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1 Biophysical Enhancement of Protein Therapeutics and Diagnostics Biophysical Enhancement of Protein Therapeutics and Diagnostics Through Engineered Linkers Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nicholas Emerson Long Graduate in The Ohio State University Biochemistry Program The Ohio State University 2018 Dissertation Committee Dr. Thomas Magliery, Advisor Dr. Christopher Jaroniec Dr. Edward Martin Jr. Dr. Richard Swenson 1 Copyrighted by Nicholas Emerson Long 2018 2 Abstract Proteins play a major role in virtually every biological process. Thus, proteins are an ideal platform for the next generation of therapeutics. Over the last few decades, technological and scientific advances in protein production and engineering have led to a new wave of protein-based biologics used in clinical settings. In this body of work, we have engineered both a protein-based cancer diagnostic and an immunotherapeutic. Antibody-based biologics are becoming one of the most widely approved drug platforms and owe their success to their versatility in binding targets, high stability, and low toxicity. The anti-TAG-72 cancer-targeting antibody, 3E8, is one such molecule that shows great potential as a diagnostic. We have designed and biophysically characterized a library of 3E8 single chain antibody fragments (scFV) with varying linker composition and length as well as domain orientations. In this library, we have found substantial variation in protein stability, binding affinity, and oligomeric states. Surprisingly, a drastic difference in the oligomeric state of these constructs was seen between conventional IMAC purification and Protein L purification. Therefore, the literature rules for scFV linker design must be updated to include the dependencies on purification method. ii A single antibody construct with optimal biophysical properties (3E8.G4S) was further characterized and subjected to in vivo pharmacokinetic studies. Due to its multimeric composition, 3E8.G4S showed a longer and more favorable clearance time compared to that of a fast clearing scFV. Xenograft mouse imaging and biodistribution studies revealed successful targeting of a colorectal tumor by 3E8.G4S with little accumulation in normal tissues. To determine the versatility of 3E8-based diagnostics and therapeutic agents, an expansive immunohistochemical analysis of TAG-72 expression was performed in over 1,500 tumors spanning 18 different cancer types. The results of this study showed enhanced staining of engineered 3E8.scFV.FLAG compared to commercially available anti-TAG-72 IgG, B72.3. We found statistically significant TAG-72 expression differences in colon, prostate, lung, cervical, ovarian, pancreatic, gastrointestinal, rectal, and breast cancers when compared to corresponding normal tissues. The intense staining of these diseased tissues by 3E8.scFV.FLAG suggests wide applicability for its clinical use as a cancer detection agent and consequently the viability of 3E8.G4S as a state of the art cancer imaging agent. Manipulation of the Notch signaling pathway is another promising therapeutic strategy for a variety of diseases. Specifically, activation of the Notch1 receptor by a clustered DLL1 ligand has been shown to enhance T-cell maturation and promote cancer cell death. This makes the engineering of a multimeric and discrete form of DLL1 a viable cancer therapy. We have identified and validated the critical binding domain of the DLL1 ligand through iii in vitro cell based and in vivo Notch inhibition studies. Additionally, the minimal multivalency for Notch activation was determined to be four DLL1 repeats. Through E. coli transgenic expression and protein refolding, we have successfully created a 4x and 6x tandem repeat of the DLL1 ligand capable of in vitro cell activation of the Notch1 pathway. Through the activation of host immune cells, this novel Notch activator has the potential to be a broadly used cancer immunotherapeutic. iv Dedication This dissertation is dedicated to my Grandfather, Earl Emerson Schneider. v Acknowledgments I would first like to thank the entire Magliery Lab for my training and making everyday lab life enjoyable. Without such a great lab, this would have been a much more difficult endeavor. Specifically, I want to thank Dr. Bandon Sullivan for monitoring me for my first few years in graduate school and allowing me to be the scientist I am today. Thank you to my advisor, Dr. Thomas Magliery, for allowing me into his lab and giving me challenging and exciting projects. Without his initial trust in my abilities, none of this work would have been possible. Thank you to Shubham Mangla, Cynthia Campbell, Callie Moore, and all the undergraduates I had the opportunity to mentor. Their young enthusiasm inspired many graduate students and made science “fun”, despite the seemingly constant frustrations that being a scientist entails. A special thank you to my undergraduate advisor Dr. Vanessa McCaffrey. With no graduate students at Albion College, it becomes reliant on the advisor to assess if an undergraduate student will be successful in graduate school. Dr. McCaffrey encouraged me to conduct research, write a senior thesis, and apply to graduate school. Here I am, five vi years later, obtaining my PhD. Thank you so much for guiding me into this career path. I owe much of my success to you. I would like to thank my friends in graduate school and from back home who pushed for me to have a social life despite my busy schedule. I look forward to the many beer fests in the future. I would like to thank my mother, father, and sister for their continued support throughout graduate school. A lot has changed in five years but your love and encouragement were a constant for which I am truly grateful. Finally, I would like to thank Kristi for her love, for her advice, for her constant support, and for being there to vent to when experiments did not go as planned. I look forward to the many bright things our future holds. vii Vita 2008…………………………………………………….…Rochester Adams High School 2012……………………………….…………….……B.A. Biochemistry, Albion College 2017………………...…Graduate Research Associate, Ohio State Biochemistry Program, The Ohio State University Publications A 3E8.scFV.Cys-IR800 Conjugate Targeting TAG-72 in an Orthotopic Colorectal Cancer Model: Gong, L., Ding, H., Long, N.E. et al. Mol Imaging Biol (2017). https://doi.org/10.1007/s11307-017-1096-4 Fields of Study Major Field: Biochemistry viii Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii Chapter 1. Introduction ....................................................................................................... 1 1.1 Preface: Protein Engineering ................................................................................ 1 1.2 The Tumor Microenvironment: A Complex Problem.......................................... 7 1.3 Cancer Immunology and Immunotherapy.......................................................... 11 1.4 Tumor Associated Antigens ............................................................................... 17 1.5 Cancer Mucins and Tumor-Associated Glycoprotein 72 ................................... 21 1.6 Advancements in Antibody Engineering ........................................................... 28 1.7 Anti-TAG72 Antibodies ..................................................................................... 37 1.8 Single Chain Antibody Fragments ..................................................................... 42 1.9 The Notch Signaling Pathway ............................................................................ 47 1.10 Notch Implication in Cancer Immunology ..................................................... 51 1.11 Thesis Summary ............................................................................................. 55 Chapter 2. Engineering linkers in anti-TAG-72 antibody fragments to optimize biophysical properties, serum half-life, and high-specificity tumor imaging ................... 56 2.1 Contributions ...................................................................................................... 56 2.2 Abstract .............................................................................................................. 57 2.3 Introduction ........................................................................................................ 59 2.4 Results ................................................................................................................ 63 2.5 Discussion .......................................................................................................... 81 ix Chapter 3. Wide-scope Immunohistochemical analysis of TAG-72 expression in solid tumors by 3E8 and B72.3 antibodies ...............................................................................
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