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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
iii
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 Tregs 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 Treg
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
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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
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tumours at 9mm2 dropped to 20% when the tumours were bigger (25mm2). When two
9mm2 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.
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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 Tregs in mesothelioma ............................................................. 22
1.7.2.4.1 Impact of Treg cell depletion on survival ....................................... 22
1.8 Combination therapies are necessary for overcoming immune suppressive
mechanisms ................................................................................................................. 25
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1.8.1 Simultaneous targeting of Tregs 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 9mm2 ...... 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
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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 intra-
peritoneal 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 Tregs 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
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3.2.3.1 Sustained depletion of Tregs 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 tumour-
bearing 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
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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 Tregs 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
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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
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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
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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.
xiv
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…..
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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.
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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
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ABBREVIATIONS
+ Immunopositive
- Immunonegative
# Number
oC Degrees (Celsius)
µg Microgram
µl Microlitre
2-ME 2-Mercaptoethanol
AON Antisense Oligonucleotides
APC Allophycocyanin
APCs Antigen Presenting Cells
ARC Animal Resource Centre
ATP Adenosine Tri-phosphate
BCG Bacillus of Calmette and Guerin
BD Becton Dickinson
BKO B-cell Knockout
CD Cluster of differentiation
Cm2 Centimetres squared
CTL Cytotoxic T Lymphocyte
CTLA-4 Cytotoxic T lymphocyte Antigen-4
DAPI 4’,6-diamidino-2-phenylindole
DC Dendritic Cell
DD Denileukin Diftitox
ddH2O Distilled deionised water
DMSO Dimethyl Sulphoxide
DNA Deoxyribonucleic acid
EDTA Ethylenediaminetetraacetic acid
xviii
ELISA Enzyme-linked immunosorbent assay
ELISPOT Enzyme-linked Immunospot
EPP Extrapleural Pneumonectomy
FACS Fluorescence-activated cell sorting
FCS Foetal Calf Serum
FDA Food and Drug Administration
FITC Fluorescein isothiocyanate
FoxP3 Forkhead box P3
FSC Forward Scatter
GCV Ganciclovir
GITR Glucocorticoid Induced TNF Factor
GM-CSF Granulocyte/Macrophage Colony-Stimulating Factor
Gy Gray (unit)
hr Hour
HRP Horseradish Peroxidase
HSV Herpes Simplex Virus
i.p Intra-peritoneal
i.t Intra-tumoural
IDO Indoleamine -2,-3 -dioxygenase
IFN Interferon
IGF Insulin-like Growth Factor
IgG Immunoglobulin G
IL Interleukin
IMRT Intensity-Modulated Radiotherapy
LCMV Lymphocytic Chorio-Meningitis Virus
mAb Monoclonal antibody
MDSC Myeloid-Derived Suppressor Cells
MFI Mean Fluorescence Intensity
xix
MHC Major histocompatibility complex
min Minute
ml Millilitre
MM Murine Mesothelioma
NK cells Null Killer cells
NKT Natural Killer T cells
OD Optical Density
PBS Phosphate Buffered Saline
PD-1 Programmed Death-1
PDGF Platelet-Derived Growth Factor
PD-L1 Programmed Death Ligand-1
PE R-Phycoerythrin
PE-Cy 7 Phycoerythrin-Cyanine 7
PerCP-Cy5.5 Peridinin-chlorophyll proteins-Cyanine5.5
pfu Plaque-forming units
RCC Renal Cell Carcinoma
RPMI Roswell Park Memorial Institute
RT Radiation Therapy
s.c Subcutaneous
SCC Squamous Cell Carcinoma
SD Standard Deviation
SMART Surgery for mesothelioma after radiation therapy
SMRP Soluble Mesothelin-Related Protein
SSC Side Scatter
SV40 Simian Virus 40
TAA Tumour Associated Antigen
TCR T Cell Receptor
TDLN Tumour Draining Lymph Node
xx
Teff Effector T cells
TGF-β Transforming Growth Factor-β
TIC Triple Immunotherapy Cocktail
tk Thymidine kinase
TNF Tumour Necrosis Factor
TPA Tissue Polypeptide Antigen
Tregs Regulatory T cells
TTI Timed Triple Immunotherapy
UT Untreated
UWA University of Western Australia
VEGF Vascular Endothelial Growth Factor
WT Wild-type
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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 Treg 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 Treg 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 Tregs 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
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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
xxiii
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 Tregs 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
xxiv
LIST OF TABLES
Table 1. 1 Monotherapies used for the depletion of Tregs 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 re-
challenge. ...................................................................................................................... 113
xxv
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
xxvi
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
xxvii
xxviii
1
Chapter 1 Literature Review
2
3
This literature review was composed subsequent to the completion of the research work
in September 2014.
1.1 Introduction
Malignant mesothelioma is a life-threatening tumour type arising from mesothelial
cells lining the pleura, peritoneum, pericardium and tunica vaginalis (Abe et al., 2002;
Hirano et al., 2002; Berry et al., 2004). The risk of malignant mesothelioma increases in
proportion to the cumulative exposure (3rd or 4th power of time since first exposure) to
asbestos (Reid et al., 2014). The disease has a latency period of 30-40 years with poor
prognosis (with a median survival of 9-12 months) from diagnosis (Robinson and Lake,
2005). The incidence of this disease has been steadily increasing since the late 90s and
is expected to continue rising for another 20 years before declining (Ismail-Khan et al.,
2006). There are currently no effective treatments for mesothelioma and diagnosis tends
to occur quite late due to the long latency period. Despite the ban on asbestos being
introduced in many developed countries; asbestos continues to be a real threat to the
general public, as a) it persists in the community due to previous widespread use and b)
developing countries continue to use asbestos (Bianchi and Bianchi, 2007; Olsen et al.,
2011; Sim, 2013). More than a quarter million deaths due to malignant mesothelioma
are estimated to occur over the next 40 years (Robinson and Lake, 2005). Development
of new treatment approaches is, therefore, necessary to improve outcomes in these
patients. Immunotherapy is one such new treatment and has been shown to be clinically
beneficial for the treatment of other malignancies (Hodi et al., 2010; Kantoff et al.,
2010; Wolchok et al., 2013). The aim of immunotherapy is to boost the patient's
immune response towards the tumour and hence lead to eradication. This treatment
4
approach can involve either release of immune suppression or direct enhancement of
immune effector activity (Pardoll, 2012).
1.2 Etiology of mesothelioma
The link between asbestos fibres and malignant mesothelioma was first evidenced
by Wagner et al in 1960; with the high incidence of malignant mesothelioma being
reported amongst asbestos miners in South Africa (Wagner et al., 1960). Asbestos was
used extensively in construction and ship building industries due to the fire retardant
and heat insulating properties of asbestos. This widespread usage was most predominant
in the United States and Europe especially from 1940s to 1979 (Ismail-Khan et al.,
2006). With the increase in awareness of carcinogenesis due to asbestos in the 1980s,
restrictions on usage of asbestos were enforced. The increased awareness eventually led
to asbestos being banned in many developed countries including Australia. Despite the
ban, incidence of mesothelioma continues to rise (Kamp, 2009), especially in
developing countries, where the restrictions are not strictly adhered to.
There are two classes of asbestos fibres, the serpentine and the amphibole.
Serpentine includes most commonly used asbestos mineral, chrysotile or white asbestos,
and the amphibole includes several fibres including crocidolite (blue asbestos) and
amosite (brown asbestos) among others (Gibbs, 1990). Of the two different fibre types,
the larger amphibole fibres were thought to be more carcinogenic (Mossman et al.,
1996; Roggli and Sharma, 2014). However, extensive review of literature carried out by
Powers et al (2002) showed that the data was so contradictory; that it was not possible
to determine whether it was the crocidolite or the chrysotile class of asbestos fibres that
were the causative agents of malignant mesothelioma (Powers and Carbone, 2002).
