Eradication of established murine mesothelioma tumours by combined immunotherapy

Eradication of established murine mesothelioma tumours by combined immunotherapy Shruti Krishnan, B.Sc (Micro), Grad Dip (ForenSc), M.Sc (Micro), M (ForSci) This thesis is presented for the degree of Doctor of Philosophy The University of Western Australia School of Pathology and Laboratory Medicine Faculty of Medicine, Dentistry and Health Sciences 2015

ABSTRACT Despite the recent progress made in applying immunotherapy for the treatment of various cancers, such a therapy has not been developed for treating mesothelioma. Mesothelioma has a latency period of 30-40 years with a median survival being 9-10 months from diagnosis. Due to the lack of early diagnostic capabilities, patients in general, present with well-advanced disease that renders many therapeutic options ineffective. Standard treatments for the management of mesothelioma are limited due to late diagnoses, significant toxicities and the occurrences of relapses. Targeted therapies such as immunotherapy are selective and are aimed at stimulating the host s immune system to produce complete tumour destruction. The work described in this thesis, and the work of others in both mouse tumour models and clinical settings has shown that simultaneous targeting of multiple immune mechanisms results in better outcomes. A major deterrent to boosting anti-tumour immune activity was the immunosuppressive environment within the growing tumours themselves. Work carried out by our laboratory, and others, defined the role of T regs and TGF-β in suppressing immune responses to growing tumours. This led to the hypothesis that overcoming immune suppression alone was not sufficient, and perhaps an additional boost to effector T cell activity would improve the outcome. Chapter 3 investigated the improved efficacy of the timed combination of three immune modulators (anti-cd25mab, anti-tgf-βmab and anti-ctla-4mab) in the AE17 model (timed triple immunotherapy, TTI). Treatment with the TTI completely eradicated tumours in 50% of the C57BL/6J mice, with maintenance of low T reg numbers nine days after treatment initiation. The cured mice also showed complete resistance to re-challenge with the same tumour cells and had an increased percentage iii

of CD4 + CD44 + T cells, indicative of immune memory creation. Increased effector T cell function was also observed during the rechallenge (Kissick et al., 2012). Chapter 4 shows that the 50% cure rate of AE17 tumours could be raised to 100% by doubling the dosage of anti-tgf-βmab in the TTI protocol. It was also found that the standard TTI treatment produced complete clearance in the non-tgf-β AB1 murine mesothelioma tumours in all BALB/c mice. Additionally, combining all three agonist antibodies into a single intra-tumoural triple immunotherapy cocktail (TIC) for injection into the established tumours was found to be as effective as the TTI in the AB1 model. Chapter 5 investigated the role of B cells in TIC and TTI mediated tumour eradication. Mice cured by the treatment showed elevated levels of tumour specific IgG antibodies. These antibodies were higher against whole live tumour cells than cell lysates. Time-course studies of tumour clearance showed; (a) that IgG levels were not elevated during tumour clearance and (b) that B cell numbers were increased in the tumour draining lymph nodes and spleens during tumour clearance. Finally, employment of B-cell knock-out mice indicated a significant role for B cells in the successful eradication of the established tumours by the TIC. Chapter 6 detailed preliminary studies examining the efficacy of the triple immunotherapy on eradicating non-mesothelioma tumours. TIC treatment tested on the murine tumours (B16 melanoma, EO771 and 4T1 breast carcinoma tumours) showed that it was ineffective in eradicating tumours. However, a transient delay in tumour growth and improvement in survival times were observed in the EO771 and 4T1 breast carcinoma models. Further optimisations of the concentration of the triple immunotherapy components, and/or the addition of a fourth component are discussed. Additional work in this chapter showed that the 100% cure rate observed against AB1 iv

tumours at 9mm 2 dropped to 20% when the tumours were bigger (25mm 2 ). When two 9mm 2 tumours were grown on each mouse and only one tumour was treated, the results showed reduced efficacy in eradication of the treated tumour and delayed growth of the untreated tumour. Overall, the findings of this thesis indicate that: a) targeting multiple immune mechanisms simultaneously can completely eradicate established tumours, b) immunity to the eradicated tumour is achieved and c) that clinical translation to mesothelioma patients (phase I) is warranted. v

