Engineering a 45-Amino Acid Protein Scaffold for Molecular Cancer Imaging A DISSERTATION SUBMITTED TO THE FACULY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Max Kruziki IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisor: Benjamin J. Hackel December 2017 © Max Kruziki 2017 Acknowledgements I am thankful for all the wonderful people I have met during my years in graduate school. They have had the biggest impact on my happiness and success both at work and in life. Katie, who will soon be my wife, has inspired me to push myself be the best that I can be, in the hopes of even approaching all she has accomplished. She has helped keep me hopeful when experiments weren’t going well, and has provided encouragement and friendship whenever I needed it most. All of my lab mates, but especially Danny, Brett, Larry, and Sadie who have shared an office with me nearly my entire time in Minnesota made each day enjoyable. We could always laugh and joke around, and yet switch to serious scientific discussion when someone had a question or needed help. The undergraduates who have worked with me; Vandon, Andrew, Lizzie, and Feifan were great people to work with and mentor, and it was fulfilling to watch their scientific knowledge grow. The entire CEMS department has been like a family to me. Especially the other students in my class, who struggled with me through early classes and long homework assignments. Without the kindness and support from them during the intense first few semesters, grad school would have been much different and much less fun. Ben, my advisor, has instilled in me the desire to do good science and be vigilant to not cut corners or take the easy way out. His optimism is contagious, and our weekly i meetings always left me feeling refreshed and encouraged to conquer what problems I may face. Finally, I want to thank my family and friends outside of CEMS. My brother, Jake, and along with my other close friends give an escape to talk about topics outside of chemical engineering and fulfill my interests outside of science. Most importantly, my parent’s unwavering belief and support is what has allowed me to accomplish all I have throughout life. ii Abstract Cancer is the second leading cause of death in the United States. Molecularly targeted cancer treatments, including monoclonal antibodies and kinase inhibitors, exhibit strong performance on a small subset of patients but are inconsistent due to tumor heterogeneity. Biopsy-based genetic and protein tumor characterization provide value but cannot address spatial or temporal variations in heterogeneity. Non-invasive methods, such as molecular imaging, to characterize cancer cells will allow for easier patient stratification and treatment monitoring. Currently, molecular imaging is limited by the modest availability of quality probes that efficiently distribute throughout the body and quantitatively localize at the site of the cancer biomarker. Engineering effective diagnostic molecular probes would provide a substantial advance in cancer characterization and personalized medicine. Protein scaffolds, which comprise a large stabilizing framework and a randomized region onto which binding interactions can be engineered, offer an efficient platform for probe engineering. More broadly, engineered binding proteins are useful in many aspects of biotechnology and medicine. In this thesis, we mined ~100,000 known protein topologies to identify candidate small protein scaffolds. We developed the 45-amino acid Gp2scaffold and evolved multiple Gp2 variants that strongly (as strong as 0.2 nM) and specifically (greater than 50:1 target:control) bind their respective target while also retaining high thermal stability (65- 80 ºC thermal desaturation midpoint) . iii A Gp2 variant that was evolved to bind with strong affinity to epidermal growth factor receptor (EGFR), a cell surface biomarker overexpressed in multiple cancer types, was more thoroughly investigated in pre-clinical studies. This variant exhibited strong (18 nM), selective binding, and was passive on normal EGFR signaling pathways, which is important to reduce off-target side effects. PET imaging of subcutaneously xenografted tumors in mice revealed effective probe localization to EGFR-high tumors while low signal was observed in EGFR-low tumors and from non-targeted control Gp2. Gp2 evolution was studied by comparing the efficacy of different combinatorial library amino acid diversity based on high throughput sequencing data, natural Gp2 homologs, structural data, and computed stability. Multiple library designs elucidated amino acid diversity that was beneficial or detrimental in different sections of the Gp2 protein, and will aid future evolution and developability of Gp2. From these libraries, high affinity Gp2 variants targeting an additional clinically-relevant cancer biomarker, programmed death-ligand 1 (PD-L1), were evolved, isolated, and characterized. Collectively this work identifies and validates Gp2 as a new potential tool for biomarker- based cancer detection and sets a strong foundation for future optimization. iv Table of Contents List of Figures ..................................................................................................................... x List of Tables ................................................................................................................... xiii Chapter 1: Introduction ....................................................................................................... 1 1.1.1 Benefits of molecular recognition ...................................................................... 1 1.1.2 Protein scaffolds as imaging agents ................................................................... 4 1.1.3 Protein evolution and design .............................................................................. 9 Chapter 2: A 45-amino acid scaffold mined from the Protein Data Bank for high affinity ligand engineering ............................................................................................................. 14 2.1. Abstract .................................................................................................................. 14 2.2. Introduction ............................................................................................................ 14 2.3 Experimental Procedures ........................................................................................ 18 2.3.1 Protein Data Bank Analysis ............................................................................. 18 2.3.2 Library Construction ........................................................................................ 19 2.3.3 Binder Selection and Affinity Maturation ....................................................... 19 2.3.4 Illumina MiSeq Analysis ................................................................................. 20 2.3.5 Affinity and Biophysical Properties................................................................. 21 2.4. Results .................................................................................................................... 22 2.4.1 Scaffold Discovery and Library Construction ................................................. 22 2.4.2 Yeast Surface Display Selection Against Model Protein Targets ................... 26 v 2.4.3 Soluble Protein Characterization ..................................................................... 30 2.4.4 EGFR-Targeting Gp2 Domains ....................................................................... 31 2.4.5 Deep Sequencing of Naïve and Binding Populations ...................................... 34 2.5 Discussion ............................................................................................................... 36 2.6 Significance............................................................................................................. 39 2.7 Acknowledgements ................................................................................................. 40 2.8 Supplemental Data .................................................................................................. 40 2.8.1 Supplemental Experimental Procedures .......................................................... 48 Chapter 3: A 64Cu-labeled Gp2 Domain for PET Imaging of Epidermal Growth Factor Receptor ............................................................................................................................ 59 3.1 Abstract ................................................................................................................... 59 3.2 Introduction ............................................................................................................. 60 3.3 Materials and Methods ............................................................................................ 62 3.3.1 Protein production and DOTA conjugation ..................................................... 62 3.3.2 Size Exclusion Chromatography...................................................................... 63 3.3.4 Cell growth....................................................................................................... 63 3.3.5 Affinity measurement ...................................................................................... 63 3.3.6 Western Blot Analysis ..................................................................................... 64 3.3.7 Internalization .................................................................................................