Apart from asbestos fibres, other factors such as erionite (mineral fibres), Simian Virus
40 (SV40), exposure to radiation, and certain genetic predispositions have also been
5
linked to the development of malignant mesothelioma (Powers and Carbone, 2002;
Carbone et al., 2007; Yang et al., 2008).
The exact mechanisms of asbestos carcinogenicity are not yet fully elucidated. With
the long latency period (34-40 years) of the disease, several proposed mechanisms are
thought to contribute to the development of malignant mesothelioma. One of them, is
thought to be linked to the constant inflammation caused by the persistence of asbestos
fibres in the pleura (Robinson et al., 2005). There is also strong evidence that reactive
oxygen species and reactive nitrogen species catalysed by the high iron content of these
asbestos fibres causes DNA damage (Kamp and Mossman, 2002). Furthermore,
mutations in the p53 gene (tumour suppressor gene) produced by crocidolite fibres have
been studied in mice (Lin et al., 2000). The link between inactivation or loss of the p53
(and other somatic mutations) and tumour development and progression are being
investigated (Altomare et al., 2005). In addition, the role of cytokines and growth
factors that promote mesothelioma proliferation are also being elucidated. These include
factors such as platelet-derived growth factor (PDGF) (Aggarwal et al., 2009),
transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF) (Liu and
Klominek, 2004).
Since the 1980s, Australia has the world’s highest incidence rate of malignant
mesothelioma per capita (Leigh and Driscoll, 2003); followed by Britain (Bianchi and
Bianchi, 2007; Robinson, 2012). In Australia, the incidence rates were 31.8 per million
in 1997 with the highest rate of 47.7 per million per year being reported in Western
Australia in the same year (Leigh and Driscoll, 2003). In 2008, the annual incidence rate
of 29 cases per million was published in Australia and these numbers are predicted to
peak between 2014 and 2021 (Robinson, 2012).
6
1.3 Importance of early diagnosis of mesothelioma
Early diagnosis, in clinical practice, is important for improving curative
treatment of tumours. However, early diagnosis of mesothelioma can be difficult. Some
of the problems faced include differentiating mesothelioma from other diseases. For
instance, epithelioid mesothelioma needs to be distinguished from benign hyperplasia
(Churg et al., 2000). In the case of pleural mesothelioma, it must be differentiated from
lung adenocarcinoma and for peritoneal mesothelioma, from serous carcinoma (Koss et
al., 1998; Baker et al., 2005). To date, biomarkers such as soluble mesothelin-related
protein (SMRP), tissue polypeptide antigen (TPA), hyaluronan and osteopontin, among
others, are being reported as possible markers for diagnosing and monitoring
progression of mesothelioma (Hedman et al., 2002; Robinson et al., 2003; Grigoriu et
al., 2007). However, their sensitivity and specificity as diagnostic markers are said to be
inadequate (Sato et al., 2014). Given the difficulties of early diagnostics and the
symptoms of the disease not appearing until 20 to 40 years after asbestos exposure;
prognosis for mesothelioma remains unsatisfactory. Current approaches to improving
early diagnosis of mesothelioma are being investigated (Fukuoka et al., 2013; Sato et
al., 2014). However, they are still in their infancy, and no standard diagnostic tool for
detecting mesothelioma exists at the moment.
1.4 Standard treatments for mesothelioma
Standard therapies involved in the management of mesothelioma include surgery,
radiation therapy and chemotherapy.
1.4.1 Surgery
Though procedures like video thoracoscopy and laparoscopy have helped in
establishing a diagnosis for mesothelioma (Waller, 2004); partial pleurectomy,
pleurodesis and pleuro-peritoneal shunting procedures were more palliative in nature
7
and aided only in relieving pleural effusions (Scherpereel et al., 2010). Only
cytoreduction by either pleurectomy or extra pleural pneumonectomy (EPP) procedures
were recommended as potential curative surgeries for mesothelioma (Stewart et al.,
2004). Such curative surgeries have been shown to reduce the bulk of the tumour
(cytoreduction) and delay disease progression (Stewart et al., 2004; Sugarbaker et al.,
2004). Pleurectomy involves the removal of the whole pleura stretching from the apex
of the lung to the diaphragm. EPP is considered a radical surgical procedure and
involves the removal of a lung along with the majority of the hemidiaphragm, parietal
and visceral pleura and a portion of phrenic nerve along with the pericardium. EPP has
achieved the greatest degree of cytoreduction, with increase in median survival by two
years with good control over local remission of the disease (Rusch, 1999). Even so,
only a few patients are eligible (Karnofsky performance status was required to be
greater than 70%) for the surgeries. The skills of the surgeon for performing such
demanding procedures and the preoperative conditions of the patients are also limiting
factors that hinder this therapeutic approach (Sterman and Albelda, 2005).
1.4.2 Chemotherapy
The current standard first-line systemic chemotherapy combination used for the
treatment of unresectable malignant mesothelioma is the combination of Pemetrexed
(500 mg/m2) and Cisplatin (75 mg/m2) (Jänne, 2003; Vogelzang et al., 2003). In a phase
III study, the median survival time for patients treated with the combination was 12.1
months compared to 9.3 months for patients who received Cisplatin alone. A similarly
weak increase in median survival was also observed for the combination of Cisplatin
(80 mg/m2) and Raltitrexed (3 mg/m2) (van Meerbeeck et al., 2005). Another
combination included Gemcitabine (617 mg/m2) and Carboplatin (80 mg/m2); though
the median survival for this combination was found to be only 66 weeks (Favaretto et
al., 2003). Other chemotherapy combinations are being tested, though none have, as yet,
8
achieved higher response/survival rates. So far, no standard second-line chemotherapy
regimens exist for the treatment of mesothelioma following the first line.
1.4.3 Radiation therapy
Most studies involving radiation therapy (RT) in the management of malignant
mesothelioma have shown no significant improvement on overall survival in patients
(Aisner, 1995). The main drawback of RT is the diffused nature of the tumour covering
majority of the pleural surface. RT treatment of such large surfaces are known to cause
radiation toxicities in the neighbouring organs (Maasilta, 1991). Nevertheless, RT
treatment studies by Boutin et al (1995) showed that administration of 7 Gy in three
daily sessions given 10-15 days post thoracoscopy, significantly decreased local
recurrence of tumour cells in all 20 treated patients (Boutin et al., 1995). It has also
been shown that it is possible to deliver doses of over 45 Gy with intensity-modulated
radiotherapy (IMRT). This is because the radiation fields are more focussed- by the
placement of markers during surgery, ergo radiation toxicities can be avoided (Stahel et
al., 2010).
Several groups have shown the importance of combining EPP with
administration of radiation or chemotherapy post-operatively to improve outcomes.
Post-operative chemotherapy with doxorubicin and cyclophosphamide, and up to 54 Gy
of RT to the hemithorax (trimodal therapy)- increased survival beyond 5 years in 15%
of the patients treated (Sugarbaker et al., 1999). IMRT doses of 50-60 Gy post-EPP
surgery were shown to annihilate local recurrence of mesothelioma tumours without
severe toxicities (Ahamad et al., 2003a; Ahamad et al., 2003b). Currently, the trimodal
treatment option is undergoing further evaluation in a phase II clinical study. The study
involved four cycles of Cisplatin and Pemetrexed in conjunction with pleurectomy/EPP
followed by IMRT targeted at the pleura (ClinicalTrials.gov Identifier NCT00715611).
9
Another promising approach involves short high-dose IMRT treatment of the
hemithoracic region followed by EPP surgery and is called SMART (surgery for
mesothelioma after radiation therapy). Results from the initial phase I/II trials showed
that cumulative survival reached 84% without any perioperative morbidity and
mortality (Cho et al., 2014).
Despite interest in the trimodal treatment options and the various combination of
chemotherapy being tested, treatment for malignant mesothelioma remains debatable
(Baud et al., 2014). Currently, novel therapies are being explored for the treatment of
mesothelioma.