TABLE OF CONTENTS ABSTRACT... iii TABLE OF CONTENTS... vi ACKNOWLEDGEMENTS... xii STATEMENT OF CANDIDATE CONTRIBUTION... xvi ABBREVIATIONS... xvii LIST OF FIGURES... xxi LIST OF TABLES... xxiv PUBLICATIONS AND PROCEEDINGS... xxv Chapter 1 Literature Review... 1 1.1 Introduction... 3 1.2 Etiology of mesothelioma... 4 1.3 Importance of early diagnosis of mesothelioma... 6 1.4 Standard treatments for mesothelioma... 6 1.4.1 Surgery... 6 1.4.2 Chemotherapy... 7 1.4.3 Radiation therapy... 8 1.5 Murine models of mesothelioma... 9 1.6 Immunosurveillance and cancer control... 10 1.7 Immunotherapy... 12 1.7.1 Enhancing anti-tumour effector activity by immunotherapy... 13 1.7.1.1 Importance of immune checkpoint blockade... 15 1.7.2 Overcoming immune suppressive mechanisms employed by growing tumours... 17 1.7.2.1 Regulatory T cells... 17 1.7.2.2 TGF-β, a key immunosuppressive cytokine... 18 1.7.2.3 Strategies targeting tumour-intrinsic evasion mechanisms... 19 1.7.2.4 Role of T regs in mesothelioma... 22 1.7.2.4.1 Impact of T reg cell depletion on survival... 22 1.8 Combination therapies are necessary for overcoming immune suppressive mechanisms... 25 vi

1.8.1 Simultaneous targeting of T regs and TGF-β is more beneficial... 27 1.8.2 Boosting effector cell function... 27 1.9 Role of B cells in immunotherapy for mesothelioma... 28 1.10 Extending combination therapy to other tumours... 29 1.11 Aims of the thesis... 29 Chapter 2 Materials and methods... 31 2.1 Murine tumour cell lines... 33 2.2 Tissue culture... 33 2.2.1 Storage of tumour cell lines... 33 2.2.2 Resuscitation of frozen stocks... 33 2.2.3 Passage of tumour cell lines... 33 2.2.4 Harvesting cells for in vivo use... 34 2.2.5 Cell counting... 34 2.3 In vivo use of tumour cell lines... 35 2.3.1 Inoculation of tumour cells... 35 2.3.2 Measurement of subcutaneous tumours... 35 2.3.3 Euthanasia of tumour bearing mice... 36 2.3.4 Treatment regimens... 36 2.3.1 Preparation of anti-cd25mab treatments... 38 2.3.2 Preparation of anti-ctla-4mab treatments... 38 2.3.3 Preparation of anti-tgf-βmab treatments... 38 2.3.4 Preparation of triple immunotherapy cocktail (TIC)... 38 2.4 Treatment administration... 38 2.4.1 Timed triple immunotherapy (TTI) treatment administration at 9mm 2... 38 2.4.2 Intra-tumoural (i.t) administration of a single dose of triple immunotherapy cocktail (TIC)... 39 2.5 Sample preparation and analysis by flow cytometry... 42 2.5.1 Preparation of lymph nodes and tumours... 42 2.5.2 Preparation of spleen cells... 42 2.5.3 Live/dead viability dye (eflour 780) staining... 43 2.5.4 Cell surface staining for flow cytometry... 43 2.5.5 Intracellular Fox P3 and Ki-67 staining... 45 2.5.6 Preparation of compensation controls for flow cytometry... 45 2.5.7 Flow cytometric analysis of samples... 46 vii

