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Title: Structural studies of IgE as a target for therapeutic intervention in allergic disease
Author: Mitropoulou, Alkistis
ISNI:       0000 0004 7656 2405
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2016
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In recent decades, the incidence of allergy, an immune disorder mediated by immunoglobulin E(IgE), has become more common. The symptoms of these allergic diseases not only cause discomfort,but may also be!fatal. Although the aetiology of allergy is debated, it is widely accepted that the interaction of IgE with its high affinity receptor FcεRI plays a pivotal role in the pathogenesis of allergic disease. The current treatments include therapeutics for both the prevention and relief of symptoms, and the intravenously administered omalizumab. The mechanism of action of this anti-IgE antibody, which is a monoclonal IgG antibody, has now been identified. In order to crystallise the omalizumab Fab/IgE-Fc complex, mutagenesis had to be performed on the Fab. This thesis reports six Fab crystal structures, of wild-type and different mutant omalizumab Fabs, and one single-chain variable fragment (scFv). The unbound Fab and! scFv structures were compared with each other (wild-type versus mutants), and also with those in the Fab/IgE-Fc complex in order to identify conformational differences. Potential small-molecule inhibitors for the IgE-FcεRI interaction were screened by X-ray crystallography and Surface Plasmon Resonance analysis. The crystal structures of a grass pollen allergen (Phl p 7) alone and in complex with a Fab fragment of a patient-derived antibody were also determined. The allergen-Fab complex crystals were susceptible to radiation damage. Multiple datasets were collected and careful merging was required to permit determination of the allergen-antibody crystal structure. This revealed that a monomeric allergen, the EF-hand Phl p 7 protein, binds simultaneously to two identical antibodies of the same specificity for Phl p 7. This discovery explains how the binding of a single monomeric allergen molecule can trigger the cross-linking of FcεRI-bound IgE by both a ‘classical’ CDR-mediated, and a framework-mediated, interaction, thereby triggering activation of a mast cell or basophil. This provides valuable new insights concerning the nature of allergenicity, and also informs the engineering of hypoallergenic vaccines for immunotherapy.
Supervisor: Sutton, Brian John ; Beavil, Andrew John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available