1.5 Murine models of mesothelioma
To assist in the development of novel therapies, pre-clinical animal models are
essential. Murine models for mesothelioma have been developed, and these models
have led to a greater understanding of the disease development and in the identification
of potential treatments for mesothelioma. Indeed, murine mesothelioma tumour model
is one of the few models that is homologous to human cancer, with the murine tumours
mimicking the human disease in the manner of the pathology, molecular biology and
clinical behaviour.
Murine mesothelioma models such as AE17, AB1 and AC29 were developed by the
intra-peritoneal inoculation of crocidolite asbestos fibres in mice (Davis et al., 1992;
Jackaman et al., 2003). Development of ascites was observed 29 weeks later and
cytology, histology and ultra-structural analyses verified the presence of mesothelioma
cells within the ascites. A continuous cell line was developed from the serial passaging
of tumour cells in vitro to achieve the immortal cell lines (Davis et al., 1992; Jackaman
et al., 2003). The AE17 cell line induces tumours in C57BL/6J mice, AB1 in BALB/c
mice and the AC29 in CBA mice. These murine models have been used in pre-clinical
10
trials for determining the efficacy and toxicity of anti-cancer therapies (Needham et al.,
2006; Anraku et al., 2010; Jackaman et al., 2012). The AE17 and AB1 cell lines are
similar, except for the fact that AE17 tumours are associated with higher levels of the
immune suppressive cytokine TGF-β (Davis et al., 1992; Fitzpatrick et al., 1994).
1.6 Immunosurveillance and cancer control
One of the central dogmas proposed by Paul Ehrlich more than a century ago, was
the concept of immunological surveillance against neoplasm. The immune system was
theorised to react to altered self-antigens expressed on cancerous or pre-cancerous cells
and destroyed the neoplasm before they caused a problem (Ehrlich, 1908; Burnet, 1969;
Möller and Möller, 1976). With the improvement in immunological models, the ability
to probe the nature of tumour immunology improved. Over the subsequent years,
vaccination studies that were conducted, have further supported the theory of
immunosurveillance (Van Pel and Boon, 1982; Barth et al., 1990).
The concept of tumour immunosurveillance was first shown by the work on in-bred
Rag2-deficient mice that lacked mature lymphocytes (T, B and NK-T cells). These mice
developed spontaneous cancers by 14-16 months of age (Shankaran et al., 2001). Work
involving the inactivation of perforin, granzyme and interferon signalling (integral part
of the immunosurveillance system) also demonstrated the susceptibility of mice to
tumourigenesis (Dunn et al., 2004a; Bui and Schreiber, 2007). These studies evidenced
the concept of immunosurveillance and the role of the immune system to engage in
tumour killing. Further evidence for immunosurveillance came from the improved
prognosis of patients whose tumours exhibited increased infiltration by lymphocytes
(Dunn et al., 2004a).
Immunosurveillance relies on cells and molecules (cytokines and chemokines)
that constitute the innate and adaptive immunity arms of the immune system. The chief
11
effector cells of the innate immune system include macrophages, natural killer T (NKT)
cells, dendritic cells (DCs) and neutrophils (de Visser et al., 2006; Ostrand-Rosenberg,
2008). In the adaptive arm, T cells are critical to detection and destruction of tumour
cells (Romagnani et al., 1997; Girardi et al., 2003) and B cells are also implicated to
some extent (DiLillo et al., 2010b; Nelson, 2010). CD4+ T cells including the subsets
Th1 and Th2 lineages are activated by exposure to the cytokines IL-12 and IL-4 and
subsequently, the Th1 cells kill tumour cells directly by secreting high levels of IFN-γ,
cytolytic granules and tumour necrosis factor (TNF)α (Becker, 2006). Girardi et al
(2003) showed that γδ T cells contribute strongly to host protection against papillomas
(Girardi et al., 2003). Destruction of tumour cells by CD8+ T cells is either direct,
through perforin/granzyme-B-mediated pathways, or through further secretion of
cytokines such as IFN-γ and IL-2 (Qin et al., 2003; Sengupta et al., 2010).
Conversely, several mechanisms exist to keep the immune system in check (known
as peripheral self-tolerance), as an excessive response to self-antigens can cause toxicity
and autoimmune reactions. Indeed, a subset of T cells known as regulatory T cells
(Tregs) are known to play a crucial role in maintaining self-tolerance for self-antigens
and preventing cytotoxicity by negatively regulating the immune system (Sakaguchi et
al., 2008). Tregs are known to occur in malignant tumours or ascites (Mougiakakos et al.,
2010). This down-regulation of the immune response by Tregs is one potential
mechanism that enables tumour cells in evading immune surveillance.
The immunoediting concept best explains the interaction between the immune
system and the developing cancer. Immunoediting is regarded as a process composed of
three phases: elimination, equilibrium and escape. The first phase is the elimination,
where immunosurveillance is highly active, and pre-cancerous cells are kept in check by
the immune system. If the elimination phase fails to stop cancer cells, the cells that were
12
not destroyed by the immune cells enter into the equilibrium phase where the cells are
maintained in a state of dormancy. Escape is when the cancer cells evade the immune
system and grow uncontrollably (Dunn et al., 2004b; Schreiber et al., 2011). The
importance of this evasion of immunosurveillance by growing tumours, is being seen as
one of the new “hallmarks of cancer” (Dunn et al., 2002; Hanahan and Weinberg,
2011).
The mechanisms used by the tumour cells to evade destruction include: reduced
immunogenicity of the tumour cells (Lengauer et al., 1998; Algarra et al., 2000),
recruitment of immunosuppressive cells and release of immunosuppressive cytokines
(Ghiringhelli et al., 2005; Drake et al., 2006; Rabinovich et al., 2007; Schreiber et al.,
2011). Immunotherapy treatments are currently being designed to overcome these
obstacles and enhance the anti-tumour immune responses.
1.7 Immunotherapy
The notion of harnessing the body’s immune system to help eradicate cancer cells is
a logical concept but not a new one. The first systematic study of immunotherapy for
the treatment of malignant tumours was started in the late 1800s by William B Coley, a
bone sarcoma surgeon. He injected cancer patients with Streptococcus pyogenes (that
causes erysipelas) and observed that the tumours disappeared. The bacterial culture
administration was based on the hypothesis that the subsequent infection would trigger
an immune response, not only against the bacteria, but also against the cancer. Over the
next forty years, more than 1000 patients with inoperable bone and soft-tissue sarcomas
were known to be treated with bacteria or bacterial toxins known as “Coley’s toxins”;
with almost half having reportedly undergone near tumour regression (Nauts and
McLaren, 1990; McCarthy, 2006). However, with the lack of reproducibility of the
13
results along with the development of other modes of cancer treatments such as
chemotherapy and radiation therapy, interest in immunotherapy waned.
With a better understanding of the tumour immunology, the interest in using the
immune system to eradicate cancer cells is once again gaining momentum. The renewed
interest in immunotherapy is especially relevant, as a) the cancer cells are specifically
targeted, b) the duration of anti-tumour response is longer, and c) there is minimal
collateral damage to healthy cells. Current immunotherapy treatment approaches can
accomplish these tasks either by a) enhancing effector activity against the growing
tumours or b) by overcoming immune suppression.
1.7.1 Enhancing anti-tumour effector activity by immunotherapy
One of the initial approaches to immunotherapy primarily revolved around
boosting the anti-cancer immune responses against tumour cells. One of the methods
applied to generating a potent anti-tumour immune response, involved boosting tumour
antigen presentation by dendritic cells (DCs): the essential initiators of adaptive
immunity. This DC priming (illustrated in Figure 1.1a) would in turn enhance cytotoxic
T lymphocyte activity against tumour cells (Banchereau and Steinman, 1998; Nefedova
et al., 2005). One such DC priming approach that has been approved by the FDA for
human treatment is the DC vaccine Sipuleucel-T. Three intra-venous treatments
administered over 2 weeks of the DC vaccine were shown to prolong the overall
survival (25.8 months with minimal toxicities) in patients with metastatic castration-
resistant prostate cancer (ClinicalTrials.gov number, NCT00065442) (Kantoff et al.,
2010).
