Addressing delivery and synthesis challenges for peptide-based cancer vaccines By Rebecca Lynn Holden B.S. Chemistry Baylor University, 2015 Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Chemistry at the Massachusetts Institute of Technology September 2020 © 2020 Massachusetts Institute of Technology All rights reserved Signature of Author: ________________________________________________ Department of Chemistry July 20, 2020 Certified by: ______________________________________________________ Bradley L. Pentelute Associate Professor of Chemistry Thesis Supervisor Accepted by: _____________________________________________________ Adam Willard Associate Professor Graduate Officer 1 2 This doctoral thesis has been examined by a committee of the Department of Chemistry as follows: Professor Elizabeth M. Nolan …………………………………………………………………. Thesis Committee Chair Ivan R. Cottrell Professor of Immunology Professor Bradley L. Pentelute………………………………………………………………….. Thesis Supervisor Associate Professor of Chemistry Catherine L. Drennan…………………………………………………………………………… Thesis Committee Member Professor of Biology and Chemistry, HHMI Investigator 3 4 Addressing delivery and synthesis challenges for peptide-based cancer vaccines By Rebecca Lynn Holden Submitted to the Department of Chemistry on July 20, 2020 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Chemistry Abstract Therapeutic peptide vaccines have the potential to elicit and direct an anti-cancer T cell response, but their clinical efficacy has been limited in part by poor delivery to the lymphatic system, inefficient cell uptake, and scalable synthesis in the case of personalized vaccines. The work presented in this thesis explores several approaches to address these challenges. First, we use our fast-flow automated synthesis technology to confront the synthesis bottleneck for patient-specific ‘neoantigen’ vaccines, which have shown early promise but are hindered by slow production. We synthesize a particularly challenging set of peptides from a previous clinical trial as a test case, demonstrating that our technology can produce the majority of sequences in sufficient quantities with comparable or higher purity than a commercial vendor in a fraction of the time. Turning towards vaccine design, we explore several approaches to address the lymphatic and intracellular delivery of peptide antigens. We demonstrate the generality and anti-tumor efficacy of vaccines containing cell-penetrating peptides (CPPs), sequences shown to enhance the cell uptake of various cargo. We characterize their mechanism and identify several unanticipated contributors, namely trafficking to the lymph nodes, serum stability, and extended presentation in vivo. We then expand on an existing approach to mediate lymphatic trafficking via binding serum albumin by exploring additional albumin binding moieties. Next, we develop a straightforward and general approach to directly quantify antigen presentation and implement this technique to explore two strategies to design more effective vaccines, including CPPs. Finally, we build on previous work using CPPs to deliver antisense oligonucleotides (ASOs), another application that we pursued in tandem with cancer vaccines. We combine amphipathic and cationic CPPs to create chimeric sequences that synergistically enhance activity of an ASO and access new routes of uptake not utilized by either parent CPP. Drawing from our experience using CPPs to deliver ASOs as well as our expertise in peptide synthesis and design, we provide insight into the rapid production of personalized vaccines and efficient delivery of vaccine antigens. This thesis represents a new area of research for our lab, one in which we will hopefully continue to apply our unique skillset and perspective. Thesis Supervisor: Bradley L. Pentelute Associate Professor of Chemistry 5 Acknowledgements The past five years have been some of the greatest years of my life but also the hardest, challenging me in ways that I could not predict. Earning a PhD is a difficult journey; and earning a PhD while coming out is a real trip. I have learned so much, grown stronger as a person, and become a better and more confident scientist. I owe a great deal of gratitude to everyone who has helped me make it through. I want to start by thanking my thesis advisor, Prof. Brad Pentelute. His enthusiasm from the moment I joined the lab set the tone for my entire grad school career. I am grateful for his guidance and mentorship, which always seemed to be exactly what I needed. He encouraged me to propose my own ideas early on and his confidence in me allowed me to grow as an independent scientist. At the same time, he provided direction and encouragement whenever I needed it. His support extended beyond scientific guidance, helping me improve my communication and presentation skills and providing invaluable professional advice. Finally, of course, I would like to thank Brad