Rational Design of Selective Protein Therapeutics for Structurally and Functionally Complex Targets

Abstract

Protein biologics have transformed modern drug discovery and development by offering high selectivity and reduced off-target effects, prolonged serum half-lives, and the ability to modulate cellular processes. However, many clinically relevant targets remain intractable due to limited structural or mechanistic information, selectivity issues, or the need to modulate multiple pathways simultaneously. This work presents two distinct case studies in engaging challenging targets using rationally designed bispecific protein therapeutics and Fc fusion proteins. In Chapter 1, I describe the successful design and characterization of the first non-EPO-based, bispecific agonists for the tissue-protective receptor, EPO-R/CD131. We generated a structural model of the EPO-R/CD131 complex using AlphaFold and available structures of EPO-R and CD131 individually bound to single chain variable fragments (scFvs). Using this model, we designed several tandem scFvs and a bispecific antibody that selectively activated EPO-R/CD131 and not the related receptor, EPO-R/EPO-R. These proteins serve as a foundation for future studies of EPO-R/CD131 biology or for the development of safer, more selective therapeutic candidates for cell-protection applications, such as neurodegenerative diseases. In Chapter 2, I detail the design and early stage testing of several mono- and bi-specific Fc fusion proteins for the treatment of dysfunctional and inflammatory pain. Using similar design principles, I generated Fc fusions to peptides that inhibit the voltage-gated sodium channels Nav1.7 and Nav1.8 and Fc fusions to neprilysin, an enzyme that degrades the neuroinflammatory peptide, substance P. Altogether, this work provides novel approaches to the modulation of these challenging targets and insights about future design considerations and possible solutions to further the development of protein therapeutics for cell-protection and chronic pain.