Antibody Fucosylation Lowers the FcγRIIIa/CD16a Affinity by Limiting the Conformations Sampled by the N162-Glycan
Abstract
Therapeutic monoclonal antibodies (mAbs) are largely based on the immunoglobulin G1 (IgG1) scaffold and many elicit a cytotoxic cell-mediated response by binding Fc γ receptors. Core fucosylation, a prevalent modification to the asparagine(N)-linked carbohydrate on the IgG1 crystallizable fragment (Fc), reduces Fc γ receptor IIIa (CD16a) binding affinity and mAb efficacy. We determined IgG1 Fc fucosylation reduced CD16a affinity by 1.7 ± 0.1 kcal/mol when compared to afucosylated IgG1 Fc, however, CD16a N-glycan truncation decreased this penalty by 1.2 ± 0.1 kcal/mol or 70%. Fc fucosylation restricted the manifold of conformations sampled by displacing the CD16a Asn162-glycan which impinges upon the linkage between the αmannose(1- 6)βmannose residues and promoted contacts with the IgG Tyr296 residue. Fucosylation also impacted IgG1 Fc structure as indicated by changes in resonance frequencies and nuclear spin relaxation observed by solution NMR spectroscopy. The effects of fucosylation on IgG1 Fc may account for the remaining 0.5 ± 0.1 kcal/mol penalty of fucosylated IgG1 Fc binding CD16a when compared to afucosylated IgG1 Fc. Our results indicated the CD16a Asn162-glycan modulates antibody affinity indirectly through reducing the volume sampled, as opposed to a direct mechanism with intermolecular glycan-glycan contacts previously proposed to stabilize this system. Thus, antibody engineeringmore »
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States)
- Sponsoring Org.:
- National Institutes of Health (NIH); USDOE
- OSTI Identifier:
- 1498328
- Resource Type:
- Accepted Manuscript
- Journal Name:
- ACS Chemical Biology
- Additional Journal Information:
- Journal Volume: 13; Journal Issue: 8; Journal ID: ISSN 1554-8929
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
Citation Formats
Falconer, Daniel J., Subedi, Ganesh P., Marcella, Aaron M., and Barb, Adam W. Antibody Fucosylation Lowers the FcγRIIIa/CD16a Affinity by Limiting the Conformations Sampled by the N162-Glycan. United States: N. p., 2018.
Web. doi:10.1021/acschembio.8b00342.
Falconer, Daniel J., Subedi, Ganesh P., Marcella, Aaron M., & Barb, Adam W. Antibody Fucosylation Lowers the FcγRIIIa/CD16a Affinity by Limiting the Conformations Sampled by the N162-Glycan. United States. https://doi.org/10.1021/acschembio.8b00342
Falconer, Daniel J., Subedi, Ganesh P., Marcella, Aaron M., and Barb, Adam W. Fri .
"Antibody Fucosylation Lowers the FcγRIIIa/CD16a Affinity by Limiting the Conformations Sampled by the N162-Glycan". United States. https://doi.org/10.1021/acschembio.8b00342. https://www.osti.gov/servlets/purl/1498328.
@article{osti_1498328,
title = {Antibody Fucosylation Lowers the FcγRIIIa/CD16a Affinity by Limiting the Conformations Sampled by the N162-Glycan},
author = {Falconer, Daniel J. and Subedi, Ganesh P. and Marcella, Aaron M. and Barb, Adam W.},
abstractNote = {Therapeutic monoclonal antibodies (mAbs) are largely based on the immunoglobulin G1 (IgG1) scaffold and many elicit a cytotoxic cell-mediated response by binding Fc γ receptors. Core fucosylation, a prevalent modification to the asparagine(N)-linked carbohydrate on the IgG1 crystallizable fragment (Fc), reduces Fc γ receptor IIIa (CD16a) binding affinity and mAb efficacy. We determined IgG1 Fc fucosylation reduced CD16a affinity by 1.7 ± 0.1 kcal/mol when compared to afucosylated IgG1 Fc, however, CD16a N-glycan truncation decreased this penalty by 1.2 ± 0.1 kcal/mol or 70%. Fc fucosylation restricted the manifold of conformations sampled by displacing the CD16a Asn162-glycan which impinges upon the linkage between the αmannose(1- 6)βmannose residues and promoted contacts with the IgG Tyr296 residue. Fucosylation also impacted IgG1 Fc structure as indicated by changes in resonance frequencies and nuclear spin relaxation observed by solution NMR spectroscopy. The effects of fucosylation on IgG1 Fc may account for the remaining 0.5 ± 0.1 kcal/mol penalty of fucosylated IgG1 Fc binding CD16a when compared to afucosylated IgG1 Fc. Our results indicated the CD16a Asn162-glycan modulates antibody affinity indirectly through reducing the volume sampled, as opposed to a direct mechanism with intermolecular glycan-glycan contacts previously proposed to stabilize this system. Thus, antibody engineering to enhance intermolecular glycan-glycan contacts will likely provide limited improvement and future designs should maximize affinity by maintaining CD16a Asn162-glycan conformational heterogeneity},
doi = {10.1021/acschembio.8b00342},
journal = {ACS Chemical Biology},
number = 8,
volume = 13,
place = {United States},
year = {Fri Jul 06 00:00:00 EDT 2018},
month = {Fri Jul 06 00:00:00 EDT 2018}
}
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Figures / Tables:
Table 1: SPR analysis of the CD16a IgG1 Fc interactions. Glycoform analysis of fuc. IgG1 Fc, fuc. IgG1 Fc, and CD16a is shown in Supplemental Figures 1 and 2.
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