Edwards, Elizabeth Jessica;
(2024)
Engineered Fc gamma receptor ligands for affinity separation of monoclonal antibody glycovariants.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Monoclonal antibodies (mAbs) are one of the greatest revenue generators in the pharmaceutical sector. Therapeutic mAbs are primarily of the IgG1 subclass and carry two N- linked glycans in their fragment crystallisable (Fc) region. The presence or absence of a core fucose on these N-glycans has a huge impact on antibody-dependent cellular cytotoxicity (ADCC), a common effector function for IgG1 mAbs. This is due to afucosylated IgG1 having a stronger affinity for the Fc gamma receptor 3a (FcgRIIIa), the immune cell receptor which mediates ADCC. This results in upregulated ADCC activity of afucosylated compared to fucosylated IgG1. Since this discovery, FcgRIIIa has been developed as a chromatographic affinity ligand for the separation of fucosylated and afucosylated IgG1. This thesis presents novel affinity ligands based on the FcgRIIIa for the separation of fucosylated and afucosylated IgG1, termed FcgR ligands. These engineered ligands comprise only the binding domain of FcgRIIIa and a single glycosylation site. This results in the FcgR ligands being smaller and less complex than FcgRIIIa, with the aim of improving the ligand density and commercial viability of a column with these ligands compared to FcgRIIIa. The results presented show that the FcgR ligands have the expected primary structure and a similar N-glycosylation pattern to what was designed. The KD obtained for the FcgRIIIa-IgG1 interaction was 1.4μM, whilst the KD values for the FcgR ligand-IgG1 interactions were ~100- fold lower at 20-60nM. The FcgR ligands show an opposite differential affinity for fucosylated and afucosylated IgG1 compared to FcgRIIIa, with a KD for the FcgRIIIa-low-fucose IgG1 interaction of 0.34μM and no observable interaction between the FcgR ligands and low-fucose IgG1. This gives a larger differential affinity of the FcgR ligands for these two glycovariants compared to FcgRIIIa. Suggestions are made for future FcgRIIIa-based ligands, along with future research needed to further understand the FcgRIIIa differential affinity for fucosylated and afucosylated IgG1.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Engineered Fc gamma receptor ligands for affinity separation of monoclonal antibody glycovariants |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2024. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10185993 |
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