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Engineering Hydrophobic Protein–Carbohydrate Interactions to Fine-Tune Monoclonal Antibodies
[Image: see text] Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is med...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2013
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788586/ https://www.ncbi.nlm.nih.gov/pubmed/23745692 http://dx.doi.org/10.1021/ja4014375 |
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author | Yu, Xiaojie Baruah, Kavitha Harvey, David J. Vasiljevic, Snezana Alonzi, Dominic S. Song, Byeong-Doo Higgins, Matthew K. Bowden, Thomas A. Scanlan, Christopher N. Crispin, Max |
author_facet | Yu, Xiaojie Baruah, Kavitha Harvey, David J. Vasiljevic, Snezana Alonzi, Dominic S. Song, Byeong-Doo Higgins, Matthew K. Bowden, Thomas A. Scanlan, Christopher N. Crispin, Max |
author_sort | Yu, Xiaojie |
collection | PubMed |
description | [Image: see text] Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein–carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan–protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man(5)GlcNAc(2)) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan–protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein–carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors. |
format | Online Article Text |
id | pubmed-3788586 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-37885862013-10-08 Engineering Hydrophobic Protein–Carbohydrate Interactions to Fine-Tune Monoclonal Antibodies Yu, Xiaojie Baruah, Kavitha Harvey, David J. Vasiljevic, Snezana Alonzi, Dominic S. Song, Byeong-Doo Higgins, Matthew K. Bowden, Thomas A. Scanlan, Christopher N. Crispin, Max J Am Chem Soc [Image: see text] Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein–carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan–protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man(5)GlcNAc(2)) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan–protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein–carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors. American Chemical Society 2013-06-07 2013-07-03 /pmc/articles/PMC3788586/ /pubmed/23745692 http://dx.doi.org/10.1021/ja4014375 Text en Copyright © 2013 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) |
spellingShingle | Yu, Xiaojie Baruah, Kavitha Harvey, David J. Vasiljevic, Snezana Alonzi, Dominic S. Song, Byeong-Doo Higgins, Matthew K. Bowden, Thomas A. Scanlan, Christopher N. Crispin, Max Engineering Hydrophobic Protein–Carbohydrate Interactions to Fine-Tune Monoclonal Antibodies |
title | Engineering Hydrophobic Protein–Carbohydrate
Interactions to Fine-Tune Monoclonal Antibodies |
title_full | Engineering Hydrophobic Protein–Carbohydrate
Interactions to Fine-Tune Monoclonal Antibodies |
title_fullStr | Engineering Hydrophobic Protein–Carbohydrate
Interactions to Fine-Tune Monoclonal Antibodies |
title_full_unstemmed | Engineering Hydrophobic Protein–Carbohydrate
Interactions to Fine-Tune Monoclonal Antibodies |
title_short | Engineering Hydrophobic Protein–Carbohydrate
Interactions to Fine-Tune Monoclonal Antibodies |
title_sort | engineering hydrophobic protein–carbohydrate
interactions to fine-tune monoclonal antibodies |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788586/ https://www.ncbi.nlm.nih.gov/pubmed/23745692 http://dx.doi.org/10.1021/ja4014375 |
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