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Improving Protein Therapeutics Through Quantitative Molecular Engineering Approaches and a Cell-Based Oral Delivery Platform University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Improving Protein Therapeutics Through Quantitative Molecular Engineering Approaches and A Cell-Based Oral Delivery Platform Ting Wun Ng University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biomedical Commons Recommended Citation Ng, Ting Wun, "Improving Protein Therapeutics Through Quantitative Molecular Engineering Approaches and A Cell-Based Oral Delivery Platform" (2013). Publicly Accessible Penn Dissertations. 784. https://repository.upenn.edu/edissertations/784 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/784 For more information, please contact [email protected]. Improving Protein Therapeutics Through Quantitative Molecular Engineering Approaches and A Cell-Based Oral Delivery Platform Abstract Proteins, with their ability to perform a variety of highly specific biological functions, have emerged as an important class of therapeutics. However, to fully harness their therapeutic potential, proteins often need to be optimized by molecular engineering; therapeutic efficacy can be improved by modulating protein properties such as binding affinity/specificity, half-life, bioavailability, and immunogenicity. In this work, we first present an introductory example in which a mechanistic mathematical model was used to improve target selection for directed evolution of an aglycosylated Fc domain of an antibody to enhance phagocytosis of tumor cells. Several aspects of directed evolution experimental methods were then optimized. A model-guided ligation strategy was developed to maximize ligation yield in DNA library construction, and this design tool is freely available through a web server. Streamlined protocols for mRNA display and ribosome display, which are powerful in vitro selection methods, were also created to allow robust selection of a variety of therapeutic proteins, including monomeric Fc domains, designed ankyrin repeat proteins, a single-chain insulin analog (SCI-57), and leptin. Anti-ICAM-1 scFv antibody fragments were also optimized for ribosome display by grafting complementarity determining regions onto a stable human framework. In addition to engineering the proteins themselves, effective delivery systems are essential for maximizing the therapeutic benefit of these proteins in a clinical setting. We therefore also developed a novel oral delivery platform based on the food-grade bacterium Lactococcus lactis. SCI-57, leptin, and SCI-57-leptin fusion proteins have been successfully secreted from this host in vitro and preliminary studies in a diabetic mouse model show reduced glucose levels after oral administration of L. lactis secreting SCI-57. We then further improved the secretion potential of this host through directed evolution of a L. lactis signal peptide. In summary, our studies have provided important advances to the field of protein engineering through the development of mechanistic mathematical models, streamlined experimental methodologies, and polypeptides with improved properties. This work has also opened up the possibility of systemic delivery of protein therapeutics using live microorganisms. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Bioengineering First Advisor Casim A. Sarkar Keywords mathematical model, oral protein delivery, protein engineering Subject Categories Biomedical This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/784 IMPROVING PROTEIN THERAPEUTICS THROUGH QUANTITATIVE MOLECULAR ENGINEERING APPROACHES AND A CELL-BASED ORAL DELIVERY PLATFORM Daphne T. W. Ng A DISSERTATION in Bioengineering Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2013 _____________________ Casim A. Sarkar, Ph.D., Assistant Professor of Bioengineering Supervisor of Dissertation _____________________ Daniel A. Hammer, Ph.D., Professor of Bioengineering Graduate Group Chairperson Dissertation Committee: Mark Goulian, Ph.D., Professor of Biology (Committee Chair) Arjun Raj, Ph.D., Assistant Professor of Bioengineering Andrew Tsourkas, Ph.D., Associate Professor of Bioengineering IMPROVING PROTEIN THERAPEUTICS THROUGH QUANTITATIVE MOLECULAR ENGINEERING APPROACHES AND A CELL-BASED ORAL DELIVERY PLATFORM COPYRIGHT 2013 Ting Wun Ng This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ To my parents This work would not have been possible without their love, support and occasional reminders :) iii ACKNOWLEDGMENTS I feel truly blessed with many amazing people throughout my PhD journey, who have given me much needed support and