2.5.7.1 Analysis of T cells... 46 2.5.7.2 Analysis of Regulatory T cells... 46 2.5.7.3 Analysis of DC cells... 46 2.5.7.4 Determination of relative expression of CD80... 46 2.5.7.5 Determination of cell numbers... 51 2.5.8 Isolation of immune cells by Fluorescence-activated cell sorting (FACS) 51 2.5.9 Adoptive transfer of immune cells into mice... 51 2.6 Tumour specific Immunoglobulin detection analysis... 52 2.6.1 Preparation of serum... 52 2.6.2 Preparation of cell lysate coated ELISA immunosorbent plates... 52 2.6.3 Detection of tumour cell lysate specific IgG in serum... 52 2.6.4 Detection of IgG in serum specific to live tumour cells... 53 2.6.5 Measurement of optical density of serum IgG specificity... 53 2.7 Statistical analysis... 54 2.8 Materials and Reagents... 54 2.8.1 Mice... 54 2.8.2 Reagents... 55 2.8.3 ELISA reagents... 56 2.9 Equipment... 56 2.10 Buffers, media and solutions... 58 CHAPTER 3 Development and characterisation of an effective triple immunotherapy in a TGF-β secreting murine mesothelioma model (AE17 model)... 61 3.1 Introduction... 63 3.2 Results... 67 3.2.1 Intra-tumoural administration of anti-cd25mab together with intraperitoneal anti-ctla-4mab and sequential anti-tgf-βmab, completely eradicates established AE17 murine mesothelioma tumours... 67 3.2.2 Administration of timed triple immunotherapy into established AE17 tumours inhibits re-accumulation of T regs in tumour draining lymph nodes... 69 3.2.2.1 TTI treatment results in greater maturation of DCs and subsequent activation of effector T cells in the TDLNs... 72 3.2.3 A specific anti-tumour memory response results from TTI treatment... 76 viii

3.2.3.1 Sustained depletion of T regs in the TDLNs of re-challenged AE17 cured mice... 78 3.2.3.2 Induction of memory T cells by the TTI treatment... 80 3.3 Discussion... 82 CHAPTER 4 Improved efficacy of the triple immunotherapy in the non-tgf-β secreting murine mesothelioma model (AB1 model)... 89 4.1 Introduction... 91 4.2 Results... 92 4.2.1 Timed administration of antibodies targeting CD25, TGF-β and CTLA-4 completely cleared established AB1 sub-cutaneous tumours in 100% of BALB/c mice.... 92 4.2.2 A combined, single administration of the triple treatment as a cocktail (TIC) is sufficient to induce complete clearance of AB1 tumours in close to 90% of animals... 94 4.2.3 Induction of systemic immune response in cured mice... 96 4.2.3.1 Susceptibility of cured mice to re-challenge with syngeneic alternate tumour type-4t1 breast carcinoma... 98 4.2.4 Incomplete neutralisation of TGF-β within the AE17 tumour microenvironment is integral to the suboptimal response generated in AE17 tumour bearing mice treated with TTI.... 100 4.2.4.1 Sub-optimal response observed in partial responders despite attempts at recovering them with additional top-up of immunotherapy treatment... 106 4.2.4.2 Increased anti-tgf-βmab dosage in the original TTI results in complete tumour eradication in AE17 tumour bearing mice... 110 4.2.4.2.1 Induction of systemic immune response in the mice cured using mtti..... 112 4.3 Discussion... 114 Chapter 5 Evidence of B cell involvement in triple immunotherapy... 121 5.1 Introduction... 123 5.2 Result... 125 5.2.1 Detection of tumour specific IgG antibodies in the serum of AE17 tumourbearing mice by ELISA... 125 5.2.2 Elevated levels of tumour specific IgG antibodies in the sera of AE17 tumour-bearing mice cured by TTI... 127 ix

