Structural Studies of IgE as a Target for Therapeutic Intervention in Allergic Disease

Student thesis: Doctoral ThesisDoctor of Philosophy


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.
Date of Award2016
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorBrian Sutton (Supervisor) & Andrew Beavil (Supervisor)

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