Humanized Chimeric Receptors in the Therapy of Multiple Sclerosis Ioana Moisini University of Tennessee Health Science Center
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University of Tennessee Health Science Center UTHSC Digital Commons Theses and Dissertations (ETD) College of Graduate Health Sciences 12-2007 Humanized Chimeric Receptors in the Therapy of Multiple Sclerosis Ioana Moisini University of Tennessee Health Science Center Follow this and additional works at: https://dc.uthsc.edu/dissertations Part of the Immune System Diseases Commons, and the Medical Sciences Commons Recommended Citation Moisini, Ioana , "Humanized Chimeric Receptors in the Therapy of Multiple Sclerosis" (2007). Theses and Dissertations (ETD). Paper 184. http://dx.doi.org/10.21007/etd.cghs.2007.0215. This Dissertation is brought to you for free and open access by the College of Graduate Health Sciences at UTHSC Digital Commons. It has been accepted for inclusion in Theses and Dissertations (ETD) by an authorized administrator of UTHSC Digital Commons. For more information, please contact [email protected]. Humanized Chimeric Receptors in the Therapy of Multiple Sclerosis Document Type Dissertation Degree Name Doctor of Philosophy (PhD) Program Pathology Research Advisor Terrence L. Geiger, MD, Ph.D. Committee Elisabeth Fitzpatrick, PhD Michael C. Levin, MD Robert B. Lorsbach, MD, PhD Edward F. Rosloniec, PhD DOI 10.21007/etd.cghs.2007.0215 This dissertation is available at UTHSC Digital Commons: https://dc.uthsc.edu/dissertations/184 HUMANIZED CHIMERIC RECEPTORS IN THE THERAPY OF MULTIPLE SCLEROSIS A Dissertation Presented for The Graduate Studies Council The University of Tennessee Health Science Center In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy From The University of Tennessee By Ioana Moisini December 2007 Copyright © 2007 by Ioana Moisini All rights reserved ii DEDICATION This dissertation is dedicated to my mom, Valeria Moisini, who gave me endless love and support, and was there for me, come rain or come shine. iii ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Terrence Geiger, for the trust, support, guidance, and most of all the patience that he has shown during my scientific struggle. He always knew how to ask things without demanding them and how to soothe my scientific frustrations. His particular way of approaching people and situations in and out of work has reshaped my character and my career. He is not only a prodigious contributor in his field, but also a very caring and considerate person. I appreciate and will always be grateful for the chance he took when he picked me out of three other colleagues to be his first graduate student. I am also extremely thankful to Drs. Lawrence Pfeffer and Edward Schneider. The first one changed my life when he sent me the acceptance letter and has been like a father figure to me ever since I came here. He and Dr. Schneider have been the most “pro-student” figures at the University of Tennessee, supporting me and always being by my side when I felt that maybe research was not my true calling. Both of them “have sent” me back to the bench and boosted my spirit when I needed it the most. Drs. Michael Levin and Elisabeth Fitzpatrick gave me a helpful and well-timed hand when my committee panel was changed; they did not hesitate for a moment to jump in and catch up with my research. I am thankful beyond words to Dr. Richard Cross. My whole project would have turned to ashes if it had not been for his help with cell sorting. But he did more than provide scientific help and advice; he was the dearest friend and the strongest shoulder I could ever ask for and lean on. iv Last, but definitely not least, I have to acknowledge my colleagues and friends. Dr. Alina Nico West, Cynthia Lancaster, Dr. Robert Borgon, Yu Fukuda, my closest friend Dr. Weili Sun, Dr. Jean-Hugues Parmentier, Dr. Noel Lenny, Dr. Rajshekhar Alli, Dr. Donald Yergeau, Dr. Michelle Hamlet, Dr. Jana Radin, Dr. Kerim Babaoglu and so many more who have been close to me and supported me throughout these years. Our St. Jude lunches, parties, “complaints sessions”, lab frustrations and so on are the best memories that I will carry on with me. I cannot thank you all enough. v ABSTRACT The role of autoreactive, antigen-specific T-cells in the development of autoimmunity has long been documented. T-cells expressing chimeric receptors are specifically redirected against such cells and have been proven to suppress autoimmune encephalomyelitis, the murine model of multiple sclerosis. We here demonstrate the ability of humanized chimeric receptors to suppress experimental autoimmune encephalomyelitis (EAE) in a humanized mouse model by redirecting T lymphocytes against autoreactive T-cells. The receptors were synthesized by linking the 84-102 epitope of human myelin basic protein (MBP) to the extracellular and transmembrane domains of the beta chain of human major histocompatibility complex (MHC) class II molecule and the cytoplasmic zeta chain of T cell receptor and pairing it to the alpha chain linked to zeta. CD8 + receptor-modified T-cells (RMTC) were able to recognize the cognate TCR receptor of antigen-specific cells and induce cytokine secretion, proliferation, and cytolysis upon engagement. Most importantly, the RMTC were able to specifically kill antigen-specific cells both in vitro and in vivo and prevent EAE disease. We hypothesize that the humanized chimeric receptors could be used as a therapeutic approach for multiple sclerosis in the future. vi TABLE OF CONTENTS Chapter 1. General introduction ......................................................................................1 1.1 Historical perspective of multiple sclerosis ...................................................................1 1.2 Clinical signs and symptoms .........................................................................................2 1.3 Diagnosis of MS ............................................................................................................3 1.4 Pathobiology of MS .......................................................................................................4 1.5 Animal models of MS....................................................................................................9 1.5.1 Current animal models of MS.............................................................................10 1.5.2 Adoptive transfer EAE........................................................................................12 1.5.3 Transgenic mice as models for MS.....................................................................13 1.6 Therapeutic approaches for MS...................................................................................15 1.6.1 Glucocorticoids...................................................................................................15 1.6.2 Cytokines ............................................................................................................16 1.6.3 Antigen-derived immunotherapies......................................................................17 1.6.4 Altered peptide ligands .......................................................................................18 1.6.5 Synthetic copolymers..........................................................................................19 1.6.6 Mucosal administration of antigen......................................................................20 1.6.7 T-cell vaccination................................................................................................20 1.6.8 Monoclonal antibodies........................................................................................21 1.6.9 Gene therapy in MS ............................................................................................21 Chapter 2. Development of chimeric receptors.............................................................24 Chapter 3. Significance of a dileucine motif in CD28-zeta (ζ)- containing chimeric receptors .........................................................................................33 3.1 Introduction..................................................................................................................33 3.1.1 [DE]XXXL[LI] signals.......................................................................................34 3.1.2 DXXLL signals...................................................................................................35 3.2 Identification of a murine CD28 dileucine motif that suppresses single-chain chimeric T-cell receptor expression and function (1)..............................36 3.2.1 Introduction.........................................................................................................36 3.2.2 Materials and methods........................................................................................38 3.2.3 Results.................................................................................................................42 3.2.4 Conclusions.........................................................................................................48 vii Chapter 4. Development and function of humanized chimeric receptors ............................................................................................................................53 4.1 Introduction to the development of humanized chimeric receptors .............................53 4.2 Materials and methods .................................................................................................55 4.2.1 Design of humanized MBP-DR2-ζ construct .....................................................55 4.2.2 Design of humanized tailless MBP-DR2