5.2.2.1 Partial cross-reactivity of IgG antibodies in TTI cured mice to B16 melanoma cell lysates and spleen cell lysates... 129 5.2.3 Development of a living whole cell ELISA for detecting serum IgG levels against AE17 tumour cells... 132 5.2.3.1 Increased reactivity of serum IgG to whole AE17 live cells in TTI long-term cured mice... 134 5.2.4 Increased levels of AB1 tumour specific IgG in sera of mice 95 days post treatment with the TTI or TIC... 136 5.2.4.1 Partial cross-reactivity of IgG antibodies in TTI and TIC cured mice to syngeneic 4T1breast carcinoma cells... 139 5.2.4.2 IgG antibodies present in the TTI and TIC cured BALB/c mice are tumour specific and not auto-reactive... 141 5.2.5 Tumour specific antibodies are not elevated during tumour eradication.. 143 5.2.5.1 Elevated B cell numbers during TIC induced tumour eradication... 145 5.2.5.2 B cells are vital to the efficacy of the combined triple immunotherapy..... 147 5.2.5.2.1 B cells are required for successful treatment of AB1 tumours by TIC... 150 5.2.5.2.2 Overcoming the immunosuppressive tumour microenvironment is critical to tumour clearance... 152 5.3 Discussion... 154 Chapter 6 Preliminary investigations of the intra-tumoural triple immunotherapy in other tumour models and its effects on distal tumours... 163 6.1 Introduction... 165 6.2 Results... 167 6.2.1 Detection of high levels of T regs within the tumour microenvironment of non-mesothelioma tumours by flow cytometry... 167 6.2.2 TIC treatment is not effective in eradicating B16 melanoma tumours in C57BL/6J mice.... 170 6.2.2.1 Increased dosage of all three components of TIC has no effect on tumour retardation.... 172 6.2.3 Transient period of EO771 breast carcinoma tumour growth retardation in C57BL/6J mice treated with TIC.... 174 x

6.2.4 Increased survival time in 4T1 tumour-bearing BALB/c mice treated with either TIC or dtic.... 176 6.2.5 Efficacy of the triple immunotherapy is diminished with an increase in tumour burden.... 179 6.2.5.1 TIC treatment was effective in generating partial concomitant immunity a preliminary study on the effect of TIC on distal tumours... 181 6.3 Discussion... 184 6.4 Conclusion and future perspectives... 192 6.4.1 Future of the triple immunotherapy... 193 6.4.1.1 Addition of more agonist antibodies to the TIC treatment to improve tumour regression... 194 Chapter 7 Bibliography... 197 xi

ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisors Assoc. Prof. Manfred Beilharz, Asst. Prof. Demelza Ireland and Dr. Haydn Kissick. Manfred, I would like to thank you for accepting me into your lab and for teaching me everything I know now. I would especially like to thank you for your patience and constant diligence when I needed help with my research. I am very grateful that I could approach you to ask questions irrespective of whether they were related to lab work, designing experiments, candidature or scholarship. Your encouragement and advice in regard to research and a future scientific career have been invaluable. I have learnt a great deal on how to further my career as a research scientist, and it is all thanks to you. Thank you for having faith in me. Dem, I am very grateful for your patient guidance and for always being there for me. You have always been very helpful with everything regarding the experimental designs, the data interpretation, critical reading of manuscripts and of course, my thesis chapters. Your insights have been invaluable, and I will always be very grateful. Haydn, you were there in the beginning when I was barely equipped with the task at hand. Thank you for patiently helping me with so many things initially when I had just started my Ph.D. All the animal work, flow cytometry, tissue culturing and data interpretation, I learnt from you. Thank you. Parts of the experimental work described in this thesis were performed by other members of the Beilharz laboratory. Specifically, Dr. Haydn Kissick assisted me with the performance and interpretation of experiments described in chapter 3 at the beginning of my Ph.D. work in March 2011. The following experimental work was conducted with the 2012 Honours student Emily Bakker: The initial TTI (see section 4.2.1) and TIC (see section 4.2.2) treatment experiments in the AB1 murine xii