In the case of malignant mesothelioma, immunotherapy is relevant as a) it has
been found to be sensitive to destruction by immunotherapies and b) patients with
mesothelioma are known to mount an immune response against the growing tumour
14
(Robinson et al., 2000; Mukherjee and Robinson, 2002). Notwithstanding, these
immune responses are known to be weak and unable to prevent the tumour growth
(Robinson et al., 2000). The first immunotherapeutic approach used for treatment of
mesothelioma involved treating 30 patients with BCG vaccine (Webster et al., 1982).
Though it was a crude treatment, improved survival was observed in the treated patients
and this response led the way for other immunotherapeutic treatments to be tested. The
first use of Bacillus of Calmette and Guerin (BCG) as a cancer vaccine was reported by
Holmgren in 1935 (Holmgren, 1935). Successful intracavitary administration of BCG
vaccine into superficial bladder tumours was conducted by Morales and colleagues in
1976. This paved the way for the BCG vaccine being accepted as an
immunoprophylactic standard treatment for bladder cancer post local surgery (Morales
et al., 1976; Alexandroff et al., 1999).
Intra-pleural treatments with immune-stimulatory cytokines such as IL-2 and
IFN-γ have also been trialled in mesothelioma patients. The treatments improved
outcomes only in patients with early disease and not in those with late-stage tumours
(Robinson et al., 1991; Boutin et al., 1994; Castagneto et al., 2001). Powell et al (2006)
showed that treatment with recombinant GM-CSF (Granulocyte/Macrophage Colony-
Stimulating Factor) combined with autologous whole-cell tumour lysates administered
as a vaccine, induced tumour-specific cellular immunity in 32% of the treated patients
(Powell et al., 2006). GM-CSF is a cytokine that activates APCs and enhances antigen
presentation by these cells to T cells in the tumour draining lymph nodes. Though some
patients were found to have a stable condition throughout the trial, an overall increase in
survival time (11.5 months) compared to historical controls (9.6 months) was observed
(Alberts et al., 1988). Such an example of using tumour vaccine for priming DC is
illustrated in Figure 1.1a.
15
1.7.1.1 Importance of immune checkpoint blockade
As described above, the immune system is regulated by inhibitory mechanisms
that enable immune evasion by growing tumours. The manipulation of these self-
tolerance pathways could potentially shift the immune response away from tumour
“escape” and move more towards “elimination” of tumour cells. Blocking of such
inhibitory signals or enhancing the activation signals (as illustrated in Figure 1.1b)
could potentially drive the immune system towards the anti-tumour effector
functionality.
Blocking of the primary negative regulatory signals (also known as immune
checkpoints) as potential immunotherapy targets is gaining interest. The first receptor to
be identified as a negative immune regulator was the cytotoxic T lymphocyte antigen-4
(CTLA-4) (Brunet et al., 1987). CTLA-4 is expressed on activated T cells and also on
Tregs and is a negative receptor for CD80/86 that are expressed on antigen presenting
cells (Greenwald et al., 2005; Quezada et al., 2006; Hodi, 2007). CTLA-4 is similar to
CD28 and is capable of binding to CD80 and CD86 (belonging to the B7 family)
present on antigen presenting cells (APCs). Binding of CD28 (which is also expressed
on T cells) to the B7 co-stimulatory molecules efficiently activates T cell responses.
However, following antigen activation of T cells, CTLA-4 expression is upregulated
(Egen et al., 2002) and this is essential for maintaining peripheral tolerance. Their role
in maintaining homeostasis was demonstrated in CTLA-4-/- deficient mice that
developed lethal hypo-lymphoproliferative disorders and multiple organ tissue
destruction (Tivol et al., 1995; Waterhouse et al., 1995).
In the tumour microenvironment, the CTLA-4 receptor binds with higher
affinity to CD80/86 on antigen presenting cells, than the CD28 receptor on T cells and
thereby, essentially shuts down T cell proliferation (Walunas et al., 1994; Schneider et
16
al., 2006). The up-regulation of CTLA-4 in effect, promotes tolerance and aids in the
immune evasion by the growing tumour cells (Vignali et al., 2008).
Promising clinical response data have been documented with blocking the
CTLA-4 inhibitory signal on T cells using Ipilimumab (Yervoy, Bristol-Myers Squibb,
New York, NY, USA). Treatment with Ipilimumab was shown to enhance patient
survival, with 15-20% of patients with advanced melanoma surviving beyond two years
(Hodi, 2010; Robert et al., 2011). In another study, twenty-nine patients with
chemotherapy-resistant advanced malignant mesothelioma were enrolled into a phase II
clinical study of anti-CTLA-4mAb treatment. Though disease control in 31% (9/29) of
patients was observed, the results seemed disappointing, as only 7% (2/29) achieved
durable partial response to the treatment (Calabrò et al., 2013).
The other well recognised regulator of suppression is the programmed death-1 (PD-1)
pathway (Duraiswamy et al., 2013; Wolchok et al., 2013). While CTLA-4 is expressed
on activated T-cells, including Tregs, PD-1 is expressed more broadly on activated T
cells and haematopoietic cells (Keir et al., 2008). The role of PD-1 blockade on effector
T cell activation is well known and has been found to increase effector T cell responses
(Topalian et al., 2012). Blockade of PD-1 by agonist antibody (BMS-936558) has
already shown varying levels of objective responses in patients with non-small-cell lung
cancer, melanoma and renal-cell carcinomas (Topalian et al., 2012). The results of these
trials strongly support further evaluation of immune checkpoint blockade as future
treatment options. On September 2014, the anti-PD-1mAb Pembrolizumab (Keytryda,
Merck, New Jersey, USA ) was approved by the FDA for the treatment of metastatic
melanoma (Najjar and Kirkwood, 2014).
17
1.7.2 Overcoming immune suppressive mechanisms employed by growing tumours
A major deterrent to enhancing anti-tumour activity is the immune evasion
mechanisms employed by the growing tumours. This leads to a highly
immunosuppressive environment within the growing tumours (Ganss et al., 2004;
Rabinovich et al., 2007; Whiteside, 2008; Shiao et al., 2011). Recruitment of
immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived
suppressor cells (MDSCs) by the growing tumour cells are known to aid in the
suppression of cytotoxic T lymphocytes (Ostrand-Rosenberg and Sinha, 2009;
Mougiakakos et al., 2010). With the secretion of immunosuppressive cytokines or
chemokines such as transforming growth factor-β (TGF-β), vascular endothelial growth
factor (VEGF), interleukin-6 (IL-6) and IL-10; the combined effects are known to
paralyse the activity of infiltrating cytotoxic T lymphocytes (CTL) and natural killer T
(NKT) cells (Shankaran et al., 2001; Lin and Karin, 2007; Rabinovich et al., 2007;
Yang et al., 2010). The down-regulation of the immune suppressive Treg cells are being
investigated as potential cancer treatment strategies.
1.7.2.1 Regulatory T cells
Regulatory T cells (Tregs) are naturally occurring CD4+CD25+FoxP3+ T cells. As
mentioned earlier, these cells play a crucial role in maintaining self-tolerance by
negatively regulating the immune system (Gershon and Kondo, 1970; Sakaguchi, 2004).
Immune tolerance is crucial for maintaining the balance between lack of response and
an excessive response to self-antigens. For a healthy host, immune tolerance is essential
to control the latter where inappropriate responses to self-antigens by auto-reactive T
cells can lead to autoimmune diseases. Indeed, Treg cell depletions have been observed
to induce spontaneous autoimmune diseases such as type I diabetes, gastritis and
thyroiditis in otherwise healthy rodents; their reconstitution preventing these diseases
(Sakaguchi et al., 1985; Sakaguchi, 2000; Singh et al., 2001).
