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Investigation of Fc modifications and their influence on mAb function

Investigation of Fc modifications and their influence on mAb function
Investigation of Fc modifications and their influence on mAb function
Monoclonal antibodies (mAbs) are therapeutic agents that have revolutionised the treatment of many diseases, particularly cancer. Most therapeutic mAbs rely on effector functions mediated by their Fc region. The work in this thesis focuses on the impact of alterations to this Fc region, and the consequence upon the efficacy of the antibody.

The role of different Fc regions on antibody binding and effector function was first analysed using different IgG subclass formats. Differential binding to target antigen for certain but not all specificities was clearly demonstrated, likely linked to the ability of the target to cluster in the membrane. Subsequently in effector assays of CDC, ADCC and ADCP, IgG1 and IgG3 were seen to be the most effective, with IgG2 and IgG4 being less able to engage direct depletion mechanisms.

To explore the potential consequences of lipid modifications on IgG functionality two different processes were examined- malondialdehyde and carboxyethylpyrrole. Both of these were performed on IgG1 mAbs in order to replicate potential in vivo processes that could occur to therapeutic mAbs. Assessment of the functional capabilities of these modified antibodies were carried out, and it was found that these lipid modified antibodies were indeed compromised in functionality.

As antibody Fc glycosylation is required for Fc effector function, and is known to vary both within health and disease as well as in vitro production, this modification was also explored using a series of enzymatic modifications. To probe their effects on Fc mediated effector mechanisms, anti-CD20 mAbs with distinct glycoforms were generated and tested for effector function. It was found that increasing the level of galactosylation and sialylation resulted in increased complement engagement, whilst not effecting FcγR mediated effector functions.

Finally, a novel method to assess the C1q binding property of mAbs was developed and characterised. This method, developed around a C1q affinity column, was shown to recapitulate the same hierarchies of C1q binding specificities as seen from cell based C1q recruitment and complement dependent killing assays using biologically relevant samples. As such, IgG3 showed the strongest binding to the C1q column, whereas IgG2 and IgG4 showed little specific binding. Furthermore, increasing levels of galactosylation and sialylation increased the binding to the column, and IgG mutants with altered C1q binding showed the anticipated shifts in column binding. These data suggest that the C1q column would be readily suited to high throughput screening of the binding of antibody mutants, subclasses or glycoforms to C1q.

Together, the work described in this thesis demonstrates the importance of the Fc region to antibody effector functions, their sensitivity to modifications, and ultimately to their efficacy as therapeutics.
University of Southampton
Marshall, Michael
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Marshall, Michael
4db088a7-c3c0-45e6-91f6-ef307517eca3
Cragg, Mark
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Beers, Stephen
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Teeling, Jessica
fcde1c8e-e5f8-4747-9f3a-6bdb5cd87d0a

Marshall, Michael (2018) Investigation of Fc modifications and their influence on mAb function. University of Southampton, Doctoral Thesis, 347pp.

Record type: Thesis (Doctoral)

Abstract

Monoclonal antibodies (mAbs) are therapeutic agents that have revolutionised the treatment of many diseases, particularly cancer. Most therapeutic mAbs rely on effector functions mediated by their Fc region. The work in this thesis focuses on the impact of alterations to this Fc region, and the consequence upon the efficacy of the antibody.

The role of different Fc regions on antibody binding and effector function was first analysed using different IgG subclass formats. Differential binding to target antigen for certain but not all specificities was clearly demonstrated, likely linked to the ability of the target to cluster in the membrane. Subsequently in effector assays of CDC, ADCC and ADCP, IgG1 and IgG3 were seen to be the most effective, with IgG2 and IgG4 being less able to engage direct depletion mechanisms.

To explore the potential consequences of lipid modifications on IgG functionality two different processes were examined- malondialdehyde and carboxyethylpyrrole. Both of these were performed on IgG1 mAbs in order to replicate potential in vivo processes that could occur to therapeutic mAbs. Assessment of the functional capabilities of these modified antibodies were carried out, and it was found that these lipid modified antibodies were indeed compromised in functionality.

As antibody Fc glycosylation is required for Fc effector function, and is known to vary both within health and disease as well as in vitro production, this modification was also explored using a series of enzymatic modifications. To probe their effects on Fc mediated effector mechanisms, anti-CD20 mAbs with distinct glycoforms were generated and tested for effector function. It was found that increasing the level of galactosylation and sialylation resulted in increased complement engagement, whilst not effecting FcγR mediated effector functions.

Finally, a novel method to assess the C1q binding property of mAbs was developed and characterised. This method, developed around a C1q affinity column, was shown to recapitulate the same hierarchies of C1q binding specificities as seen from cell based C1q recruitment and complement dependent killing assays using biologically relevant samples. As such, IgG3 showed the strongest binding to the C1q column, whereas IgG2 and IgG4 showed little specific binding. Furthermore, increasing levels of galactosylation and sialylation increased the binding to the column, and IgG mutants with altered C1q binding showed the anticipated shifts in column binding. These data suggest that the C1q column would be readily suited to high throughput screening of the binding of antibody mutants, subclasses or glycoforms to C1q.

Together, the work described in this thesis demonstrates the importance of the Fc region to antibody effector functions, their sensitivity to modifications, and ultimately to their efficacy as therapeutics.

Text
MM Final Thesis 2019 - Version of Record
Restricted to Repository staff only until 3 June 2022.
Available under License University of Southampton Thesis Licence.

More information

Published date: September 2018

Identifiers

Local EPrints ID: 436543
URI: http://eprints.soton.ac.uk/id/eprint/436543
PURE UUID: a9b9afec-4a6e-4ef3-87da-f082b674045e
ORCID for Mark Cragg: ORCID iD orcid.org/0000-0003-2077-089X
ORCID for Stephen Beers: ORCID iD orcid.org/0000-0002-3765-3342
ORCID for Jessica Teeling: ORCID iD orcid.org/0000-0003-4004-7391

Catalogue record

Date deposited: 12 Dec 2019 17:30
Last modified: 18 Feb 2021 16:56

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Contributors

Author: Michael Marshall
Thesis advisor: Mark Cragg ORCID iD
Thesis advisor: Stephen Beers ORCID iD
Thesis advisor: Jessica Teeling ORCID iD

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