mesothelioma model; initial experiments on IgG detection following the cure of AB1 tumours (see section 5.2.4) and the single tumour burden experiment described in section 6.2.5. The following year, a new Honours student, Cassandra Lee assisted in the following experiments: the IgG detection experiments in sections 5.2.4.1 and 5.2.4.2 and the time-course experiments described in 5.2.5 and 5.2.5.1. I would also like to thank Dr. Sara Greay, Dr. Cornelia Hooper and Dr. Erika Bosio for their support and assistance throughout the difficult times faced during my candidature. I would not be the person or a scientist that I am today if it had not been for you. Not only have you guys been there for support during this whole experience but you have also shared your knowledge as young successful scientists and have given me the confidence that I could make a good researcher as well. Chandelle, you have been a great office buddy- thanks for cheering me up in the laboratory when it seemed impossible to smile. Thank you for sharing my enthusiasm for the goodies available at conferences and for distracting me from my nervousness for public speaking. Cassandra and Milly, thank you for being such exceptionally good students! Your input into the project has been invaluable. I will miss all of you, and I wish, wherever life takes us, we will always find a way to stay in touch (Thank god for social networking sites!!). I would like to thanks the Dust Disease Board of NSW for funding part of the research presented in my thesis. I am very grateful to the University of Western Australia for awarding me with a Scholarship for International Research Fees (SIRF), University Postgraduate Award for International Students (UPAIS) and the UWA Travel Award. Without the financial support provided, none of this would have been possible. I would also like to acknowledge the School of Pathology and Laboratory Medicine (UWA) for allowing me to undertake my research within the school. xiii

Furthermore, I would like to acknowledge some of our collaborators. I would like to thank Tracy Lee-Pullen, Irma Larma and Matthew Linden from CMCA for their excellent assistance with operating the flow cytometer, designing my antibody panels and data analyses on FlowJo. A special thanks to Kathy Heel for patiently teaching me the importance of maintaining gating strategies for all samples being analysed. I appreciate the assistance provided by Sandy Goodin, Kelly Hunt, Neill Wilson and the rest of the staff at M block, Animal Care Unit of the University of Western Australia for assisting with animal monitoring. Thank you! I feel incredibly blessed to have so many amazing people in my life. I would like to take this opportunity to thank my friends and family for their love and support; when I needed them the most. Dibakar, Paritosh and Prashant: you guys are the best! You had been there to lend an ear when I needed someone to talk to and your humour, it astonishes me but yet, I do not think I have ever laughed so much. Thanks, also for having me over at your houses and for taking time to teach me how to cook. I would also like to thank all the new friends I made since I first arrived in Australia and for all the good times we have had together. Thanks for being such great friends. Chloe and Jerome, you guys have been the best housemates, and I am extremely blessed to have you guys in my life. You welcomed me into your home and made me feel like I was part of your family. Thank you for all the fun moments we have had together: the yummy salads, pasta, interesting conversations, Zombie marathon, a shared passion for star wars, dancing, the dinner parties, the list is endless.. xiv

To my most treasured family: Amma, Appa, Vasanth, Santosh and Chitra. All of you have been integral to making me a strong and independent person. If it were not for your love and support, none of this would have been possible. To my dear husband Vasanth, you have made me a better person from the moment you came into my life. You are definitely, the person who has motivated and inspired me the most; and made me try harder to achieve my goals and to never give up hope. You are unquestionably my better half, and I love you for that. Thanks for being there for me. Amma and Appa, what can I say: you have inspired and encouraged me every step of the way! I am forever grateful to you for always believing in me and for the unconditional love you have been giving me. I honestly believe that I could not have finished my candidature without my parents and my husband. Love you guys. My thoughts also go to my beloved great-uncle Seetharaman, who believed in me long before I believed in myself. xv

