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A Dissertation Entitled Synthesis and Study of MUC1-Based Anti-Tumor A Dissertation entitled Synthesis and Study of MUC1-Based Anti-tumor Vaccines by Partha Karmakar Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry _________________________________________ Dr. Steven J. Sucheck, Committee Chair _________________________________________ Dr. Katherine A. Wall, Committee Member _________________________________________ Dr. Jianglong Zhu, Committee Member _________________________________________ Dr. Donald Ronning, Committee Member _________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo December 2015 Copyright 2015, Partha Karmakar This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Synthesis and Study of MUC1-Based Anti-tumor Vaccines by Partha Karmakar Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in <Enter name of your Discipline or Program as it appears in your degree audit> The University of Toledo Chemistry It is important to obtain CD8+ T cell activation in the course of developing a potent anti-tumor vaccine. CD8+ T cell response to extracellular antigen is generated by processing of the extracellular antigen by antigen presenting cells (APCs). This step is followed by cross presentation of the corresponding epitope to the CD8+ T cell receptors via MHC class I molecules. Cross presentation can be facilitated by efficient antigen uptake via immune-complex-mediated maturation of the APCs. It is well known that vaccination with tumor associated cancer antigen (TACA)-containing MUC1 peptide with the variable number tandem repeat (VNTR) sequence can break self-tolerance in humanized MUC1 transgenic mice. We hypothesized that a MUC1 sequence TSAPDT(GalNAc)RPAPGSTAPPAHGV that contains a CD8+ T cell epitope delivered on a targeted liposome surface could enhance antigen uptake. Anti-rhamnose antibodies are some of the most abundant naturally occurring antibodies found in humans. Our liposomes contain L-Rhamnose (Rha) epitopes displayed on their surface that can facilitate the natural antibody-dependent immune-complex formation and antigen uptake mechanism for better antigen presentation. To test this hypothesis, synthesis of a 20 iii amino acid MUC1-Tn sequence, TSAPDT(GalNAc)RPAPGSTAPPAHGV was performed by solid phase peptide synthesis (SPPS) and a Toll-like receptor ligand (TLRL) was covalently attached to it by Cu(I)-assisted click chemistry. The 20 amino acid MUC1 sequence contained B cell, CD4+ T cell, and CD8+ T cell epitopes. The TLRL-MUC1-Tn vaccine was formulated onto liposomes that consisted of TEG- cholesterol or Rha-TEG-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with a total lipid concentration of 30 mM. The vaccine was tested on groups of female C57BL/6 mice. Some of the groups of mice were immunized with Rha-Ficoll prior to vaccination, in order to generate anti-Rha antibodies in those mice. On vaccination, a 10 fold higher antibody production was observed against TLRL-MUC1-Tn by the anti-Rha expressing mice group that received the Rha-vaccine compared to the other groups of mice. The CD8+ T cell responses for the anti-Rha antibody expressing mice group that received the Rha-vaccine were higher compared to the others when they were evaluated by measuring CD8+ T cell proliferation, IFNγ production and cytotoxicity against a cancer cell line. These results indicate that antigen uptake was facilitated by the anti-Rha dependent immune complex formation that resulted efficient antigen uptake and the CD8+ T cell epitope was processed and presented by the APCs. iv Acknowledgements My whole graduate research work is the aftermath of great support, inspiration and generosity of many people. First, I would like to pay gratitude to my research advisor Dr. Steven J. Sucheck for giving me an opportunity to work in his esteemed group and in very interesting projects. His motivation and mentoring inspired me through every challenge that came in my way throughout my graduate study. I deeply thank my co- advisor Dr. Katherine A. Wall for training and educating me in the fields of immunology that enabled me to perform all the immunological studies on the animal model. I highly appreciate the helpful suggestions and inspiration that I received from my committee members Dr. Jianglong Zhu and Dr. Donald Ronning. I also acknowledge my past and present group members for the constructive discussions, and for keeping an extremely friendly environment in the work place. I am highly thankful to Dr. Yong Wah Kim for the training and help that I received from him in NMR and ESI-MS experiments. I am extremely grateful to my family, especially to my mother and father, my younger brother and sister in law and my wife. I would like to mention my mother once more because of her inspiration during my graduate studies. I deeply acknowledge my friends for their constant encouragement and support throughout my study which converted the tedious graduate research into a joyful event of life. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of contents ................................................................................................................ vi List of tables……………………………………………………………………………... ix List of figures .……………………………………………………………………..…...... x List of schemes ………………………………………………………………………… xii List of abbreviations ........................................................................................................ xiii 1 Carbohydrate-based anti-cancer vaccine development: background and significance....................................................................................1 1.1 Introduction ......................................................................................1 1.2 Immune system overview ………………………………………...2 1.3 Tumor-associated carbohydrate antigens ….……………………...8 1.4 Improved TACA-based vaccines ………………………………..13 1.5 MUC1-based cancer vaccine .........................................................14 1.6 Multicomponent vaccines ..............................................................16 1.7 Immunocomplex mediated vaccine internalization; role of anti- rhamnose antibodies …..…………...…………………………………………….17 1.8 References …………………………………………………….…18 vi 2 Synthesis of a liposomal MUC1 glycopeptide-based immunotherapeutic and evaluation of the effect of L-rhamnose on cellular immune response …..….24 2.1 Project summary …………...……………………………………25 2.2 Introduction………………………………………………….…..26 2.3 Results and discussions ………………………………………….31 2.4 Immunological results…………………………………………....38 2.5 Significance………………………………………………………46 2.6 Experimental procedure .....………………………………………47 2.7 References………………………………………………………..57 Supplementary data: Appendix A…………...…………………….…..…99 3 Mixed phase synthesis of gycopeptides: An extended use of N-peptidyl- 2,4-dinitrobenzene sulfonamide-thio acid ligation strategy in ..............................64 3.1 Project summary …...……………………...…………………….64 3.2 Introduction ....................................................................................65 3.3 Results and discussion ...................................................................67 3.4 Significance....................................................................................73 3.5 Experimental procedure …………………………………………73 3.6 References …………………………………………………...…..83 Supplementary data: Appendix B………………………………...….…106 4 Synthesis of L-rhamnosyl ceramide and evaluation of it’s binding to anti- rhamnose antibodies: Use of rhamnose ceramide as tumor marker……………..………87 4.1 Project summery ………………………………………………..87 4.2 Introduction …………………………………………………….89 vii 4.3 Results and discussion ………………………………….……….90 4.4 Significance ……………………………………………………..94 4.5 Experimental procedure ………………………………………...95 4.6 References ……………………………………………………...98 viii List of Tables 1.1 Major classes of lymphocytes in Chapter 1…………………………...…………..5 2.1 Conditions for Cu(I)-assisted click reaction for synthesis of Pam3Cys-MUC1-Tn glycopeptide 4 in Chapter 2 ...............................................................................................37 2.2 Vaccination plan for the groups of mice in Chapter 2 ...........................................40 3.1 N-Peptidyl-2,4-dinitrobenzenesulfonamides in Chapter 3..……………….……..69 ix List of Figures 1-1 Schematic representation of mechanism of antigen processing and presentation in MHC II pathway ……………………………….…………………………………7 1-2 Schematic representation of mechanism of antigen processing and presentation in MHC I pathway …………………………………………………………………..8 1-3 Glycoprotein associated Tn, TF and STn cancer antigens…………………………………………………………………………....9 1-4 Examples of ganglioside glycolipid associated TACAs……………….……..….10 1-5 : Glycolipid-associated TACAs from Globo-serie ……...…………….………...11 1-6 TACAs of lacto-series or the Lewis antigens ……………………………….......12 2-1 Anti-rhamnose antibody-mediated enhanced presentation of liposomal vaccine and generation of cellular immuneresponse .…………………………………………………………………………..……………….24 2-2 Mechanism of anti-rha-mediated enhancement of cellular and humoral immuneresponse
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