for cultivating a supportive and collaborative lab culture that has significantly shaped both my research and my entire experience at MIT. I could not have asked for a more invested and supportive thesis committee chair than Prof. Liz Nolan. I want to thank her for being an amazing mentor. It was always evident during our meetings that she genuinely cared about my research and my growth as a scientist. I appreciate her many insightful questions and thoughtful suggestions over the years. I am especially grateful more recently for her postdoc application and general career advice. Than you to Prof. Cathy Drennan, who has been another incredible mentor. I appreciated her encouragement throughout grad school, particularly during my second- and third-year exams. Her advice for my postdoc search and her support with fellowship applications has been invaluable, and I am extremely grateful. I am thankful to my collaborators in the Irvine lab, beginning with Prof. Darrell Irvine, who has provided a great deal of advice and scientific guidance for many of the projects described in this thesis. I am also grateful to Coralie, whose insight and advice over the last few years helped me to be a better scientist and whose sincerity, thoughtfulness, and humor made her a great colleague and friend. Thank you to Kelly, who was both a collaborator and a mentor during my first couple of years in grad school. I am grateful for her organization and positivity getting several of these projects up and running. I would also like to thank Dan, Josetta, and Naveen for their contributions to our work. I would like to express my gratitude to Prof. Cathy Wu and Prof. Nir Hacohen for a productive and exciting collaboration on their neoantigen vaccines. I have learned a lot working with them and look forward to learning more in the future. I would also like to thank Keerthi, Sisi, Matt, Zhuting, and the whole Neovax subgroup. I cannot imagine what my experience in grad school would have been without my amazing colleagues and friends in the Pentelute lab. Thank you to my best friend Azin, whose compassion, vivacity, and sarcasm always brightened my day. I am grateful to have worked with someone so supportive, who was always willing to listen and help with anything going on in my research or personal life. Thank you to Nina, whose cheerfulness and style brightened our space. I am grateful for her thoughtfulness, generosity, and mentorship. I know I can turn to her any time in the future. I would like to thank Colin for being my first person in the lab and teaching me so much. He was a delight to work with and helped me through some of the hardest moments of grad school. Thank you to Justin for being an easygoing, practical, and encouraging friend and collaborator. I am grateful to Alex M. for being a willing teacher, a caring friend, and a fun roommate. He truly is the definition of precious. I would also like to thank Ethan for his commiseration and for the scientific input he happily provided anytime I asked. Thank you to Charlotte for being dependably upbeat and always willing to help. I owe a great deal of 6 gratitude to Anthony Q. for his tireless efforts to keep the Orbitrap running, as well as his down- to-earth kindness and his many, many, many jokes. I am thankful to Carly for being amazing to work with and a brilliant sand volleyball team captain. Thank you to Alex L. and Tuang for being my comrades throughout the grad school process, especially second- and third-year exams. I would like to thank Nick for being an enthusiastic collaborator and for organizing our immunotherapy super-group meetings. And finally, I would like to thank all of my other friends and colleagues who have made the past five years so enlightening and enjoyable: Katie, Jessie, Rachael, Zak, Mette, Diómedes, Joe, Aaron, Deborah, Surin, and everyone else in the Pentelute lab. I am also grateful to Christine, Katherine, and Alex (and Eli) from the Kiessling lab for their scientific input and friendship. I want to extend a huge thank you to all of the friends that I made outside of lab. In particular, I would like to thank Lindsey, who became an immediate friend and who has been a source of endless support, commiseration, and sangria. I also want to thank Marty, who was one of the first friends I made in Cambridge and whose kindness and originality have been welcome constants throughout grad school. I enjoyed our trips together almost as much as I enjoyed being roommates. Thank you to Sheena, the gentlest spirit I know; Dmitro, one of my favorites; Zach, who will be relieved to know that he is also one of my favorites; and Brendan, who is a treasure. Thank you to Alex, Aubrey, Ashley, and Chad—Candace’s amazing friends who not only gave me their stamp of approval but became my friends, too. I am thankful to Erin for their care and wisdom. I also want to thank my comrades from Boston Feminists for Liberation, who have taught me so much.