STATEMENT OF CANDIDATE CONTRIBUTION I hereby declare that all of the work described within this thesis was performed by myself. Exceptions are the few experimental contributions from other members of the Beilharz laboratory detailed in the acknowledgements. Shruti Krishnan A/Prof Manfred Beilharz Candidate Co-ordinating supervisor April 2015 xvi

ABBREVIATIONS + Immunopositive - Immunonegative # Number o C Degrees (Celsius) µg Microgram µl Microlitre 2-ME AON APC APCs ARC ATP BCG BD BKO CD Cm 2 CTL CTLA-4 DAPI DC DD ddh 2 O DMSO DNA EDTA 2-Mercaptoethanol Antisense Oligonucleotides Allophycocyanin Antigen Presenting Cells Animal Resource Centre Adenosine Tri-phosphate Bacillus of Calmette and Guerin Becton Dickinson B-cell Knockout Cluster of differentiation Centimetres squared Cytotoxic T Lymphocyte Cytotoxic T lymphocyte Antigen-4 4,6-diamidino-2-phenylindole Dendritic Cell Denileukin Diftitox Distilled deionised water Dimethyl Sulphoxide Deoxyribonucleic acid Ethylenediaminetetraacetic acid xvii

ELISA ELISPOT EPP FACS FCS FDA FITC FoxP3 FSC GCV GITR GM-CSF Gy hr HRP HSV i.p i.t IDO IFN IGF IgG IL IMRT LCMV mab MDSC MFI Enzyme-linked immunosorbent assay Enzyme-linked Immunospot Extrapleural Pneumonectomy Fluorescence-activated cell sorting Foetal Calf Serum Food and Drug Administration Fluorescein isothiocyanate Forkhead box P3 Forward Scatter Ganciclovir Glucocorticoid Induced TNF Factor Granulocyte/Macrophage Colony-Stimulating Factor Gray (unit) Hour Horseradish Peroxidase Herpes Simplex Virus Intra-peritoneal Intra-tumoural Indoleamine -2,-3 -dioxygenase Interferon Insulin-like Growth Factor Immunoglobulin G Interleukin Intensity-Modulated Radiotherapy Lymphocytic Chorio-Meningitis Virus Monoclonal antibody Myeloid-Derived Suppressor Cells Mean Fluorescence Intensity xviii

MHC min ml MM NK cells NKT OD PBS PD-1 PDGF PD-L1 PE Major histocompatibility complex Minute Millilitre Murine Mesothelioma Null Killer cells Natural Killer T cells Optical Density Phosphate Buffered Saline Programmed Death-1 Platelet-Derived Growth Factor Programmed Death Ligand-1 R-Phycoerythrin PE-Cy 7 Phycoerythrin-Cyanine 7 PerCP-Cy5.5 pfu RCC RPMI RT s.c SCC SD SMART SMRP SSC Peridinin-chlorophyll proteins-cyanine5.5 Plaque-forming units Renal Cell Carcinoma Roswell Park Memorial Institute Radiation Therapy Subcutaneous Squamous Cell Carcinoma Standard Deviation Surgery for mesothelioma after radiation therapy Soluble Mesothelin-Related Protein Side Scatter SV40 Simian Virus 40 TAA TCR TDLN Tumour Associated Antigen T Cell Receptor Tumour Draining Lymph Node xix

T eff TGF-β TIC tk TNF TPA T regs TTI UT UWA VEGF WT Effector T cells Transforming Growth Factor-β Triple Immunotherapy Cocktail Thymidine kinase Tumour Necrosis Factor Tissue Polypeptide Antigen Regulatory T cells Timed Triple Immunotherapy Untreated University of Western Australia Vascular Endothelial Growth Factor Wild-type xx

