Induction of anti tumor response by dendritic cell based vaccination

INDUCTION OF ANTI-TUMOR RESPONSE BY DENDRITIC CELL-BASED VACCINATION FONG CHOY LEN B.Sc (Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDGEMENTS I am especially grateful to my supervisor, Professor Hui Kam Man, for his guidance throughout the project. I would like to express my gratitude to Dr Hwang Le-Ann and Dr Wu Xia Feng for their technical assistance in the project. I would also thank Dr Mok Chen-Lang for writing the manuscript. The supportive roles played by my colleagues in the laboratories are highly appreciated. I especially thank my husband, Mr Lim Yee Ghee, for being supportive, kind, considerate and tolerant for my study. Last but not least, I thank my family for their moral support. i PUBLICATIONS Fong CL, Mok CL, Hui KM Intramuscular immunization with plasmid co-expressing tumour antigen and Flt3L results in potent tumour regression. Gene Ther 2005, in press Fong CL, Hui KM. Generation of potent and specific immune responses via in vivo stimulation of dendritic cells by pNGVL3-hFLex plasmid DNA and immunogenic peptides. Gene Ther. 2002, 9(17):1127-38. Khoo HE, Fong CL, Yuen R, Chen D. Stimulation of haemolytic activity of sea anemone cytolysins by 8-anilino-1naphthalenesulphonate. Biochem Biophys Res Commun. 1997, 232(2):422-6. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS i PUBLICATIONS ii TABLE OF CONTENTS iii SUMMARY ix LIST OF TABLES xi LIST OF FIGURES xii ABBREVIATIONS xv CHAPTER 1: INTRODUCTION 1.1 Dendritic cells (DC) in tumor immunotherapy 1.2 1.2.1 1.2.1.1 1.2.1.2 Parameters of DC-based vaccines Subsets of DC Subsets of murine DC Subsets of human DC 3 1.2.2 Maturation stages of DC 1.2.3 Antigen capturing, processing and presentation of DC 11 1.2.4 Antigen-loading onto DC 13 1.2.5 Migratory and homing properties of DC 15 1.2.6 DC and the development of cancer vaccines 16 1.3 1.3.1 1.3.2 1.3.3 1.3.4 1.3.5 FLT-3 ligand (FL) and cancer immunotherapy FL and DC Structure and expression of FL FL in normal cells and tissues Effects of FL on T, B, and NK cell development Structure and expression of Flt-3 18 18 19 21 22 22 1.4 1.4.1 Tumor associated antigens (TAA) Overview 24 24 1.5 MUC-1 25 iii 1.5.1 1.5.2 1.5.3 1.5.4 1.5.5 1.5.6 1.5.7 Discovery of MUC-1 Molecular structure of MUC-1 O-glycosylation in mammalian cells Mouse homolog of human MUC-1 Cellular functions of MUC-1 MUC-1 and cancer MUC-1 and immunotherapy 25 27 28 29 30 31 33 1.6 1.6.1 1.6.2 Overview of Project Peptide-based immunization DNA-based immunization 37 38 41 CHAPTER 2: MATERIALS AND METHODS 43 2.1 Animals and cell lines 44 2.2 Peptides 44 2.3 2.3.1 2.3.2 2.3.3 Preparation of bone marrow-derived DC (BM-DC) Culturing of BM-DC Purification of BM-DC Purity analysis of BM-DC by flow cytometry 44 44 45 46 2.4 Wright-Giemsa staining 46 2.5 Preparation of muc-1-pulsed BM-DC 46 2.6 2.6.1 Immunization protocol for peptide-based strategy Immunization of mice with muc-1-pulsed BM-DC and/or muc-1 peptide Immunization of mice with muc-1 pulsed BM-DC and hFlex-DC Immunization of mice by in vivo stimulation with pNGVL3-hFLex plasmid DNA and muc-1 peptide 47 47 2.6.2 2.6.3 48 49 2.7 2.7.1 2.7.2 In vivo delivery of muc-1 peptide Intrasplenic injection of muc-1 peptide Intravenous delivery of muc-1 peptide 49 49 50 2.8 Preparation of spleen cells 50 2.9 Proliferation assay 50 2.10 2.10.1 2.10.2 Purification of allogeneic T cells (H-2d) Preparation of nylon wool T cell isolation from non-adherance to nylon wool 51 51 51 iv 2.11 Allostimulation assay 52 2.12 2.12.1 2.12.2 IL-2-conditioned media Preparation of IL-2 conditioned media (Con-A-sup) Determination of optimal IL-2-conditioned media (Con-A-sup) concentration for CTLL-2 cell line 52 52 53 2.13 2.13.1 2.13.2 2.13.3 CTL assay Preparation of effector cells Preparation of chromium-51 (Cr-51)-labeled target cells Cr-51 release assay 53 53 54 54 2.14 Determination of CTLp frequency 54 2.15 Detection of anti-muc-1 antibody by ELISA 55 2.16 Measurement of IL-2 using CTLL-2 cell line 56 2.17 Determination of IFN-γ and IL-10 concentration by ELISA 56 2.18 2.18.1 2.18.2 Gene delivery system Hydrodynamic-based tail-vein injection of plasmid DNA Intramuscular injection (i.m.) of plasmid