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Throughput Phage Display and Building the Foundations of a Novel High-Throughput Intrabody Pipeline

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Throughput Phage Display and Building the Foundations of a Novel High-Throughput Intrabody Pipeline The Generation of Affinity Reagents Using High- throughput Phage Display and Building the Foundations of a Novel High-throughput Intrabody Pipeline by Nicolas Odysseas Economopoulos A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Molecular Genetics University of Toronto © Copyright by Nicolas Economopoulos 2011 The Generation of Affinity Reagents Using High-throughput Phage Display and Building the Foundations of a Novel High- throughput Intrabody Pipeline Nicolas Odysseas Economopoulos Master of Science Graduate Department of Molecular Genetics University of Toronto 2011 Abstract Phage display technology has emerged as the dominant approach in antibody engineering. Here I describe my work in developing a high-throughput method of reliably generating intracellular antibodies. In my first data chapter, I present the first known high-throughput pipeline for antibody-phage display libraries of synthetic diversity and I demonstrate how increasing the scale of both target production and library selection still results in the capture of antibodies to over 50% of targets. In my second data chapter, I present the construction and validation of a novel scFv-phage library that will serve as the first step in my proposed intrabody pipeline. Antibodies obtained from this library will be screened for functionality using a novel yeast-two- hybrid approach and have numerous downstream applications. This high-throughput pipeline is amenable to automation and can be scaled up to thousands of domains, resulting in the potential generation of many novel therapeutic reagents. ii Acknowledgments It is surreal to think that I have reached the end of a journey that started three years ago. Although my next chapter in life will be away from the benchtop, my time in the Sidhu Lab has been an unforgettable experience that I will always cherish. I have been blessed to work with a remarkable group of individuals and gained friendships that will last a lifetime. First and foremost, I want to thank my supervisor Dev Sidhu for being such a great mentor. Not only was Dev patient, encouraging and supportive of my endeavours but his genuine care for me as a person and not just as a student meant a lot to me. It was not easy to think about my future path and I will always be grateful for Dev‟s understanding. I also want to thank Peter Roy for being there for me in the beginning and helping me navigate through the uncertainties of choosing a lab. If it weren‟t for Peter, I would have never met Dev or been part of such a fantastic group. I would also like to thank my committee members Charlie Boone and Jeff Wrana for their guidance and insight throughout my studies. I also want to thank all of the Sidhu Lab members from 2008-2011. If I wrote out all of your names, my thesis would be twice as long! It was an honor to work alongside all of you and I am truly grateful for our time together. In particular, I want to give a big thank you to Bryce Nelson, Raffi Tonikian, Helena Persson, Andreas Ernst and Bernard Liu. Our conversations about life and work gave me some much-needed perspective and helped me weather the storms and bask in the successes of grad school. Most importantly, I want to thank my family for always believing in me and for their unconditional love and support. I am forever indebted to my parents, Marita and Panos, who sacrificed so much for me and are a constant source of inspiration and strength. I also want to thank my brother Aris, who has always been there for me not only as a loving brother but as a true friend. Lastly, I want to thank my wonderful girlfriend Amanda, whose incredible love and encouragement added California sunshine to each and every day. iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Appendices .......................................................................................................................... x Chapter 1 Introduction .................................................................................................................... 1 1.1 Exploring Protein Function ................................................................................................. 2 1.2 Antibodies as Scaffolds for Protein Engineering ................................................................ 2 1.2.1 Antibody Structure .................................................................................................. 2 1.2.2 The Antigen Binding Site ....................................................................................... 4 1.2.3 Engineering Antibodies .......................................................................................... 5 1.3 Therapeutic Applications of Antibodies ............................................................................. 6 1.3.1 Extracellular Applications ...................................................................................... 6 1.3.2 Intracellular Applications ........................................................................................ 7 1.4 Hybridoma Technology for Antibody Generation .............................................................. 8 1.5 Phage Display and Antibody Engineering .......................................................................... 9 1.5.1 Displaying Proteins on Phage Particles .................................................................. 9 1.5.2 Site-directed Mutagenesis and Phage Library Construction ................................. 12 1.5.3 Construction of Antibody Libraries ...................................................................... 13 1.5.4 Fab-Phage Library F ............................................................................................. 13 1.5.5 Performing Selections ........................................................................................... 15 1.5.6 Determining Enrichment and Specificity During Selections ................................ 17 1.6 Increasing the Scale of Phage Display .............................................................................. 17 1.7 Issues with Intracellular Stability of Antibodies ............................................................... 18 iv 1.8 Intein-Yeast-Two-Hybrid: a Novel Approach .................................................................. 20 1.8.1 Intein-mediated Cyclization of Proteins Can Enhance Stability ........................... 20 1.8.2 Cyclization of scFvs .............................................................................................. 20 1.9 Thesis Rationale and Objectives ....................................................................................... 21 Chapter 2 Generating High Affinity Antibodies using a High-Throughput Pipeline for Phage Display ..................................................................................................................................... 23 2 Generating High Affinity Antibodies using a High-Throughput Pipeline for Phage Display . 24 2.1 Validation of Library F against a set of 20 high-quality SH2 antigens ............................ 25 2.2 Affinity Maturation of anti-SH2 clones results in improved affinities ............................. 25 2.3 GST-SH3 constructs as candidates for HTP selections .................................................... 26 2.4 Selection in a 96-well HTP format yields unique Fabs against 60% of purified targets .. 26 2.5 Affinity Maturation Libraries were generated against a subset of SH3 domains ............. 28 2.6 Selection of Affinity Maturation Libraries yielded clones with improved affinities ........ 36 2.7 Purified Fabs can be used as detection reagents ............................................................... 38 2.8 Construction of Chimeric IgGs as a more direct comparison of detection ....................... 41 2.9 Discussion ......................................................................................................................... 42 2.10 Materials and Methods ...................................................................................................... 44 2.10.1 Strains and constructs ........................................................................................... 44 2.10.2 High-throughput Expression and Purification of Protein Targets ........................ 45 2.10.3 Construction of Libraries ...................................................................................... 46 2.10.4 Amplification and Preparation of phage libraries/supernatants before and during selections ................................................................................................... 46 2.10.5 SH2 Mictrotiter Plate Selections ........................................................................... 47 2.10.6 SH3 High-throughput Selections .......................................................................... 48 2.10.7 Selection of Affinity Maturation Libraries ..........................................................
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