Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.745561
Title: Specific targeting of FcγRIIIa using artificial scaffold protein variants
Author: Waterhouse, Mark Peter
ISNI:       0000 0004 7225 662X
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2018
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Thesis embargoed until 01 Jul 2023
Access from Institution:
Abstract:
Fcγ Receptors (FcγRs) are cell-surface receptors for IgG that are expressed on immune cells including macrophages, monocytes and natural killer (NK) cells. Upon binding to IgG-containing immune complexes, FcγRs trigger cell-mediated effector functions that lead to the clearance of pathogenic material and immune homeostasis. However, aberrant activation of these pathways can result in autoimmune susceptibility and, as such, FcγRs are implicated in the pathogenesis of several autoimmune disorders. For example, FcγRIIIa, expressed on macrophages and NK cells, has been functionally and genetically linked to susceptibility for rheumatoid arthritis and is a valid target for several FcγR-mediated autoimmune disorders. Despite this, FcγRIIIa is currently under-exploited due to a lack of FcγRIIIa-specific agents, which results from the expression of the near identical FcγRIIIb on neutrophils. However, several FcγRIIIa-specific Adhirons (an engineered protein scaffold) have recently been described, which inhibit IgG binding and effector functions (phagocytosis and cytokine release) of THP-1 cells. This thesis aimed to fully characterise and enhance the properties of one of these recently described Adhirons (known as AdG3) through site-directed mutagenesis (SDM); Molecular Dynamics (MD) simulations; and fusion to the IgG1- or IgG2-Fc. SDM identified several key binding residues in the AdG3 variable regions through screening of AdG3 mutants by surface plasmon resonance (SPR). SPR also identified three mutants with enhanced affinity for FcγRIIIa and showed that AdG3 possesses background binding to the highly homologous FcγRIIIbNA1, but not FcγRIIIbNA2, which was supported by MD simulations that revealed the exquisite mode of AdG3 specificity for these receptors, which was mediated through the Arg18Ser polymorphism in the AdG3 binding site. Fusion to the IgG-Fc was not detrimental to the FcγRIIIa-AdG3 interaction, and fusion to the IgG2-Fc was observed to potentially enhance the affinity and specificity of AdG3 for FcγRIIIa. These findings could subsequently be utilised for further improvement of AdG3 affinity and specificity, as well as other clinically relevant parameters such as serum half-life.
Supervisor: Robinson, James I. ; Baxter, Euan W. ; Tomlinson, Darren C. ; Morgan, Ann W. Sponsor: Ann Wilkes Memorial Fund
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.745561  DOI: Not available
Share: