Development of a Treatment for Eosinophil-Mediated Allergic Inflammatory Diseases Utilizing a Neutralizing Agent Targeting Eosinophil Granule Major Basic Protein

ABSTRACT The eosinophil is a peripheral blood leukocyte containing an abundance of cytoplasmic granules, rich in cationic protein toxins. Among these, the most abundant on a molar basis is the major basic protein-1, eMBP1. eMBP1 kills helminths, bacteria, and numerous cells, such as respiratory epithelium, but also activates cells, including basophils and mast cells. Studies of human diseases show that eMBP1 is present in secretions from patients with eosinophil-mediated diseases, including asthma, chronic rhinosinusitis, and gastrointestinal diseases, and it is deposited on damaged targets. These studies show that the eosinophil mediates its damage to parasites and tissues by discharging its toxin rich granules onto microbial targets and tissues. eMBP1 is synthesized as a precursor, pro-eMBP1, composed of eMBP1 and a remarkably acidic pro-piece sequence. Developing eosinophils synthesize pro-eMBP1, and the pro-piece is removed during granule maturation. Analyses of pro- piece in different models show that it can neutralize the toxic eMBP1 effect and also the toxicity of the eosinophil cationic protein (ECP). This project proposes that neutralization of eMBP1 can be a treatment to mitigate tissue damage in eosinophil-related diseases. It will develop a pro-piece product for treatment of eosinophil-mediated diseases by neutralization of eMBP1 and other granule toxins. Currently, no therapies for these diseases address the neutralization of granule proteins. In this project, the pro-piece will be expressed and associated glycans will be modified to identify the optimal form of the molecule for binding to and neutralizing eMBP1. These tests will determine whether glycosylation of pro-piece importantly alters its binding to eMBP1 by analyzing whether N- or O- glycans and the glycosaminoglycan at S62 modify its activities. The project will identify the most important pro-piece regions for binding eMBP1 and neutralizing it by creating overlapping peptides and creating mutants with increased amino acid sequences characteristic of active regions. Evaluation of the pro-piece panel for binding to and inhibiting eMBP1 will be evaluated here in several in vitro models. The goal of this project is to identify an active form of the pro-piece with optimal inhibition effects on eMBP1 and other granule toxic proteins. This form will be further investigated and developed in future projects.