Designing an anti-allergy biologic for the clearance of IgE

Abstract

Immunoglobulin E (IgE), pivotal for the allergic response, is an important and proven drug target for allergic diseases, including asthma. Recent insights into the molecular properties of IgE, such as the allosteric regulation of binding to the FceRI and CD23 receptors and the calcium dependence of the interaction with CD23, together with recent innovations in biological therapeutics that exploit the natural pathway for IgG and albumin recycling through the neonatal Fc receptor (FcRn), suggest an alternative mechanism for eliminating IgE. A panel of novel anti-IgE fusion proteins was developed that combines the ability of IgE to bind CD23 with the FcRn binding properties of IgG. The fusion proteins thus have the potential to remove IgE and be recycled to target more IgE antibodies. This thesis describes biophysical techniques and cellular-based assays that investigate the properties and potential mechanisms of action of these novel anti-IgE fusion proteins. Surface plasmon resonance (SPR) was used to assess the stoichiometries and kinetics for the interactions between the novel anti-IgE molecules and IgE. Basophil degranulation assays suggested that these molecules do not induce cellular degranulation, nor do they induce degranulation of cells in which IgE is pre-bound to FceRI, therefore these anti-IgE molecules are unlikely to pose a safety risk. The novel molecules do, however, prevent IgE binding to the cell. Furthermore, flow cytometry revealed that CD63 and CD203c, biomarkers of inflammation, were not upregulated by IgE when in complex with the anti-IgE molecules, and the interaction between IgE and FceRI was inhibited in a concentration-dependent manner. A novel cell-based recycling assay was developed to evaluate the ability of the anti-IgE molecules to be recycled to the extracellular supernatant, after delivering IgE to a cellular degradation pathway. This assay confirmed recycling of the anti-IgE molecules into the extracellular supernatant, with intracellular retention of IgE. Transcytosis assays also revealed that IgE could not transcytose a cell monolayer when in complex with the anti-IgE molecules, and was instead retained intracellularly. Finally, the intracellular fate of IgE was tracked using confocal microscopy, which revealed that IgE was delivered to the lysosomal degradation pathway. It is anticipated that such reagents could be a starting point to inform the design of therapeutics able to catalyse the degradation of IgE without themselves being consumed.

Date of Award	1 Jan 2021
Original language	English
Awarding Institution	King's College London
Supervisor	James McDonnell (Supervisor)