DNA 57 57 57 2.19 58 2.19.1.8 2.19.1.9 2.19.1.10 2.19.1.11 2.19.2 Molecular Cloning of pNGVL3-Flex-muc-1 and pNGVL3-muc-1 DNA vaccines Construction of chimeric pNGVL3-FLex-muc-1 DNA vaccine Restriction of pNGVL3-hFLex and pNGVL3-hFLex-Trail plasmid DNA Purification of restricted pNGVL3 plasmid DNA using QIAquick Gel extraction kit Removal of 5’ phosphate group from the plasmid vector QIAquick PCR purification kit Chemical synthesis and annealing of synthetic muc-1 oligonucleotides Ligation Preparation of transformation competent DH5α E.coli (Rubidium chloride) Transformation Analysis of MUC-1+ bacterial clones PCR reaction QIAprep Spin Miniprep kit Construction of pNGVL3-muc-1 DNA vaccine 63 64 64 65 65 2.20 In vitro transfection 66 2.21 Isolation of plasmid DNA-EndoFree Plasmid Maxi kit 66 2.19.1 2.19.1.1 2.19.1.2 2.19.1.3 2.19.1.4 2.19.1.5 2.19.1.6 2.19.1.7 58 58 59 60 60 61 62 62 v 2.22 UV spectrophotometric quantitation of DNA 68 2.23 Electrophoresis of DNA and PCR Product 68 2.24 Immunofluorescence staining for confocol microscopy 68 2.25 Determination of protein mass by Western Blot 69 2.26 Collection of mouse sera and determination of FL protein in sera 70 2.27 Preparation of muscle tissues 70 2.28 Immunization of mice with pNGVL3-FLex-muc-1 and pNGVL3-muc-1 DNA vaccines via hydrodynamic-based tail-vein injection and intramuscular injection 70 2.29 Tumor protection assay 71 2.30 Immunohistochemistry (IHC) of muscle sections 72 2.31 Hematoxylin staining 72 CHAPTER 3: RESULTS 74 3.1 Generation of potent and specific immune responses 75 via in vivo stimulation of dendritic cells with pNGVL3-hFLex plasmid DNA and immunogenic peptides 3.1.1 Generation of bone marrow-derived DC (BM-DC) 75 3.1.2 Muc-1-pulsed DC could act as APC to antigen specific CTL 79 3.1.3 Induction of CD11c+ DC via in vivo immunization with pNGVL3-hFLex plasmid DNA 86 3.1.4 Cell surface phenotypes of bone marrow-derived DC 89 (BM-DC) and splenic DC induced by the pNGVL3-hFLex plasmid DNA (hFlex-DC) were identical 3.1.5 hFlex-DC are immune competent 92 3.1.6 hFlex-DC are capable of processing the muc-1 antigenic peptide in vivo to generate potent anti-muc-1 CTL responses 94 vi 3.1.7 Tumor-specific immune response induced by in vivo stimulation with pNGVL3-hFLex plasmid DNA and muc-1 peptide 99 3.2 Expansion and recruitment of DC in situ potentiate the immunonogenicity of DNA vaccine encoding the antigenic epitope fused to human Flt-3L 101 3.2.1 Generation and in vitro expression of pNGVL3-hFLex-muc-1 DNA construct 101 3.2.2 Hydrodynamic-based intravenous immunization of pNGVL3-hFLex-muc-1 induces expansion of CD11c+ DC in vivo 105 3.2.3 DC generated by intravenous immunization are immune competent but fail to induce a tumor response 113 3.2.4 Intramuscular immunization of pNGVL3-hFLex-muc-1 induces antigen specific CTL response and results in anti-tumor protection 116 3.2.5 Recruitment of DC to intramuscular immunization sites by 123 pNGVL3-hFLex-muc-1 DNA vaccine CHAPTER 4: DISCUSSION 126 4.1 Generation of potent and specific cellular immune responses via in vivo stimulation of dendritic cells by pNGVL3-hFLex plasmid DNA and immunogenic peptides 127 4.2 Expansion and recruitment of DC in situ potentiate the immunogenicity of DNA vaccine encoding the antigenic epitope fused human Flt-3L 137 4.2.1 Routes of DNA immunization 146 4.2.2 CpG motif 148 4.2.3 Immunization regimen 150 4.3 DC-based peptide vaccine and DNA vaccine 150 4.4 Prophylactic and therapeutic tumor immunity 152 4.5 Conclusion 152 vii 4.6 Future studies REFERENCES 153 154 viii SUMMARY Dendritic cells (DC) are the most potent professional antigen-presenting cells (APC) with exquisite capacity to interact with T cells to initiate strong primary cellular immune response. 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J Biol Chem. 274(9):5550-6. 183 [...]