Improving methods for cytokine immunotherapy of cancer

Cytokine therapy can activate potent antitumor responses, yet collateral toxicity often limits dosages. Although immunocytokines have been designed with the intent to localize cytokine activity, systemic dose-limiting side effects are not fully ameliorated by attempted tumor targeting. In the first part of this work, we used the B 1 6F 10 melanoma model to demonstrate that a nontoxic dose of IL-2 immunocytokine synergized with tumor-specific antibody to significantly enhance therapeutic outcomes compared to monotherapy with immunocytokine, concomitant with increased tumor saturation and intratumoral cytokine responses. Examination of cell subset biodistribution showed that the immunocytokine associated mainly with IL-2Rexpressing innate immune cells, with more bound immunocytokine present in systemic organs than in the tumor microenvironment. More surprisingly, immunocytokine antigen specificity and FEcyR interactions did not appear necessary for therapeutic efficacy or biodistribution patterns, as immunocytokines with irrelevant specificity and/or inactive mutant Fc domains behaved similarly to tumor-specific immunocytokine. IL-2-IL-2R interactions, rather than antibodyantigen targeting, dictated immunocytokine localization; however, the lack of tumor targeting did not preclude successful antibody combination therapy. This study presents a safe, straightforward strategy for augmenting immunocytokine efficacy via supplementary antibody dosing and explores underappreciated factors that can subvert efforts to purposefully alter cytokine biodistribution. Numerous studies have identified cancer immunotherapy combinations that exhibit synergistic antitumor activity, but surprisingly, these studies rarely consider the effects of relative dose timing. In the second part of this work, using established syngeneic tumor models, we found that staggering IFNa administration after, rather than simultaneously with, serum-persistent IL-2 and tumor-specific antibody significantly increased long-term survival and generated immunological memory. Successful combination therapy required IFNa-induced activation of cross-presenting CD8a+ DCs following release of antigenic tumor debris by the IL-2-and-antibody-mediated immune response. Due to decreased phagocytic ability post-maturation, DCs activated too early captured much less antigen and could not effectively prime CD8+ T cells. Temporally programming DC activation to occur after tumoricidal activity enhanced tumor control by multiple combination immunotherapies that act through distinct mechanistic pathways, presenting a facile strategy for augmenting efficacy in the combinatorial treatment setting and highlighting dose schedule as an overlooked factor that can profoundly affect the success of multi-component immunotherapies. Thesis Supervisor: K. Dane Wittrup Title: C.P. Dubbs Professor of Chemical Engineering & Biological Engineering 3 ACKNOWLEDGEMENTS It truly takes a village to get through a Ph.D., and there are a fair number of people whom I must acknowledge for their contributions to the successful completion of this thesis. First, I would like to offer my profound gratitude to my advisor, Dane Wittrup, for his exceptional mentorship and unwavering support these past six years. Additional thanks go to my thesis committee members Dr. Darrell Irvine, Dr. Jianzhu Chen, and Dr. Willem Overwijk, who have offered me nothing but continuous encouragement and invaluable advice and to M.I. T. , the National Science Foundation, the Koch Institute Ludwig Center for Molecular Oncology, and the Siebel Scholars Foundation, whose funding helped make this work possible. I am also indebted to all of the members of the Wittrup lab (past, present, and honorary) with whom I had the fortune to interact, in particular Nicole Yang, Cary Opel, Byron Kwan, Monique Kauke, Kelly Moynihan, Eric Zhu, Ryan Kelly, and Naveen Mehta, who helped directly with various pieces of this project; Annie Gai, David Liu, Jordi Mata-Fink, Jamie Spangler, Chris Pirie, John Rhoden, Xiaosai Yao, Tiffany Chen, and Seymour de Picciotto, who welcomed me into the world of protein engineering; and Jim Van Deventer, Alessandro Angelini, Katie Maass, Michael Traxlmayr, Adrienne Rothschilds, Alison Tisdale, Byong Kang, and Noor Momin, who have maintained the collaborative and friendly spirit of the lab. Ryan Sullivan and Max Vasquez were instrumental collaborators on portions of this work as well. Next, I would like to thank the staff of the Swanson Biotechnology Center at the Koch Institute, especially the Flow Cytometry Core, for technical assistance. I additionally thank my brother, Tony Tzeng, for providing online company whenever it was needed, and my fiance Kenneth Danton, for patiently commuting between Cleveland and Boston just to accompany me through the toils and tribulations of research. My final and greatest thanks go to my parents, whose coaching, persistence, and blessing inspired me to embark on this academic journey.