Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.706541
Title: Fc receptor interactions of a monoclonal antibody produced in plants
Author: Stelter, Szymon
ISNI:       0000 0004 6057 6829
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2016
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Abstract:
Plants provide a cost-effective and scalable technology for production of therapeutic antibodies, with the potential for precise engineering of antibody glycosylation. Altering glycan structures in the antibody Fc region influences binding properties to Fc receptors and thus opens up new opportunities for modulation of antibody effector functions. To test the impact of plant glycosylation and effects of glycan modification on binding to human Fc receptors, different glycovariants of VRC01, a broadly-neutralising HIV antibody, were generated in Nicotiana benthamiana using glycoengineering tools and characterised. These include glycovariants lacking plant characteristic [alpha]l,3-fucose and [beta]l,2-xylose residues and extended with terminal [beta]l,4-galactose. Several surface plasmon resonance-based assays were inspected for kinetic/affinity evaluation of antibody - FcγR interactions. Protein A capture of antibodies was found to provide appropriate assay conditions and revealed that antibodies with typical plant glycosylation have a limited capacity to engage FcγRI, FcγRIla, FcγRIIb and FcγRIIIa, however the binding characteristics can be restored and even improved with glycoengineering. Functional consequences of distinct FcγR-binding activities were further investigated in cell-based assays using flow cytometry. Fluorescent latex beads were coated with HIV-1 gpl40 antigen and different VRC01 glycovariants were compared for their ability to mediate phagocytosis of the beads by THP-1 human monocytes. The results demonstrate that typical plant glycosylation had a negative effect on binding of antibody-coated antigen-beads to cell surface receptors, and consequently may limit their uptake by the cells. However, plant glycoengineered antibodies are equally effective in enhancing cell-mediated phagocytosis as a human cell-made variant. Furthermore, a novel SPR-based assay was developed to investigate the influence of plant glycosylation on antibody affinity to neonatal Fc receptor (FcRn), which is involved in determining IgG half-life in the blood. The results suggest that FcRn binding is independent of plant glycosylation, however all plant-made glycovariants had a slightly lower affinity to the receptor, which correlates with higher oxidation levels of methionine residues involved in binding. Plant-based production and purification procedures, that may promote antibody oxidation leading to lower affinity to FcRn, point towards a need for process optimisation to control oxidation levels and improve the quality of plant-produced antibodies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.706541  DOI: Not available
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