... in the form of vaccination A crucial step for mounting an effective anti- tumor response is the capturing, processing and presentation of TAA to cognate T cells by professional antigen-presenting cells (APC) Dendritic cells (DC) are professional antigen presenting cells (APC) with exquisite capability to activate naive T cells and hence induction of primary immune response DC, loaded with antigen ex... non-MHC-restricted recognition of NKT cells [57] NKT cells orchestrate the production of large amounts of cytokines from several cell types and have the capacity to act as adjuvants for T cell mediated immunity The versatile of DC in antigen presentation definitely requires further investigation to generate desired immune response 12 NK CD8+ CTL DC Destruction of tumor cells CD4+ CTL NKT Figure 1.1 Cell- mediated immunity... MAGE-3 specific CTL response was generated where DC acquire MAGE-3 antigen from apoptotic tumor cells [74] The capture of apoptotic bodies is likely through the cross-presentation mechanism An attempt to employ tumor cell- DC cell hybrid as vaccine has sucessfully induced regression of renal cell carcinoma in patients [75] However, the generation and maintainance of tumor cell- DC cell hybrid is laborious... several ways: 1) by in vivo increasing of DC number by stimulating DC replication or survival signal by Flt-3 ligand (FL) [82] 2) by recruitment of DC in situ by Flt-3L or chemokines/chemokine receptors 3) by enhancing vaccine capturing and processing by DC 4) by promoting DC maturation and 5) by targeting antigen to specific DC subset 17 In our study, we hypothesize that the expansion of DC in situ will... exerted by hFL in a SCID mouse model in 1997 Massive infiltration of DC was found within the tumor site as revealed by immunohistochemistry staining, suggesting a role of DC in generating the anti- tumor response Soon following the above 18 report, several studies further demonstrated the tumor suppression capability of hFL in an immuno-competent murine cancer model [86] The anti- tumor effect of hFL... Production of cytokines 85 41 xii 3.1.7 Kinetics of induction of DC via in vivo immunization with pNGVL3-hFLex plasmid DNA 88 3.1.8 Comparison of the cell surface phenotypes of purified BM-DC and hFlex-DC 90 3.1.9 hFlex-DC are functionally competent 93 3.1.10 Generation of muc-1 specific immune responses via in vivo stimulation of DC with pNGVL3-hFLex plasmid DNA and muc-1 peptide 96 3.1.11 Induction of muc-1... properties are also altered by remodeling of DC chemokine receptors and ligands profile [50] 1.2.3 Antigen capturing, processing and presentation of DC One of the unique features of DC is the ability to present exogenous Ag to class I pathway [51-53] This process is termed cross-presentation or indirect presentation Indirect presentation of class I-restricted antigens by professional APC is an important... CTL responses but fails to confer tumor protection 3.2.8 Intramuscular administration of pNGVL3-hFLex-muc-1 119 DNA vaccine generates muc-1 specific cellular responses 3.2.9 Intramuscular immunization with pNGVL3-l-hFLex-muc-1 121 DNA vaccine induces a potent anti- tumor response xiii 3.2.10 Advance systemic DC expansion prior to immunization with DNA vaccines does not elicit a more potent anti- tumor response. .. eradicate tumor The most exciting aspect of stimulating an endogenous immune response is its potential in initiating long-term immunological memory This may provide a permanent cure for the cancer patients whereby a long-lived anti- tumor immune response can be elicited For the past decades, many efforts have been attempted to understand the induction and regulation of tumor immunity The basic premise of immunotherapy... expressed by pNGVL3hFLex-muc-1 DNA vaccine In conclusion, we devised two strategies of inducing DC to prime muc-1 specific anti- tumor response in vivo It is suggested that the availability of DC at the antigen site (in situ) is a critical factor to enhance the immunogenicity of both peptide and DNA vaccines x LIST OF TABLES Page 1.1 Members identified in mucin family 26 3.1 Surface phenotypes of BM cells . processing and presentation of TAA to cognate T cells by professional antigen-presenting cells (APC). Dendritic cells (DC) are professional antigen presenting cells (APC) with exquisite capability. INDUCTION OF ANTI- TUMOR RESPONSE BY DENDRITIC CELL- BASED VACCINATION FONG CHOY LEN B.Sc (Hons). activate naive T cells and hence induction of primary immune response. DC, loaded with antigen ex vivo, have been shown to induce potent anti- tumor response [1-2]. The generation of clinically