encouragement in one form or the other. I am also truly grateful for the opportunity to explore my scientific curiosity in the intellectual environment provided by the University of Pennsylvania over the past few years. First, I am most fortunate for having the opportunity to learn from my advisor and mentor, Dr. Casim Sarkar. I am extremely grateful for his patience and support as he witnessed my growth as a scientist. I find myself being continually inspired as his enthusiasm and passion in the subject matter is highly contagious. His encouragement on creativity and hard work has consistently pushed me to reach further than what I thought I could accomplish. I am also indebted to my thesis committee, Dr. Mark Goulian, Dr. Andrew Tsourkas and Dr. Arjun Raj for their guidance and helpful suggestions throughout my dissertation. Their generous support and encouragement both during and outside of formal committee meetings are very much appreciated. Some of the most interesting work would not have been possible without wonderful collaborators whom I had the pleasure to work with. I would like to thank Dr. George Georgiou, Sang Taek and Will for the opportunity in aglycosylated IgG modeling; Dr. Ali Naji and Ming for the help and expertise in diabetic mice experiments; iv and Dr. Vladimir Muzykantov and Colin for the discussions regarding the anti-ICAM-1 scFv project. My dissertation was truly enjoyable thanks to my incredible and talented colleagues in the Sarkar lab: Najaf, Pam, Santhosh and Ellen. I greatly appreciate their support, help and all the lively discussions. They have never failed to amaze me with their scientific vigor and generosity. To all my friends and family who have walked alongside me, I am forever grateful for your support, inspiration and encouragement. Thank you for listening to my tears and laughter, and for sharing a part of your lives with me. To my parents, thank you for showering me with love and never-ending support. Although far away, your patience, support and faith have motivated me every day and shaped me throughout my academic career. Last but not least, I must thank my best friend and my love, Hubert, for your company, support, encouragement, patience, love, and for keeping me happy and sane. v ABSTRACT IMPROVING PROTEIN THERAPEUTICS THROUGH QUANTITATIVE MOLECULAR ENGINEERING APPROACHES AND A CELL-BASED ORAL DELIVERY PLATFORM Daphne T. W. Ng Casim A. Sarkar, Ph.D. Proteins, with their ability to perform a variety of highly specific biological functions, have emerged as an important class of therapeutics. However, to fully harness their therapeutic potential, proteins often need to be optimized by molecular engineering; therapeutic efficacy can be improved by modulating protein properties such as binding affinity/specificity, half-life, bioavailability, and immunogenicity. In this work, we first present an introductory example in which a mechanistic mathematical model was used to improve target selection for directed evolution of an aglycosylated Fc domain of an antibody to enhance phagocytosis of tumor cells. Several aspects of directed evolution experimental methods were then optimized. A model-guided ligation strategy was developed to maximize ligation yield in DNA library construction, and this design tool is freely available through a web server. Streamlined protocols for mRNA display and ribosome display, which are powerful in vitro selection methods, were also created to allow robust selection of a variety of therapeutic proteins, including monomeric Fc domains, designed ankyrin repeat proteins, a single-chain insulin analog (SCI-57), and vi leptin. Anti-ICAM-1 scFv antibody fragments were also optimized for ribosome display by grafting complementarity determining regions onto a stable human framework. In addition to engineering the proteins themselves, effective delivery systems are essential for maximizing the therapeutic benefit of these proteins in a clinical setting. We therefore also developed a novel oral delivery platform based on the food-grade bacterium Lactococcus lactis. SCI-57, leptin, and SCI-57-leptin fusion proteins have been successfully secreted from this host in vitro and preliminary studies in a diabetic mouse model show reduced glucose levels after oral administration of L. lactis secreting SCI-57. We then further improved the secretion potential of this host through directed evolution of a L. lactis signal peptide. In summary, our studies have provided important advances to the field of protein engineering through the development of mechanistic mathematical models, streamlined experimental methodologies,
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