cell-mediated cytotoxicity, and C1q binding, but also FcRn binding, thermal stability and propensity for protein aggregation.

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Section: Effector Functions and Affinity Characteristics To Fc Receptorssupporting

confidence: 92%

Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms

Wada

Matsui

Kawasaki

2018

mAbs

128

115

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“…Several research groups have correlated this effect to a reduced affinity for FcγRIIIa; others associated sialylation to increased conformation flexibility of the IgG Fc, which would in turn favour binding to DC-SIGN [ 14 , 53 ]. However, the effects of sialylation are still debated, with others showing that α2,6-sialylation has no significant influence on the IgG-FcγRIIIa interactions (compared to terminal galactosylation) and that the observed anti-inflammatory activity was independent of sialylation [ 46 , 54 , 55 , 56 , 57 ]. On the other hand, terminal galactosylation increases the affinity between IgG and FcγRIIIa [ 14 , 27 , 46 , 54 , 56 ].…”

Section: Igg-fcγr Interactionmentioning

confidence: 99%

“…Finally, the use of endoglycosidases being specific to the whole glycan tree rather than only one residue was also explored. In this approach, an endoglycosidase is able to remove the N-glycans of a protein while another endoglycosidase transfers a new already-assembled glycan [ 53 , 55 , 56 , 88 ]. However, these in vitro engineering approaches remain expensive, long, and poorly scalable as of today [ 54 ].…”

Section: Toward a Better Understanding Of The Igg-fcγr Interactionmentioning

confidence: 99%

On the Use of Surface Plasmon Resonance Biosensing to Understand IgG-FcγR Interactions

Forest-Nault

Gaudreault

Henry

et al. 2021

IJMS

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Surface plasmon resonance (SPR)-based optical biosensors offer real-time and label-free analysis of protein interactions, which has extensively contributed to the discovery and development of therapeutic monoclonal antibodies (mAbs). As the biopharmaceutical market for these biologics and their biosimilars is rapidly growing, the role of SPR biosensors in drug discovery and quality assessment is becoming increasingly prominent. One of the critical quality attributes of mAbs is the N-glycosylation of their Fc region. Other than providing stability to the antibody, the Fc N-glycosylation influences immunoglobulin G (IgG) interactions with the Fcγ receptors (FcγRs), modulating the immune response. Over the past two decades, several studies have relied on SPR-based assays to characterize the influence of N-glycosylation upon the IgG-FcγR interactions. While these studies have unveiled key information, many conclusions are still debated in the literature. These discrepancies can be, in part, attributed to the design of the reported SPR-based assays as well as the methodology applied to SPR data analysis. In fact, the SPR biosensor best practices have evolved over the years, and several biases have been pointed out in the development of experimental SPR protocols. In parallel, newly developed algorithms and data analysis methods now allow taking into consideration complex biomolecular kinetics. In this review, we detail the use of different SPR biosensing approaches for characterizing the IgG-FcγR interactions, highlighting their merit and inherent experimental complexity. Furthermore, we review the latest SPR-derived conclusions on the influence of the N-glycosylation upon the IgG-FcγR interactions and underline the differences and similarities across the literature. Finally, we explore new avenues taking advantage of novel computational analysis of SPR results as well as the latest strategies to control the glycoprofile of mAbs during production, which could lead to a better understanding and modelling of the IgG-FcγRs interactions.

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“…Moreover, Shivatare et al found that the T138Q mutant of Endo-S2 also reduced hydrolytic activity, though it maintained transglycosylation activity, and generated antibodies with new glycoforms of various high-mannose, hybrid and complex type oligosaccharides. 48) A residue in the proximity of the catalytic domain like Thr-138 might also modulate the glycosynthase activity. These results stimulated further studies on the remodeling of whole bodies of therapeutic mAbs and analyses of their biofunctions.…”

Section: Use Of Transglycosylation Activity Of Endoglycosidases For Igg Remodelingmentioning

confidence: 99%

Innovative Preparation of Biopharmaceuticals Using Transglycosylation Activity of Microbial Endoglycosidases

Katoh

Yamamoto

2021

J. Appl. Glycosci.

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Most functional biopharmaceuticals such as antibodies are glycoproteins carrying N-linked oligosaccharides (N-glycans). In animal cells, these glycans are generally expressed as heterogeneous glycoforms that are difficult to separate into a pure form. The structure of these glycans directly affects several biological aspects of the glycoproteins, especially binding affinity. Therefore, the preparation of glycoproteins with well-defined and homogeneous glycoforms is necessary for functional studies and improved efficacy, particularly for biopharmaceuticals. This review describes the recent remarkable progress in the development and production of biopharmaceutical glycan-modified antibodies, through the use of glycan remodeling using microbial endoglycosidases and sophisticated glycoengineering techniques utilizing microbial enzymatic reaction mechanisms.

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Development of glycosynthases with broad glycan specificity for the efficient glyco-remodeling of antibodies

Cited by 33 publications

References 38 publications

Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms

Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms

On the Use of Surface Plasmon Resonance Biosensing to Understand IgG-FcγR Interactions

Innovative Preparation of Biopharmaceuticals Using Transglycosylation Activity of Microbial Endoglycosidases

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