LIST OF FIGURES Figure 1. 1 Major mechanisms by which potent anti-tumour immune responses can be generated... 21 Figure 2. 1 Timed Triple Immunotherapy (TTI) experiment protocol.... 41 Figure 2. 2 Gating strategy for analysing CD4 + and CD8 + T cells.... 48 Figure 2. 3 Gating strategy for T reg estimation in TDLNs and tumours.... 49 Figure 2. 4 Gating strategy for analysing dendritic cells.... 50 Figure 3. 1. Improved survival with complete tumour eradication in 46% of mice treated with timed triple immunotherapy (TTI) a significant improvement over double immunotherapy (DI)... 68 Figure 3. 2. Sustained depletion of T reg cells in TDLNs by TTI treatment even 3 days post treatment completion.... 71 Figure 3. 3 Enhanced CD80 expression by dendritic cells and increased effector T cell levels in mice treated with TTI.... 75 Figure 3. 4 Sustained depletion of T regs in the TDLNs of re-challenged cured mice.... 79 Figure 3. 5 Induction of memory T cells in mice cured of AE17 murine mesothelioma by TTI treatment.... 81 Figure 4.1 Complete tumour eradication in 100% of AB1 tumour bearing BALB/c mice treated with TTI... 93 Figure 4. 2 Tumour growth kinetics and survival of AB1 tumour bearing BALB/c mice following treatment with the timed triple immunotherapy (TTI) or the triple immunotherapy cocktail (TIC)... 95 xxi

Figure 4. 3 Tumour growth of partial responders to the TTI treatment is not significantly different from the DI treated mice.... 103 Figure 4. 4 Failed recovery of partial responders despite attempts with secondary round of immunotherapy.... 109 Figure 4. 5 Complete tumour eradication in 100% of AE17 tumour bearing C57BL/6J mice treated with mtti.... 111 Figure 5. 1 Elevated AE17 cell lysate specific IgG antibodies detected in the sera of AE17 end-point tumour bearing untreated mice.... 126 Figure 5. 2 Elevated levels of tumour specific IgG observed in sera of TTI cured mice compared to untreated controls.... 128 Figure 5. 3 Partial cross-reactivity of IgG antibodies in the serum of TTI long-term cured mice to syngeneic B16 melanoma tumour cell lysates.... 131 Figure 5. 4 Detection of increased reactivity of serum IgG in TTI cured mice to whole live AE17 cells.... 133 Figure 5. 5 Increased reactivity of serum IgG to whole AE17 live cells in TTI cured mice compared to AE17 cell lysates.... 135 Figure 5. 6 Elevated levels of tumour specific IgG antibodies detected in combined immunotherapy (TTI and TIC) treated mice.... 138 Figure 5. 7 Partial cross-reactivity high against live syngeneic 4T1 tumour cells in TTI and TIC cured mice.... 140 Figure 5. 8 Low levels of auto-reactive antibodies in TTI and TIC cured mice.... 142 Figure 5. 9 No significant change in serum IgG levels in TIC treated mice up to 20 days post treatment.... 144 Figure 5. 10 Changes in B cell percentage post TIC treatment.... 146 Figure 5. 11 Successful tumour eradication with TIC requires B cells.... 149 xxii

Figure 5. 12 Anti-tumour efficacy of TIC treatment is augmented by B cells.... 151 Figure 5. 13 Manipulation of tumour microenvironment is important for successful clearance of established tumours.... 153 Figure 6. 1 Detection of T regs within non-mesothelioma tumours grown in both C57BL/6J and BALB/c mice.... 169 Figure 6. 2 Growth of melanoma tumours in C57BL/6J mice is unhindered by TIC treatment.... 171 Figure 6. 3 Double the dosage of all three components in the TIC has no effect on melanoma growth in C57BL/6J mice.... 173 Figure 6. 4 Treatment with TIC or dtic did not significantly improve survival of mice bearing EO771 tumours.... 175 Figure 6. 5 Improved survival with TIC or dtic in BALB/c mice bearing 4T1 tumours, compared to mice left untreated.... 177 Figure 6. 6 Central zone of tumour clearance observed in mice post treatment with dtic.... 178 Figure 6. 7 Tumour eradication efficacy of TTI treatment lowered with increase in tumour burden in AB1 tumour bearing BALB/c mice.... 180 Figure 6. 8 TIC treatment of single primary tumour in mice co-challenged simultaneously with AB1 tumours was effective in generating partial concomitant immunity to secondary tumours.... 183 xxiii