18
However, in response to tumour growth, Tregs have been found to abrogate
tumour specific T cell immunity. Selective expansion of Tregs in the periphery (tumour
draining lymph nodes) and tumour microenvironment have been demonstrated
previously in different human cancers (Woo et al., 2001; Liyanage et al., 2002; Wolf et
al., 2003; Curiel et al., 2004; Ghiringhelli et al., 2005). These Tregs that occur within the
tumours were found to inhibit the cytotoxic capacity of CD8+ T-cells as well as NKT
cells in a TGF-β dependent manner (Smyth et al., 2006). Suppression of CD4+ T cells
by Tregs has also been found to be through granzyme B dependent cell-to-cell contact
(Wang et al., 2004; Gondek et al., 2005). Glucocorticoid-induced TNF factor (GITR)
and CTLA-4 have also been reported to play critical roles in regulating T cell function
through Tregs (Wing et al., 2008; Friedline et al., 2009).
1.7.2.2 TGF-β, a key immunosuppressive cytokine
TGF-β is a ubiquitous signalling polypeptide and influences multiple processes
such as embryogenesis, carcinogenesis and immune responses (Elliott and Blobe, 2005;
Li and Flavell, 2008). With regards to malignancies, TGF-β is thought to have dual
roles: as a tumour promoter (Kehrl et al., 1986), as well as a suppressor (Markowitz and
Roberts, 1996). But it now seems that the role of TGF-β as a tumour suppressor occurs
earlier on in tumour progression while its role as promoter of tumour growth occurs at
later stages of cancer (Gold, 1998; de Caestecker et al., 2000; Wakefield and Roberts,
2002; Biswas et al., 2011).
This cytokine is produced by some tumours and high levels have also been
shown to contribute to the metastasis of the disease (Friedman et al., 1995; Penafuerte
and Galipeau, 2008; Wang et al., 2013). TGF-β was also reported to be responsible for
the accumulation of the immunosuppressive Tregs within the tumour microenvironment.
Additionally, TGF-β can promote the conversion of naïve CD4+ T cells into Tregs,
19
thereby contributing further to immunosuppression (Chen et al., 2003; Ghiringhelli et
al., 2005). TGF-β is also known to promote immune tolerance by a) partially mediating
the induction of MDSCs by the growing tumour cells and b) inhibiting the maturation
and production of IFN-γ by APCs (Borkowski et al., 1996; Geissmann et al., 1999;
Xiang et al., 2009).
The implication of TGF-β in progression of cancer has been widely reported
(Elliott and Blobe, 2005). There is growing interest in developing cancer therapeutics
that target the immunosuppressive cytokine TGF-β specifically.
1.7.2.3 Strategies targeting tumour-intrinsic evasion mechanisms
Strategies targeting attenuation of the Tregs and neutralization of the
immunosuppressive cytokines mentioned above are being investigated (as illustrated in
Figure 1.1c). Denileukin Diftitox (DD), a recombinant fusion protein consisting of
peptide sequences of diphtheria toxin and human IL-2; is a FDA approved drug which
targets and kills cells expressing IL-2 receptors (also known as CD25). Phase III clinical
trials of DD for treating patients with late stage cutaneous T-cell lymphoma resulted in
20% of patients with partial response and 10% (7/71) with complete response to the
treatment. Also, reduction in circulating Tregs with the corresponding increase in
cytotoxic capacity of CD8+ T cells was observed in the treated patients (Olsen et al.,
2001). Depletion of Tregs with Denileukin Diftitox (DD) (ONTAK, Ligand
pharmaceuticals Inc., San Diego, CA, USA) has also shown some success in enhancing
immune function in human clinical trials against renal cell carcinoma, T-cell non-
Hodgkin lymphoma, melanoma and non-small cell lung cancer (Dannull et al., 2005;
Dang et al., 2007; Mahnke et al., 2007; Gerena-Lewis et al., 2009).
Therapeutic inhibition of the immune suppressive cytokine TGF-β activity is
also being investigated and includes a) using antisense oligonucleotides to prevent the
20
translation of TGF-β mRNA, b) inhibition of activity of TGF-β or c) using soluble
receptors or antibodies to neutralize TGF-β (Yingling et al., 2004; Hawinkels and Dijke,
2011). Targeting TGF-β secretion by glioma cells using the antisense oligonucleotide
AP12009 was tested in Phase I/II clinical trials for treating patients with anaplastic
astrocytoma or glioblastoma multiforme. 2/24 (8%) underwent complete tumour
regression, and stable disease was observed in 7/24 (29%) of the treated patients (Hau et
al., 2007).
Immunotherapies involving inhibition of TGF-β have shown great promise in
murine models. IN-1330, a TGF-β type-1 receptor kinase inhibitor was found to inhibit
lung metastasis of breast cancer in mice (Park et al., 2014). Neutralisation of TGF-β
using 1D11 (anti-TGF-βmAb) has been shown to reduce tumour burden in mouse
models of mesothelioma and breast cancer (Saunier and Akhurst, 2006; Kissick et al.,
2010; Biswas et al., 2011; Hawinkels and Dijke, 2011; Kissick et al., 2012).
Similar to the anti-TGF-βmAb, monoclonal antibodies (mAbs) targeting other
immune suppressive cytokines and growth factors, are being actively investigated. A
neutralizing mAb to VEGF, Bevacizumab, was shown to induce tumour regression by
almost 30% in one out of five patients with rectal adenocarcinoma and a significant
prolongation in disease progression was observed in patients with metastatic renal-cell
carcinoma (Yang et al., 2003; Willett et al., 2004). IL-10 is another potent negative
regulator of the immune response. The neutralising anti-IL-10 antibody is currently
being investigated and has shown promise in murine models (Newton et al., 2014).
21
Figure 1. 1 Major mechanisms by which potent anti-tumour immune responses can be generated a) Priming of dendritic cells to enhance antigen presentation can be achieved by multiple methods. These include i) injecting irradiated whole tumour cells that continue secreting cytokines, ii) re-infusing dendritic cells loaded with tumour cells and matured ex vivo, back into the patients and iii) injecting dead tumour cells (killed by chemotherapy). b) Enhancing cytotoxicity of Effector T cells by i) preventing transduction of negative regulators such as PD-1 and CTLA-4 using antibodies to block these receptors and ii) injecting agonist co-stimulatory antibodies to receptors 4-1BB and GITR to boost effector activity. c) Overcoming immune suppression by i) depleting immunosuppressive cells such as Tregs and MDSCs and ii) neutralizing immunosuppressive cytokines to promote tumour destruction. GITR: Glucocorticoid induced TNF factor, MHC: Major histocompatibility complex and TCR: T cell receptor. Reproduced from Vanneman and Dranoff, 2012.
22
In order to develop an effective immunotherapy treatment, it is important to
understand the mechanisms employed by the growing tumour to evade destruction by
the immune system. The specific research conducted by our laboratory into the search
for immunotherapeutic targets elucidated the critical roles of Tregs and TGF-β in
immune suppression in murine mesotheliomas.
1.7.2.4 Role of Tregs in mesothelioma
As described above, immune suppression by Tregs is one potential mechanism
that assists tumour cells in evading immune-mediated destruction. Our laboratory was
the first to identify the presence of these Tregs within the AE17 murine mesotheliomas in
C57BL/6J mice (Needham et al., 2006). This model was chosen as it has been shown to
have high levels of Tregs within the tumour microenvironment and represents an
aggressive tumour akin to their human counterpart (Davis et al., 1992; Hegmans et al.,
2006; Needham et al., 2006). These Tregs that were detected within the tumour
microenvironment of the AE17 tumours were found to increase as a percentage of total
CD4+ T cells. as the tumours grew (Needham et al., 2006).
