Rational Engineering of Erythropoietin for Smarter Protein Therapeutics: Structure–Function Relationships and Molecular Geometry

Protein therapeutics exploit the biology of naturally occurring proteins to deliver desired pharmacological activities to the patient’s body, and their use has increased dramatically over the past three decades. They have the advantages of reducing off-target effects, thanks to their higher structural diversity, and more specific target protein interactions compared to small molecule drugs. Despite their similarity to natural molecules and low off-target interactions, protein therapeutics can cause undesired effects by signaling on wrong tissues, triggering pleiotropic actions, or activating immune responses. This dissertation aims to demonstrate strategies to rationally engineer proteins to maximize drug efficacy while minimizing such side effects, using erythropoietin (EPO) as a model protein. Chapter 2 presents a bifunctional AND-gate ligand that directs EPO activity to target cells and away from other cells that also bear EPO receptors. Tissue-targeted activation of EPO receptors was achieved by modulating receptor–ligand binding strength and quantitatively analyzing the molecular geometry of protein domains on the target cell surface. Chapter 3 describes EPO fusion proteins that are designed to exhibit a subset of the pleiotropic activities of EPO based on its structure–function relationships in different signaling contexts, such that only desired effects can be selectively exploited for a given medical condition. Through this method, EPO fusion proteins with one or both of erythropoietic and tissue-protective effects were constructed. Chapter 4 delineates protein resurfacing approaches to reduce immune responses to EPO and prevent adverse consequences mediated by neutralizing anti-drug antibodies that are cross-reactive with its endogenous counterpart. Taken together, the present work shows that structural and spatial information about proteins can drive smarter therapeutic designs, as well as contributes to expanding the methods of protein engineering in drug development and construction of protein-based synthetic-biological systems.