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Cited by 84Monoclonal and Polyclonal Antibodies ResearchGlycosylation and Glycoproteins ResearchCarbohydrate Chemistry and Synthesis

Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies

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Yu, Xiaojie, Baruah, Kavitha, Harvey, David J., Vasiljevic, Snezana, Alonzi, Dominic S., Song, Byeong-Doo, Higgins, Matthew K., Bowden, Thomas A. +2 more·2013·Journal of the American Chemical Society
DOI 10.1021/ja4014375PMC PMC3788586
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Abstract

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 (Man5GlcNAc2) 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.

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  • CHO-expressed IgG1-Fc with four mutations (F241A/F243A/V262E/V264E) has a more open conformation, which increased accessibility and processing by galactosyl- and sialyltransferases.

    “CHO-expressed IgG1-Fc with four mutations (F241A/F243A/V262E/V264E) has a more open conformation, which increased accessibility and processing by galactosyl- and sialyltransferases [11].”

    From Glycoengineering Chinese hamster ovary cells: a short history (2021) · cites this paper as [11]

    The Abstract and Results and Discussion report increased galactosylation and sialylation for the listed interface mutations, and discuss greater glycan accessibility to glycosyltransferases. But the HPLC Analysis section describes separate mutants expressed in HEK cells, not a four-mutation IgG1-Fc expressed in CHO cells. The proposed shift toward a more accessible conformation is also presented as a possibility, not an established structural finding for that construct; page numbers are unavailable in our copy. (LLM, conf=0.90)

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Cited by

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Authors
Yu, X., Baruah, K., Harvey, D. J., Vasiljevic, S., Alonzi, D. S., Song, B.-D., Higgins, M. K., Bowden, T. A., Scanlan, C. N., & Crispin, M.
Affiliations
  • University of Oxford (GB)
  • Scripps Korea Antibody Institute (KR)
  • Centre for Human Genetics (GB)
Corresponding author
Crispin, Max
ORCID
Venue
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Published
2013-06-25
Volume
135
Issue
26
Pages
9723-9732
ISSN
  • 0002-7863 (ISSN-L)
  • 1520-5126
DOI
10.1021/ja4014375
PMC
PMC3788586
OpenAlex
W1975380390
Cited by
84
References
2
Citations by year
2026: 2 · 2025: 3 · 2024: 3 · 2023: 1 · 2022: 5 · 2021: 11 · 2020: 5 · 2019: 8 · 2018: 11 · 2017: 8 · 2016: 5 · 2015: 14 · 2014: 7 · 2013: 1
FWCI
6.99
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of the American Chemical Society (publishedVersion, other-oa)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • ePrints Soton (University of Southampton) (acceptedVersion)
Funders
Wellcome Trust (089026/Z/09/Z, 090532)
Topics
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
Keywords
IgG1 Fc, carbohydrate-protein interactions, hydrophobic interactions, Fc glycan, FcγRIIIa, antibody-dependent cellular cytotoxicity, galactosylation, sialylation, glycan engineering, site-directed mutagenesis, Fc receptor binding, monoclonal antibodies, Fc effector functions
MeSH terms
Antibodies, Monoclonal, Immunoglobulin G, Immunoglobulin Fc Fragments, Models, Molecular, Polysaccharides, Protein Engineering, Crystallography, X-Ray, Hydrophobic and Hydrophilic Interactions

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