© Copyright 2018 Dennis Ryan Goulet
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© Copyright 2018 Dennis Ryan Goulet Biophysical Approaches for the Development of Stable, Long-Lived, Multi-Functional, and Potent Antibody Therapeutics Dennis Ryan Goulet A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2018 Reading Committee: William M. Atkins, Chair Abhinav Nath Kelly K. Lee Program Authorized to Offer Degree: Pharmacy – Medicinal Chemistry University of Washington Abstract Biophysical Approaches for the Development of Stable, Long-Lived, Multi-Functional, and Potent Antibody Therapeutics Dennis Ryan Goulet Chair of the Supervisory Committee: William M. Atkins Department of Medicinal Chemistry 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. TABLE OF CONTENTS Page List of figures .......................................................................................................................... iv List of tables ............................................................................................................................ vi Chapter 1: Considerations for the design of antibody-based therapeutics ............................. 1 1.1 Introduction to therapeutic antibodies ......................................................................... 1 1.2 Antibody structure and function .................................................................................. 2 1.3 Antigen specificity ..................................................................................................... 10 1.4 Class, subclass, and allotype ...................................................................................... 17 1.5 Identification of variable regions ............................................................................... 29 1.6 Expression system ...................................................................................................... 35 1.7 Post-translational modifications................................................................................. 37 1.8 Fragmentation ............................................................................................................ 42 1.9 Multimerization.......................................................................................................... 46 1.10 Conjugation .............................................................................................................. 48 1.11 Multispecificity ........................................................................................................ 57 1.12 Protein engineering .................................................................................................. 64 1.13 Biophysical properties ............................................................................................. 67 1.14 Summary .................................................................................................................. 75 1.15 References ................................................................................................................ 82 Chapter 2: Diffusion of soluble aggregates of antibodies in serum ..................................... 97 2.1 Introduction ................................................................................................................ 97 2.2 Experimental procedures ........................................................................................... 99 2.2.1 Proteins and other materials .............................................................................. 99 2.2.2 Generation of disulfide cross-linked THIOMAB ............................................. 99 2.2.3 Generation of glutaraldehyde cross-linked Abs .............................................. 100 2.2.4 Purification and characterization of antibody oligomers ................................ 101 2.2.5 Preparation of viscosity-matched buffers ....................................................... 101 2.2.6 Fluorescence correlation spectroscopy ........................................................... 102 2.3 Results ...................................................................................................................... 103 2.3.1 Generation of oligomeric standards ................................................................ 103 2.3.2 Benchmarks of viscosity ................................................................................. 106 2.3.3 Diffusion of Ab oligomers in buffer and serum.............................................. 106 i 2.4 Discussion ................................................................................................................ 109 2.5 References ................................................................................................................ 124 Chapter 3: A combinatorial approach to the prediction of antibody pharmacokinetics ..... 127 3.1 Introduction .............................................................................................................. 127 3.2 Experimental procedures ......................................................................................... 129 3.2.1 Proteins and other materials ............................................................................ 129 3.2.2 Biolayer interferometry ................................................................................... 129 3.2.3 Differential scanning calorimetry ................................................................... 130 3.2.4 Literature reports of antibody pharmacokinetics ............................................ 131 3.2.5 Regression analysis ......................................................................................... 131 3.3 Results ...................................................................................................................... 132 3.3.1 Review of literature on antibody pharmacokinetics ....................................... 132 3.3.2 Antibody binding to the neonatal Fc receptor at endosomal pH .................... 132 3.3.3 Antibody binding to the neonatal Fc receptor at physiological pH ................ 133 3.3.4 Antibody thermal stability .............................................................................. 133 3.3.5 Multiple regression to identify combinations of PK predictors ...................... 134 3.3.6 Testing the predictive power of the model ..................................................... 136 3.4 Discussion ................................................................................................................ 137 3.5 References ................................................................................................................ 160 Chapter 4: Mechanism of controlled Fab-arm exchange to form bispecific antibodies .... 164 4.1 Introduction .............................................................................................................. 164 4.2 Experimental procedures ......................................................................................... 167 4.2.1 Proteins and other materials ............................................................................ 167 4.2.2 Hydrophobic interaction chromatography .....................................................