Dataset Information

Effects of terminal galactose residues in mannose ?1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies.

ABSTRACT: Typical crystallizable fragment (Fc) glycans attached to the CH2 domain in therapeutic monoclonal antibodies (mAbs) are core-fucosylated and asialo-biantennary complex-type glycans, e.g., G2F (full galactosylation), G1aF (terminal galactosylation on the Man ?1-6 arm), G1bF (terminal galactosylation on the Man ?1-3 arm), and G0F (non-galactosylation). Terminal galactose (Gal) residues of Fc-glycans are known to influence effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (CDC), but the impact of the G1F isomers (G1aF and G1bF) on the effector functions has not been reported. Here, we prepared four types of glycoengineered anti-CD20 mAbs bearing homogeneous G2F, G1aF, G1bF, or G0F (G2F mAb, G1aF mAb, G1bF mAb, or G0F mAb, respectively), and evaluated their biological activities. Interestingly, G1aF mAb showed higher C1q- and Fc?R-binding activities, CDC activity, and Fc?R-activation property than G1bF mAb. The activities of G1aF mAb and G1bF mAb were at the same level as G2F mAb and G0F mAb, respectively. Hydrogen-deuterium exchange/mass spectrometry analysis of dynamic structures of mAbs revealed the greater involvement of the terminal Gal residue on the Man ?1-6 arm in the structural stability of the CH2 domain. Considering that mAbs interact with Fc?R and C1q via their hinge proximal region in the CH2 domain, the structural stabilization of the CH2 domain by the terminal Gal residue on the Man ?1-6 arm of Fc-glycans may be important for the effector functions of mAbs. To our knowledge, this is the first report showing the impact of G1F isomers on the effector functions and dynamic structure of mAbs. Abbreviations: ABC, ammonium bicarbonate solution; ACN, acetonitrile; ADCC, antibody-dependent cell-mediated cytotoxicity; C1q, complement component 1q; CDC, complement-dependent cytotoxicity; CQA, critical quality attribute; Endo, endo-?-N-acetylglucosaminidase; FA, formic acid; Fc, crystallizable fragment; Fc?R, Fc? receptors; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GST, glutathione S-transferase; HER2, human epidermal growth factor receptor 2; HDX, hydrogen-deuterium exchange; HILIC, hydrophilic interaction liquid chromatography; HLB-SPE, hydrophilic-lipophilic balance-solid-phase extraction; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; Man, mannose; MS, mass spectrometry; PBS, phosphate-buffered saline; SGP, hen egg yolk sialylglycopeptides.

SUBMITTER: Aoyama M

PROVIDER: S-EPMC6601563 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies.

Aoyama Michihiko M Hashii Noritaka N Tsukimura Wataru W Osumi Kenji K Harazono Akira A Tada Minoru M Kiyoshi Masato M Matsuda Akio A Ishii-Watabe Akiko A

mAbs 20190508 5

Typical crystallizable fragment (Fc) glycans attached to the CH2 domain in therapeutic monoclonal antibodies (mAbs) are core-fucosylated and asialo-biantennary complex-type glycans, e.g., G2F (full galactosylation), G1aF (terminal galactosylation on the Man α1-6 arm), G1bF (terminal galactosylation on the Man α1-3 arm), and G0F (non-galactosylation). Terminal galactose (Gal) residues of Fc-glycans are known to influence effector functions such as antibody-dependent cell-mediated cytotoxicity and ...[more]

PMID: 30990348

Similar Datasets

Project description:Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as host susceptibility factors. GII.13 and GII.21 huNoVs form a unique genetic lineage that emerged from mainstream GII NoVs via development of a new, nonconventional glycan binding site (GBS) that binds Lea antigen. This previous finding raised the question of whether the new GII.13/21 GBS really has such a narrow glycan binding spectrum. In this study, we provide solid phenotypic and structural evidence indicating that this new GBS recognizes a group of glycans with a common terminal β-galactose (β-Gal). First, we found that P domain proteins of GII.13/21 huNoVs circulating at different times bound three glycans sharing a common terminal β-Gal, including Lec, lactose, and mucin core 2. Second, we solved the crystal structures of the GII.13 P dimers in complex with Lec and mucin core 2, which showed that β-Gal is the major binding saccharide. Third, nonfat milk and lactose blocked the GII.13/21 P domain-glycan binding, which may explain the low prevalence of GII.13/21 viruses. Our data provide new insight into the host interactions and epidemiology of huNoVs, which would help in the control and prevention of NoV-associated diseases.IMPORTANCE Evidence from both phenotypic binding assay and structural study support the observed interactions of human noroviruses (huNoVs) with histo-blood group antigens (HBGAs) as receptors or attachment factors, affecting their host susceptibility. GII.13 and GII.21 genotypes form a unique genetic lineage that differs from the mainstream GII huNoVs in their unconventional glycan binding site. Unlike the previous findings that GII.13/21 genotypes recognize only Lea antigen, we found in this study that they can interact with a group of glycans with a common terminal β-Gal, including Lec, lactose, and mucin core 2. However, this wide glycan binding spectrum in a unique binding mode of the GII.13/21 huNoVs appears not to increase their prevalence, probably due to the existence of decoy glycan receptors in human gastrointestinal tract limiting their infection. Our findings shed light on the host interaction and epidemiology of huNoVs, which would impact the strategy of huNoV control and prevention.

Project description:Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (Fc?Rs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for Fc?R and complement activity. Bisection had no effect on Fc?R or C1q-binding, and sialylation had no- or little effect on Fc?R binding. We confirmed that hypo-fucosylation of IgG1 increased binding to Fc?RIIIa and Fc?RIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for Fc?RIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for Fc?R- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for Fc?RIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.

Project description:Effective characterization of protein-based therapeutic candidates such as monoclonal antibodies (mAbs) is important to facilitate their successful progression from early discovery and development stages to marketing approval. One challenge relevant to biopharmaceutical development is, understanding how the stability of a protein is affected by the presence of an attached oligosaccharide, termed a glycan. To explore the utility of molecular dynamics simulations as a complementary technique to currently available experimental methods, the Fc fragment was employed as a model system to improve our understanding of protein stabilization by glycan attachment. Long molecular dynamics simulations were performed on three Fc glycoform variants modeled using the crystal structure of a human IgG1 mAb. Two of these three glycoform variants have their glycan carbohydrates partially or completely removed. Structural differences among the glycoform variants during simulations suggest that glycan truncation and/or removal can cause quaternary structural deformation of the Fc as a result of the loss or disruption of a significant number of inter-glycan contacts that are not formed in the human IgG1 crystal structure, but do form during simulations described here. Glycan truncation/removal can also increase the tertiary structural deformation of CH2 domains, demonstrating the importance of specific carbohydrates toward stabilizing individual CH2 domains. At elevated temperatures, glycan truncation can also differentially affect structural deformation in locations (Helix-1 and Helix-2) that are far from the oligosaccharide attachment point. Deformation of these helices, which form part of the FcRn, could affect binding if these regions are unable to refold after temperature normalization. During elevated temperature simulations of the deglycosylated variant, CH2 domains collapsed onto CH3 domains. Observations from these glycan truncation/removal simulations have improved our understanding on how glycan composition can affect mAb stability.

Dataset Information

Effects of terminal galactose residues in mannose ?1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies.

Publications

Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets