CD74 targeted nanoparticles as Dexamethasone Delivery System for B lymphoid malignancies DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Georgia Triantafillou, MPH Integrated Biomedical Sciences Graduate Program The Ohio State University 2011 Dissertation Committee John C. Byrd, MD, advisor Natarajan Muthusamy, PhD co-advisor Robert J.Lee, PhD, co-advisor Mitch A.Phelps, PhD, co-advisor Guido Marcucci, MD committee member Susheela Tridandapani, PhD committee member Copyright page Georgia Triantafillou 2011 ABSTRACT Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia and is not curable with standard therapy. In CLL the B lymphocytes appear morphologically mature but immunologically express less mature B cell makers. Specifically, the CLL B cells co-express surface antigens including CD19, CD20, CD5, CD23. Due to nonspecific delivery of the drugs to malignant as well as normal cells, patients experience numerous side effects. Targeted drug delivery to cancer cells is a desirable goal for all anti cancer strategies. Corticosteroids have been proven to be a beneficial and well established part of CLL treatment. Dexamethasone(DEX) is frequently used in CLL therapies as well as other malignancies and antiinflammatory diseases. However, DEX nonspecifically enters all tissues inducing various toxicities to patients. Therefore, DEX treatment is often discontinued or not administered to certain patients. We aimed to deliver this efficient clinical agent via a targeted delivery vehicle and examine its effect on its target B-CLL cells. The main scope of the work presented is the creation of a sterically stable nanoparticle, an ii immunoliposome (ILP), that has DEX encapsulated in its interior and a monoclonal antibody(Mab) in its exterior targeting CLL B cells expressing the surface antigen that is recognized by the Mab. Chapter 1 is an indroduction to CLL, other B cell malignancies and Mabs as well as DEX in disease treatment and management. The chapter also presents background on targeted delivery via liposomes and immunoliposomes Chapter 2 describes CD74 as the antibody of choice to target the nanoparticle and details the evaluation of the CD74 liposome without drug in vitro . This nanoparticle was shown to be cytotoxic in vitro in cell lines and B CLL cells and mimick abilities of anti-CD74 in vivo. CD74 liposome showed high binding to malignant cells expressing the antigen and induction of leukemic cell apoptosis.This gave us a platform to continue with forming dexamethasone liposome (DEX LIP) particles . In chapter 3 a methodoly was developed to quantitate DEX in vitro and in vivo using Liquid Chromatography tandem Mass Spectometry( LC/MS-MS). The method was adjusted to measure encapsulated DEX inside the liposome in plasma. In this chapter we also describe a pharmacokinetic study in mice comparing DEX as free drug and DEX LIP. Chapter 4 focuses on the evaluation of CD74 DEX LIP in vitro and in vivo. The liposomal DEX targeted with anti- CD74 was evaluated in vitro in cell lines and primary CLL B cells and showed potent cytotoxicity . Immunoliposome efficacy was subsequently, tested in vivo in mouse model iii of lymphoid disease and showed that it significantly increased survival of diseased mice compared to controls.Overall, the study showed therapeutic effect of CD74 DEX LIP and advantage of encapsulating the drug into immunoliposome .Also another in vivo study in mice is described showing that nontargeted DEX LIP has protective effect against non desirable effects of free DEX. Chapter 5 summarizes and discusses the work presented here , as well as presents future directions steming from the studies. The work presented provides evidence that DEX immunoliposomes can have therapeutic effect in B cell malignancies with the possibility to be applied in other disease in which corticosteroids are administered. iv Dedicated to my family v ACKNOWLEDGEMENTS I express my gratitude to Dr. John C. Byrd, my advisor, who is a constant inspiration to strive for the best. I admire his diligence, his energy and his unparalleled memory. He is a brilliant physician and scientist and a great benefit to our society.I also feel privileged for working and learning in his laboratory and having access to clinical samples. I am gratefult to Dr. Natarajan Muthusamy that has continuously guided me and challenged me throughout the last five years. He is an enthusiastic scientist, an inspiring professor and at times he has stood as a father figure. Without his persistence and help this work would have never been completed. I would also like to thank Dr. Mitch Phelps for his guidance and encouragement. I had no idea about mass spectrometry when I started the work in his laboratory and now I feel like the knowledge acquired in his laboratory is invaluable. Also thank you for all the support and understanding, I always felt that I could be open about everything in our meetings.Thanks to all the lab members of the Phelps lab that have been critical in completing my work vi Also I am grateful to Dr.Robert J.Lee for introducing me to the world of nanoparticles ,guding me and giving me access to techniques and equipment to design and complete the formulations.The collaboration with Dr.Lee was crucial in completing this work I also want to thank Dr.Guido Marcucci and Dr. Susheela Tridandapani for being my committee members and helping me complete my goals. All of the individuals mentioned above have contributed into making my work truly diverse and integrated. In Dr.Byrd’s lab I want to thank all the former and present members that have worked with me because they have all helped me in some ways. In particular, I would like to thank Dr.Rosa Lapalombella for her help and her friendship that have been very supportive throughout the years. Also, Asha Ramannuni who has been a good friend. In addition, I would like to thank Frank Frissora and Carolyn Cheney for being the first people to teach me techniques in the lab and for always being supportive and helping regardless of the time requirements. I want to thank William Towns and Matt Stefanovski for making mouse work enjoyable and creating a great working environment.I will miss our conversations . I am grateful to Dr. Erin Hertlein for her help , her conversations and her support.In addition, I want to give special thanks to all the graduate students in the Byrd lab since we all had to deal with similar situations and you have all helped me in different ways. vii Special thanks to Dr. Bo Yu that has helped me with the formulations of the nanoparticles and for the great conversations. Also I am grateful for the help and great friendship of Yicheng Mao that has been crucial in completing this work. Thank you to Dr. Lapo Allinari, a remarkable physician scientist and good friend. I want to express my deepest gratitude to all the patients that have suffered from CLL and have so kindly given consent so we can improve science, achieve translational research and possibly one day beat this disease. I would like to thank all my friends around the world that have kept in touch and have supported me throughout my PhD pursuit. Your friendship is invaluable. I am always grateful to my fiance, Jon, for all the years of encouragement and support. Thank you for always believing in me. No matter what your schedule is and even when you are on call you always put me first. Your love and dedication has been a driving force to complete this work and enjoy our lives together. To my parents, thank you for raising me with good values , believing in me and helping me accomplish my dreams. You have been incredibly supportive of any decision I have ( rationally) made and even though I have been so far I always felt close to you. I could have never completed my studies if I did not know I always had you by my side. There are not enough words to express viii my love and appreciation. My sister, my nieces and nephew are a constant source of joy for me.Sometimes when everything else seemed wrong just a laughter from you was all it took to make things just fine.Vaso you are my best friend. I cannot imagine my life without you.Thank you to everyone that has helped me emotionally, physically and professionally. ix VITA September ,1982………...................................................Born-Athens, Greece 2004………………………………………………………………………BS, Biology Kent State University 2005………..............................................................................MPH, Ohio State University 2006 to present…………...……….......................................Graduate Research Associate Integrated Biomedical Graduate Sciences program, The Ohio State University x PUBLICATIONS 1.Triantafillou G, Mao Y, Hertlein E, Towns W, Stefanovski M Jarjoura D., Phelps M.A , Marcucci G, Lee, RJ, , Muthusamy N., Byrd JC . Milatuzumab Immunoliposomes as Dexamethasone Carriers to CLL B Cells Extend Survival of Diseased Mice and Minimize Drug Associated Side Effects. Blood, 2011, in preparation 2. Triantafillou G. He L, Lee, RJ, Muthusamy N., Byrd JC ,Phelps M.A. Method for detection of encapsulated liposomal dexamethasone in mice using liquid chromatography tandem mass spectrometry. J.Chrom.B ,2011, in preparation 3. Chan KK, Liu Z, Xie Z, Chiu M, Wang H, Chen P, Dunkerson S, Chiu M, Liu S, Triantafillou G, Garzon R, Croce CM, Byrd JC, Muthusamy N, Marcucci G. A Novel Ultrasensitive Hybridization-Based ELISA Method for 2- Methoxyphosphorothiolate MicroRNAs and Its In vitro and In vivo Application. AAPS J. 2010 Dec;12(4):556-68. 4. Hertlein E*, Triantafillou G*, Sass EJ, Hessler JD, Zhang X, Jarjoura D, Lucas DM, Muthusamy N, Goldenberg DM, Lee RJ, Byrd JC. Milatuzumab immunoliposomes induce cell death in CLL by promoting accumulation of CD74 on the surface of B cells.