1.7.2.4.1 Impact of Treg cell depletion on survival
With the elucidation of the immune suppression mediated by Tregs in the AE17
murine mesothelioma model, Treg depletion studies were carried out in the same AE17,
as well as other murine mesothelioma tumour models. These studies involved the use of
monotherapies such as anti-CD25 monoclonal antibodies (mAb) and cyclophosphamide
for depleting Tregs (Table 1.1). Work carried out by Anraku et al (2010) showed that a
significantly prolonged survival with no development of tumour could be achieved
when the anti-CD25mAb treatment was administered prior to tumour challenge (Anraku
et al., 2010). However, clinical translation of such data is difficult. In our laboratory,
low dose intra-tumoural (i.t) treatment with anti-CD25mAb was shown to inhibit
23
tumours successfully in 100% of the cases. However, this inhibition of AE17 tumour
growth was only limited to approximately ten days (Needham et al., 2006).
24
Table 1. 1 Monotherapies used for the depletion of Tregs in murine mesothelioma models
Mouse strain and tumour
Treg cell depletion strategy Survival Outcome References
C57BL/6J s.c. AE17
Low dose i.t. anti-CD25 mAb (PC61)to established tumours
10 days tumour growth inhibition
Needham et al., 2006
High dose i.t. anti-CD25 mAb to established tumours
No change in tumour growth
Jackaman et al., 2009
BALB/c with i.p. AB1
Cyclophosphamide in drinking water
10 days increase in survival
Veltman et al, 2010
CBA with i.t. AC29
High-dose systemic anti-CD25 mAb prior to tumour challenge
Significantly prolonged survival
Anraku et al., 2010
High-dose systemic anti-CD25 mAb to established tumours
No improvement in survival
Anraku et al., 2010
CBA with s.c. AC29
High dose anti-CD25 mAb to established tumours
Short delay in tumour growth
Wu et al., 2011
(modified from Ireland et al., 2012)
25
Despite the fact that these immunotherapeutic agents (and many others)
prolonged survival time or caused transient tumour growth inhibition, complete tumour
clearance was never achieved. This lack of clearance of tumours could be due to the
existence of compensatory pathways or other mechanisms of tolerance that may also be
simultaneously at work (Takeda et al., 2010). There is also the possibility that
monotherapies act as selective pressures that further enhance tumour evasion. This led
to the hypothesis that two or more agents used in combination will a) narrow the
window for the tumour to escape immune eradication and b) combination
immunotherapies that target different aspects may improve efficacy over administration
of monotherapies (Takeda et al., 2010).
1.8 Combination therapies are necessary for overcoming immune suppressive
mechanisms
Improved outcomes with combination therapies have now been shown in several
mouse tumour models. The combined treatment of murine B16 melanoma tumours with
recombinant murine IFN-γ and recombinant human IL-2 had improved tumour
eradication in tumour-bearing mice than the mice that received the respective therapies
separately (Silagi et al., 1988). In the same murine melanoma tumour model, treatment
with anti-CTLA-4mAb in combination with GM-CSF was shown to be effective in
eradicating established tumours in 68/85 of the treated mice, whilst the monotherapies
had little or no effect on tumour growth retardation (van Elsas et al., 1999).
A triple therapy combination termed “trimAb” consisting of anti-death receptor
(DR)-5mAb, anti-CD40mAb and anti-CD137mAb was shown to completely eradicate
close to 80% of 4T1 breast carcinoma tumours in mice (Uno et al., 2006). Treatment
with agonist antibodies to CD40 and CD137 (4-1BB) have been shown to activate
antigen presentation by APCs and hence enhance cytotoxic T cell activity against
26
tumours such as lymphoma, sarcoma and mastocytoma in mice (Melero et al., 1997;
French et al., 1999). In a recent study published by Dai et al (2014), combination of
monoclonal antibodies targeting CD137/CTLA-4/PD-1 or the 4mAb combinations
(CD137/PD-1/CTLA-4/CD19) were trialled in murine models of B16 and SW1
melanoma and the TC1 lung carcinoma (Dai et al., 2014). Addition of CD19 to the
3mAb combination in the above study produced complete tumour regression in 50% of
the mice with large tumours. Increase in long-term memory CD8 T cells and increased
production of IFN-γ and TNF-α was also achieved. Tumour regression was achieved
with the 3mAb, though it was to a lesser extent than the 4mAb combination. The
authors showed that the efficacy of the immunotherapy to treat large tumours could be
improved by combining mAbs that targeted different mechanisms of action. As detailed
previously, anti-CTLA-4mAb and anti-PD-1mAb treatments block the immune
regulatory checkpoints, thereby enhancing tumour activity. Agonist antibodies targeting
CD137 (also known as 4-1BB) expressed on APCs and T cells, enhances the co-
stimulation required for anti-tumour immunity (May et al., 2002). In contrast, the anti-
CD19mAb used in their 4mAb combination was to counteract the B cells and dendritic
cells that were involved in promoting tumour growth and inducing immune tolerance,
respectively (Baban et al., 2005; de Visser et al., 2006).
In the AE17 murine mesothelioma model, complete regression of large tumours
were observed in 87% of the mice that received multiple intra-tumoural treatments with
the combination of the cytokine IL-2 and anti-CD40 antibody delivered intra-
tumourally (Jackaman et al., 2008). More recently, the combination of anti-CTLA-
4mAb and the chemotherapy agent gemcitabine was used to treat BALB/c mice
inoculated with AB1-HA mesothelioma tumour cells. The treatment led to tumour
clearance in close to 60% of treated mice compared to 13% and 8% found in the groups
that received anti-CTLA-4mAb or gemcitabine alone (Lesterhuis et al., 2013).
27
A clinical trial with a combination of Ipilimumab targeting CTLA-4 and
Nivolumab that targets PD-1 administered intra-venously was tested in patients with
stage III or IV unresectable melanoma tumours. Though adverse events were observed
in 53% of the patients, rapid and deep regression of the melanoma tumours by 80% or
more were observed in 50% of the treated patients (Wolchok et al., 2013). This
evidence shows that combining one or more agents is more effective in mounting an
anti-tumour immune response than single therapies.
1.8.1 Simultaneous targeting of Tregs and TGF-β is more beneficial
Given that combination therapies that target multiple mechanisms were more
beneficial than monotherapies, as reported above, combined depletion of Tregs with
neutralisation of TGF-β (double immunotherapy) was, therefore, examined in our
laboratory. The additional neutralisation of TGF-β was included, as TGF-β was found to
be a master regulator of Treg function within the AE17 murine mesothelioma tumours
(Kissick et al., 2009). The combined depletion of Tregs using anti-CD25mAb and timed
neutralization of TGF-β using anti-TGF-βmAb was tested on the established AE17
tumours in our laboratory. An effective anti-tumour immune response with a transient
regression in tumour growth was generated in the treated mice treated with the timed
double immunotherapy (DI). However, complete tumour eradication was not achieved
(Kissick et al., 2009). This led to the notion that, the therapeutic efficacy of the timed
DI could be improved by perhaps simultaneously boosting the immune response.
1.8.2 Boosting effector cell function
Given that the blockade of the immune checkpoints CTLA-4 and PD-1 on the
surface of Teff cells improved tumour-destructive T cell responses, it was hypothesised
that their addition to the double immunotherapy would improve therapeutic efficacy.
Blockade of CTLA-4 and PD-1 by antibodies have been extensively tested in mouse
28
tumour models and were shown to improve anti-tumour T cell activity (Hirano et al.,
2005; Zou, 2006). The clinical benefits of using antibodies to block CTLA-4 (Hodi,
2010; Robert et al., 2011), PD-1 (Topalian et al., 2012) and the combination of CTLA-4
and PD-1 (Wolchok et al., 2013) have been proven. More recently, the relationship
between Tregs and PD-1 was elucidated in an article published in JEM, where the studies
involving Treg depletion in mice with chronic lymphocytic chorio-meningitis virus
(LCMV) showed that an up-regulation of PD-L1 on T cells occurred, leading to
inhibition of signals and failure to decrease viral load (Penaloza-MacMaster et al.,
2014). The authors also found that treatment combinations of Treg depletion and PD-L1
blockade resulted in a more significant reduction in viral titres than when the treatments
were administered as monotherapies.
