Biophysical Approaches for the Development of Stable, Long-Lived, Multi-Functional, and Potent Antibody Therapeutics

Abstract

Antibody-based proteins have become an important class of biologic therapeutics, due in large part to the stability, specificity, and adaptability of the antibody framework. Indeed, antibodies not only have the inherent ability to bind both antigens and endogenous immune receptors, but they have also proven extremely amenable to protein engineering. Thus, several derivatives of the monoclonal antibody format, including bispecific and multispecific antibodies, antibody-drug conjugates, and antibody fragments, have demonstrated efficacy for treating human disease, particularly in the fields of immunology and oncology. Presented here is a thorough examination of the design of antibody-based therapeutics, and a description of four projects that each use different combinations of biophysical methods to characterize clinically relevant properties of antibodies. Chapter 1 reviews several aspects of therapeutic antibody design, including therapeutic mechanisms, isotype selection, and engineering strategies. Chapter 2 explores the behavior of soluble antibody oligomers, using fluorescence correlation spectroscopy to monitor diffusion properties in buffer and serum. This work revealed that antibody aggregation is dependent not only on oligomer size, but also on environment. In Chapter 3, a multiple regression method to predict antibody pharmacokinetic parameters is investigated. It was determined that combinations of neonatal Fc receptor binding parameters determined by biolayer interferometry and thermal stability parameters measured by differential scanning calorimetry allow for improved prediction of half-life and clearance. The kinetic mechanism of controlled Fab-arm exchange is presented in Chapter 4. A Förster resonance energy transfer method allowed for real-time monitoring of bispecific antibody formation and revealed conditions that accelerate the reaction. Finally, Chapter 5 explores the effect of Fc multimerization on Fc receptor binding and functionality. A novel antibody construct containing two Fc domains was generated and shown to exhibit multivalent binding to Fc gamma receptors and the neonatal Fc receptor. Collectively, this work demonstrates the utility of biophysical techniques in developing antibody therapeutics with increased conformational stability, longer half-life, multiple antigen-binding functionality, and higher-avidity Fc receptor binding.