The Impact of Nonideality on the Biophysical Properties of Therapeutic Antibodies in Physiological Environments

Abstract

Antibody-based therapeutics are some of the best-selling drugs on the market with indications in several aggressive cancers, chronic autoimmune conditions, and other disease states. They provide several advantages over traditional small molecule drugs but factors controlling the pharmacokinetics (PK) and pharmacodynamics (PD) are less understood. A key contributing factor is a lack of understanding of how proteins are affected by complex, crowded biological environments such as serum and plasma. This is largely because techniques used in the biopharmaceutical industry are poorly suited for neat ex vivo samples. For this reason, the complex effects of crowding in the therapeutic context of efficacy and clearance have not been documented. The second virial coefficient (B2) parameter quantifies such weak interactions and can be determined by a variety of techniques; however, probing nonideality in complex biological fluids remains challenging. Fluorescence correlation spectroscopy (FCS) is a technique capable of measuring the diffusive properties of proteins directly in biological fluids. Therefore, the focus of this dissertation is to utilize FCS to explore nonideality and antibody-target interactions directly in undilute serum. Chapter 1 introduces important topics related to antibody structure and function, therapeutic antibody optimization, and macromolecular crowding. Chapter 2 presents the development and validation of a novel in-serum FCS approach for probing nonideality via determination of apparent second virial coefficients (B2,app). The findings revealed that nonideality effects in serum are antibody dependent. In Chapter 3, the in-serum FCS approach is utilized to further characterize the origins of nonideality. Here, B2,app measurements were used to identify the components of human serum responsible for non-ideal interactions with mAbs and Fab fragments. Most notably, attractive interactions were observed with serum IgGs in Fab domains. Therefore, Chapter 4 utilizes FCS to investigate the impact of nonideality on antigen binding. As a preliminary assessment, a model system was used to determine antibody-antigen affinity in buffer and serum via FCS. In addition, correlations between B2,app and serum-induced changes in binding affinity were explored. This provided initial insight into the significance of the magnitude of B2,app values, where slight attraction in the model system did not result in functional consequences to antigen binding. Together, this work demonstrates the potential utility of FCS in the biopharmaceutical industry and provides the foundation for investigating the impact of nonideality on the biophysical properties of therapeutic antibodies in physiological environments.