At the time when the research was commenced (March 2011), anti-CTLA-4mAb
(Ipilimumab) was the first among monoclonal antibody treatments, to be approved by
the FDA for clinical treatment of melanoma. The work described in this thesis involved
the addition of anti-CTLA-4mAb to the timed DI regime for the treatment of murine
mesothelioma. In order to differentiate between the previous double immunotherapy
(DI) and the novel combination of using three mAbs against Tregs, TGF-β and CTLA-4
respectively, the new treatment was therefore called timed triple immunotherapy (TTI).
1.9 Role of B cells in immunotherapy for mesothelioma
There is some evidence in the literature that has suggested that B cells can serve as
APCs and thereby enhance cellular immune responses. This enhancement of cellular
responses is initiated by the activation of tumour specific cytotoxic T cells and by the
production of antibodies (antibodies have also been shown to modestly contribute to
anti-tumour activity) (Manson, 1994; Crawford et al., 2006; DiLillo et al., 2010b;
Jackaman et al., 2010; Molnarfi et al., 2013). In the context of pre-clinical
29
mesothelioma, previous findings by Jackaman et al (2010) suggested a role for B cells
for mounting an effective anti-tumour immune response; in their partially successful
CD40 based immunotherapy against AE17 and AB1 murine mesotheliomas (Jackaman
et al., 2010). The work in my thesis detailing the efficacy of the novel triple
immunotherapy treatment in eradicating murine mesothelioma tumours also included
studies on the role of B cells and whether they play an agonistic or antagonistic role.
1.10 Extending combination therapy to other tumours
The role of Tregs and TGF-β in suppressing immune responses to growing tumours
have been documented not just in pre-clinical models of mesothelioma, but in several
other murine tumour models as well (Onizuka et al., 1999; Shimizu et al., 1999). The
secretion of TGF-β by growing tumours has also been confirmed in other tumour
models such as melanoma and hepatocellular carcinoma among others (Penafuerte and
Galipeau, 2008; Wang et al., 2013). From the literature detailed in the above sections, it
is also clear that the combination immunotherapy being trialled in our laboratory may
have relevance in other tumour models.
1.11 Aims of the thesis
The initial work in this thesis investigated the additional blockade of the negative
regulator CTLA-4 in conjunction with the removal of intra-tumoural immune
suppression by Tregs and TGF-β (as TTI) in the AE17 murine mesothelioma tumour
model. Previously, in our laboratory, the re-accumulation of Tregs 7-10 days post-DI
treatment had been observed (Kissick et al., 2010). With the addition of anti-CTLA-
4mAb to the previous DI treatment protocol, the Treg cell population during the TTI
treatment period was to be investigated. The subsequent success of this approach led to
investigating the efficacy of this triple immunotherapy in a murine mesothelioma that
does not secrete TGF-β (AB1). The importance of timing was also examined by
30
combining all three agonist antibodies (anti-CD25mAb, anti-TGF-βmAb and anti-
CTLA-4mAb) into a single cocktail for intra-tumoural injection. The previously
reported role of B cells (Jackaman et al., 2010) during tumour clearance in response to
immunotherapy was also investigated in relation to the novel triple immunotherapy
trialled in our laboratory. The goal of the experiments detailed in this thesis was to
develop an effective treatment option to modulate the tumour microenvironment to
overcome immune suppression and enhance tumour destruction. The achievement of
this goal will enable their rapid translation to human trials for the treatment of
mesothelioma.
31
Chapter 2 Materials and methods
32
33
2.1 Murine tumour cell lines
The C57BL/6J tumour cell lines are the AE17 murine mesothelioma, B16
melanoma and EO771 breast carcinoma (Dunham and Stewart, 1953; Fidler, 1973;
Jackaman et al., 2003). The BALB/c cell lines are the AB1 murine mesothelioma and
the 4T1 breast carcinoma (Dexter et al., 1978; Davis et al., 1992).
2.2 Tissue culture
All tissue culture procedures were carried out in a class 2 biological laminar flow
hood using aseptic techniques.
2.2.1 Storage of tumour cell lines
Tumour cells were frozen (-80oC) in 1 ml aliquots containing cell numbers
ranging from 1 × 106 to 1 × 107 in freezing media (Section 2.10). Following freezing,
they were stored in liquid nitrogen.
2.2.2 Resuscitation of frozen stocks
Frozen tumour cells were taken out of the liquid nitrogen storage and thawed at
room temperature and slowly resuspended in 10ml of supplemented RPMI media. Cell
suspension was centrifuged at 350 × g for 5 minutes and supernatant discarded. The
pellet was resuspended in 5 ml of supplemented media and transferred to a 75 cm2
tissue culture flask containing 10 ml of supplemented RPMI medium to bring the total
volume up to 15ml. Cells were incubated at 37°C over-night in 5% CO2 incubator. The
media was replaced the following day with fresh RPMI media, to remove remaining
DMSO and non-adherent dead cells.
2.2.3 Passage of tumour cell lines
Cell lines were passaged when the flasks reached 70-80% confluency. The
RPMI media was discarded, and the cells were washed by adding 10 ml of PBS to the
flask. 1 ml of trypsin was added to the flask to cover the cells completely and then
34
incubated at 37°C for 2 minutes. The flask was tapped firmly to dislodge cells, and the
trypsin was inactivated by the addition of 10ml of supplemented RPMI media. Cells
were mixed well by pipetting and transferred to a 50 ml centrifuge tube for
centrifugation at 350 × g for 5 minutes. The pelleted cells were resuspended in 10 ml of
supplemented RPMI media. Aliquots of this suspension containing 7.5 × 105 were then
added to 225 cm2 tissue culture flasks containing 30 ml of supplemented RPMI media
and incubated at 37°C.
2.2.4 Harvesting cells for in vivo use
When cells reached 70% confluency, they were considered ready for in vivo use.
The cells for in vivo inoculation were harvested as mentioned above and then washed
twice with PBS by resuspension and centrifugation.
2.2.5 Cell counting
The final cell pellet was resuspended in 10 ml of PBS and a cell count
performed using trypan blue exclusion method by applying a one to one ratio of 0.4%
trypan blue solution to the cell suspension. 10 µl of the trypan blue/cell suspension were
loaded onto a Neubauer haemocytometer by capillary action. Numbers of viable cells
(clear) were counted using a light microscope in the central 5 × 5 square grid as the
dead cells stain blue. The total viable cell number in the sample was determined using
the below equation.
Total live cells= LC × DF × 1 × 104 × ml
LC= Live cells
DF= Dilution factor
Ml= Number of ml of suspended cells
35
Cell counts were performed in duplicate, and the mean of the two sample counts
was used to determine the cell concentration. Following cell concentration estimation,
cells were pelleted and re-suspended in the required volume of sterile PBS to give the
necessary concentration of tumour cells required for injection.
2.3 In vivo use of tumour cell lines
2.3.1 Inoculation of tumour cells
6-8 weeks old C57/BL6J and BALB/c mice were injected sub-cutaneously (s.c)
on the right flank above the ribcage with the required cell numbers suspended in PBS
using a MICROLITRE Hamilton syringe mounted with a 26g needle. The final
concentrations required were 1 × 107 cells /100µl for AE17 mesothelioma cells, 1 × 106
cells/50µl for AB1 mesothelioma cells and 5 × 105 cells/50µl for B16 melanoma,
EO771 breast carcinoma and 4T1 breast carcinoma cell lines. The cells were injected
into the respective mice within 30 minutes of harvesting.
All animal experiments were approved by The University of Western Australia
Animal Ethics Committee and were documented in the Animal Ethics applications
RA/3/100/498 and RA/3/100/1184 respectively.