LIST OF TABLES Table 1. 1 Monotherapies used for the depletion of T regs in murine mesothelioma models... 24 Table 2. 1 Mono-clonal antibodies... 37 Table 2. 2 Antibodies used for flow cytometry... 44 Table 3. 1 TTI cured mice are resistant to re-challenge with original inoculum (AE17) and partially resistant to syngeneic B16 melanoma re-challenge.... 77 Table 4. 1 BALB/c mice cured of established AB1 tumours with TTI or TIC are resistant to re-challenge.... 97 Table 4. 2 Immunological memory generated in BALB/c mice cured of established AB1 tumours with TIC are tumour specific.... 99 Table 4. 3 No significant improvement in immune cell numbers in partial responders.... 105 Table 4. 4 Mice cured of established AE17 tumours with mtti are resistant to rechallenge.... 113 xxiv

PUBLICATIONS AND PROCEEDINGS Publication arising from Ph.D. candidature 1. Kissick, H. T., Ireland, D. J., Krishnan, S., Madondo, M. & Beilharz, M. W. 2012. Tumour eradication and induction of memory against murine mesothelioma by combined immunotherapy. Immunology and Cell Biology, 90, 822-826. (This paper covers the data described in chapter 3 and represents the early stages of the development of the successful timed triple immunotherapy (TTI) for the treatment of subcutaneous murine mesothelioma) 2. Krishnan, S., Bakker, E., Lee, C., Kissick, H. T., Ireland, D. J. & Beilharz, M. W. 2014. Successful combined intra-tumoural immunotherapy of established murine mesotheliomas requires B cell involvement. Journal of Interferon & Cytokine Research, (In press). (This paper covers the data described in chapters 4 and 5 of this thesis which concerns the development and characterisation of the triple therapy cocktail (TIC) and also includes the role of B cells in TIC mediated tumour eradication) Conference proceedings 1. Krishnan, S., Ireland, D. J., Kissick, H. T. & Beilharz, M. W. (2011) Improved success rate with a triple immunotherapy for the treatment of mesothelioma. The Australian Society for Medical Research, Western Australian scientific symposium, Australia. Oral 2. Krishnan, S., Ireland, D. J., Kissick, H. T. & Beilharz, M. W. (2011) Characterisation of a successful triple immunotherapy for the treatment of mesothelioma. Combined Biological Science Meeting, Australia. Poster xxv

3. Krishnan, S., Ireland, D. J., Kissick, H. T. & Beilharz, M. W. (2012) Tumour eradication and induction of memory against murine mesothelioma by combined immunotherapy. Australian Society for Medical Research, Western Australian scientific symposium, Australia. Oral 4. Krishnan, S., Bakker, E., Ireland, D. J., Kissick, H. T. & Beilharz, M. W. (2012) Tumour eradication and induction of memory against murine mesothelioma by combined immunotherapy. Combined Biological Science Meeting, Australia. Oral 5. Krishnan, S., Bakker, E., Lee, C., Kissick, H. T., Ireland, D. J. & Beilharz, M. W. (2014). Successful combined intra-tumoural immunotherapy of established murine mesotheliomas requires B cell involvement. International Cytokine and Interferon Society, Cytokines Down Under in 2014: From Bench to Beyond. Australia. Poster xxvi