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Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity

Monoclonal antibodies (mAbs) comprise an essential type of biologic therapeutics and are used to treat diseases because of their anti-cancer and anti-inflammatory properties, and their ability to protect against respiratory infections. Its production involves post-translational glycosylation, a bios...

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Autores principales: Hatfield, Grayson, Tepliakova, Lioudmila, Gingras, Genevieve, Stalker, Andrew, Li, Xuguang, Aubin, Yves, Tam, Roger Y.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9596277/
https://www.ncbi.nlm.nih.gov/pubmed/36304448
http://dx.doi.org/10.3389/fimmu.2022.972168
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author Hatfield, Grayson
Tepliakova, Lioudmila
Gingras, Genevieve
Stalker, Andrew
Li, Xuguang
Aubin, Yves
Tam, Roger Y.
author_facet Hatfield, Grayson
Tepliakova, Lioudmila
Gingras, Genevieve
Stalker, Andrew
Li, Xuguang
Aubin, Yves
Tam, Roger Y.
author_sort Hatfield, Grayson
collection PubMed
description Monoclonal antibodies (mAbs) comprise an essential type of biologic therapeutics and are used to treat diseases because of their anti-cancer and anti-inflammatory properties, and their ability to protect against respiratory infections. Its production involves post-translational glycosylation, a biosynthetic process that conjugates glycans to proteins, which plays crucial roles in mAb bioactivities including effector functions and pharmacokinetics. These glycans are heterogeneous and have diverse chemical structures whose composition is sensitive to manufacturing conditions, rendering the understanding of how specific glycan structures affect mAb bioactivity challenging. There is a need to delineate the effects of specific glycans on mAb bioactivity to determine whether changes in certain glycosylation profiles (that can occur during manufacturing) will significantly affect product quality. Using enzymatic transglycosylation with chemically-defined N-glycans, we show that galactosylation at a specific location of N-glycans in an afucosylated anti-viral mAb is responsible for FcγRIIIA binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. We report a facile method to obtain purified asymmetric mono-galactosylated biantennary complex N-glycans, and their influence on bioactivity upon incorporation into an afucosylated mAb. Using ELISA, surface plasmon resonance and flow cytometry, we show that galactosylation of the α6 antenna, but not the α3 antenna, consistently increases FcγRIIIA binding affinity. We confirm its relevance in an anti-viral model of respiratory syncytial virus (RSV) using an adapted ADCC reporter assay. We further correlate this structure-function relationship to the interaction of the galactose residue of the α6 antenna with the protein backbone using 2D-(1)H-(15)N-NMR, which showed that galactosylation of at this location exhibited chemical shift perturbations compared to glycoforms lacking this galactose residue. Our results highlight the importance of identifying and quantifying specific glycan isomers to ensure adequate quality control in batch-to-batch and biosimilar comparisons.
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spelling pubmed-95962772022-10-26 Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity Hatfield, Grayson Tepliakova, Lioudmila Gingras, Genevieve Stalker, Andrew Li, Xuguang Aubin, Yves Tam, Roger Y. Front Immunol Immunology Monoclonal antibodies (mAbs) comprise an essential type of biologic therapeutics and are used to treat diseases because of their anti-cancer and anti-inflammatory properties, and their ability to protect against respiratory infections. Its production involves post-translational glycosylation, a biosynthetic process that conjugates glycans to proteins, which plays crucial roles in mAb bioactivities including effector functions and pharmacokinetics. These glycans are heterogeneous and have diverse chemical structures whose composition is sensitive to manufacturing conditions, rendering the understanding of how specific glycan structures affect mAb bioactivity challenging. There is a need to delineate the effects of specific glycans on mAb bioactivity to determine whether changes in certain glycosylation profiles (that can occur during manufacturing) will significantly affect product quality. Using enzymatic transglycosylation with chemically-defined N-glycans, we show that galactosylation at a specific location of N-glycans in an afucosylated anti-viral mAb is responsible for FcγRIIIA binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. We report a facile method to obtain purified asymmetric mono-galactosylated biantennary complex N-glycans, and their influence on bioactivity upon incorporation into an afucosylated mAb. Using ELISA, surface plasmon resonance and flow cytometry, we show that galactosylation of the α6 antenna, but not the α3 antenna, consistently increases FcγRIIIA binding affinity. We confirm its relevance in an anti-viral model of respiratory syncytial virus (RSV) using an adapted ADCC reporter assay. We further correlate this structure-function relationship to the interaction of the galactose residue of the α6 antenna with the protein backbone using 2D-(1)H-(15)N-NMR, which showed that galactosylation of at this location exhibited chemical shift perturbations compared to glycoforms lacking this galactose residue. Our results highlight the importance of identifying and quantifying specific glycan isomers to ensure adequate quality control in batch-to-batch and biosimilar comparisons. Frontiers Media S.A. 2022-10-12 /pmc/articles/PMC9596277/ /pubmed/36304448 http://dx.doi.org/10.3389/fimmu.2022.972168 Text en Copyright © 2022 Hatfield, Tepliakova, Gingras, Stalker, Li, Aubin and Tam https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Hatfield, Grayson
Tepliakova, Lioudmila
Gingras, Genevieve
Stalker, Andrew
Li, Xuguang
Aubin, Yves
Tam, Roger Y.
Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity
title Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity
title_full Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity
title_fullStr Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity
title_full_unstemmed Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity
title_short Specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its FcγRIIIA binding affinity
title_sort specific location of galactosylation in an afucosylated antiviral monoclonal antibody affects its fcγriiia binding affinity
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9596277/
https://www.ncbi.nlm.nih.gov/pubmed/36304448
http://dx.doi.org/10.3389/fimmu.2022.972168
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