2.3.2 Measurement of subcutaneous tumours
Following the inoculation of mice with tumour cells, swelling and redness at the
site of injection was observed, and the mass was soft and was difficult to measure its
size accurately. However, the swelling soon regresses and established hard tumours
could be palpated and measured. The two largest diameters of the palpable tumour were
measured at right angles to each other using Vernier callipers that are multiplied to
obtain tumour area in mm2. Mice bearing tumours were monitored daily for tumour size
measurements, weight loss and changes in overall health.
36
2.3.3 Euthanasia of tumour bearing mice
Humane culling of the tumour bearing mice were carried out when tumour areas
reached 100mm2 or if there was an overall deterioration in the health of the mice
(symptoms included weight loss, lethargy and seclusion). Cervical dislocation
euthanized mice following anaesthesia with methoxyflurane for 3-5 minutes.
2.3.4 Treatment regimens
Reagents used for the treatment are outlined in Table 2.1. The preparation for
the treatments is outlined below.
37
Table 2. 1 Mono-clonal antibodies
Compound Source (City,
State, Country)
Catalogue Number
Anti-CD25mAb
(PC61)
eBioscience (San
Diego, CA, USA)
14-0251-86
Anti-CTLA-4mAb
(9H10)
AbSolutions
(Perth, WA,
Australia)
N/A
Anti-TGF-βmAb
(1D11)
R&D Systems
(Minneapolis,
MN, USA)
MAB1835
38
2.3.1 Preparation of anti-CD25mAb treatments
The anti-CD25mAb stock solution (0.5 mg/ml) was diluted in sterile PBS to
obtain a final concentration of 2 µg/50 µl for AE17 treatments and 2 µg/30 µl for AB1
tumour treatments respectively.
2.3.2 Preparation of anti-CTLA-4mAb treatments
Anti-CTLA-4mAb stock (2mg/ml) was diluted with sterile PBS to have a final
concentration of 1 µg/µl for both AE17 and AB1 treatments.
2.3.3 Preparation of anti-TGF-βmAb treatments
Stock solution of anti-TGF-βmAb (0.5 mg/ml) was diluted in sterile PBS to
obtain a solution of 1 µg/50 µl for AE17 treatment and 1 µg/ 30 µl for AB1 treatments
respectively.
2.3.4 Preparation of triple immunotherapy cocktail (TIC)
Stock solution of the three triple therapy components was diluted with sterile PBS
to make a solution containing 2 µg of anti-CD25mAb, 2 µg of anti-CTLA-4mAb and 2
µg of anti-TGF-βmAb administered at a final volume of 50 µl for AE17 treatments and
30 µl for the other tumour models respectively.
2.4 Treatment administration
The procedures used for the administration of the TTI and TIC treatments are
outlined below
2.4.1 Timed triple immunotherapy (TTI) treatment administration at 9mm2
Murine tumours were allowed to reach an established size of 9mm2 before
treatment was commenced. The TTI protocol as outlined in Figure 2.1 was then
initiated. For intra-peritoneal (i.p) treatments, tumour bearing mice were held securely
with ventral abdomen exposed and head pointed slightly downward. Animals were
39
injected into the right lower abdominal quadrant with a 0.5 ml insulin syringe mounted
with 29g needle. Intra-tumoural (i.t) treatments were injected into the centre of the
established tumours in mice using a 0.5 ml insulin syringe mounted with 29g needle.
2.4.2 Intra-tumoural (i.t) administration of a single dose of triple immunotherapy
cocktail (TIC)
Mice receiving the triple immunotherapy cocktail (TIC) received 2 µg of each of
the three components as a single i.t injection in 30 µl of PBS into 9mm2 tumours using a
0.5 ml insulin syringe fitted with 29g needle.
40
41
Figure 2. 1 Timed Triple Immunotherapy (TTI) experiment protocol. 6-8 week old mice were inoculated s.c with tumour cells and TTI treatment was initiated when the tumours reached 9mm2. The timed triple immunotherapy (TTI) consisted of single intra-tumoural (i.t) injection of anti-CD25mAb with one intra-peritoneal (i.p) injection of anti-CTLA-4mAb on the same day, followed by seven daily i.t injections of anti-TGF-ßmAb. Mice were monitored daily for tumour growth and tumour sizes were measured using microcalipers.
42
2.5 Sample preparation and analysis by flow cytometry
The procedures used for the preparation and analyses of samples for flow
cytometry are presented below
2.5.1 Preparation of lymph nodes and tumours
Mice were humanely euthanized, and the tumour and tumour draining lymph
nodes (TDLNs) were surgically removed and placed in 1.5 ml microfuge tubes
containing 500 µl of un-supplemented RPMI media. The tissues were homogenised by
mechanically mincing with scissors for 5 minutes and then enzymatically digested by
the addition of 12.5 µl of Liberase blendzyme III (1.4 units/100 µl) and 12.5 µl of
DNase I (2000 kU/100 µl) and mixed on a rotating wheel for 30 minutes. The mixture
was passed through a spoon sieve with the help of a 3 ml syringe plunger. The sieve
was rinsed with additional 1 ml of un-supplemented RPMI media to remove any
remaining cells. This sieved solution containing the single cell suspension was collected
back in the 1.5 ml microfuge tube and centrifuged at 1500 × g for 3 minutes. The
supernatant was discarded and the pellet was washed once with FACS wash buffer and
once in 1 ml of sterile PBS before being suspended in 100 µl of PBS ready to be stained
with the live/dead viability dye.
2.5.2 Preparation of spleen cells
Spleens were surgically removed and placed in 1.5 ml microfuge tubes
containing 500 µl of un-supplemented RPMI media. They were mechanically digested
by chopping them finely with scissors and further homogenised by enzymatically
digesting them with 12.5 µl Liberase blendzyme III and 12.5 µl DNase I on a rotating
wheel for 30 minutes. The mixture was passed through fine wire mesh spoon sieve with
a 3 ml syringe plunger. The sieve was rinsed with 1 ml of un-supplemented RPMI
media to remove any remaining cells, and this solution was collected in a 1.5 ml
43
microfuge tube and centrifuged at 1500 × g for 3 minutes. The supernatant was
removed, and the pellet was resuspended in 1 ml of red cell lysis buffer for 5 minutes.
The solution was centrifuged, and the supernatant was discarded. The pellet was washed
once with FACS wash buffer and washed once again with 1 ml of sterile PBS and the
pellet was resuspended in 100 µl of PBS and ready to be stained with the live/dead
viability dye.
2.5.3 Live/dead viability dye (eFlour 780) staining
As a single stain control for the live/dead viability dye, a small aliquot (50 µl) of
the single cell suspension was taken in an 1.5 ml microfuge and killed by 3 × freeze-
thaw cycles using liquid nitrogen. These dead cells were mixed with a further 50 µl of
live cells from the single cell suspension. The samples and the single stain live/dead
viability control tube were incubated for 30 minutes at 4°C with the addition of the
live/dead viability dye eFlour780 (1 µl/ml of sample containing 1-10 × 106 cells/ml).
2.5.4 Cell surface staining for flow cytometry
After the live/dead cell staining step, the cells were washed twice with 500 µl of
FACS wash and the pellet was resuspended in 50 µl of master mix containing the
appropriate fluorescently labelled antibodies at the concentrations recommended by the
manufacturers in FACS wash buffer. The antibodies used for flow cytometry are
presented in Table 2.2. The samples were incubated for 30 minutes at 4°C, washed twice
with FACS wash buffer before being resuspended in 500 µl of FACS wash buffer, ready
to be analysed by flow cytometry (FCM).
44
Table 2. 2 Antibodies used for flow cytometry
Antibody Clone Conjugate Supplier Catalogue #
CD3e 145-2C11 FITC eBioscience 11-0031
CD4 GK1.5 eFlour 450 eBioscience 48-0041
CD4 GK1.5 APC eBioscience 17-0041
CD8a 53-6.7 PE-Cy 7 eBioscience 25-0081
CD8a 53-6.7 PE eBioscience 12-0081
CD25 PC61.5 APC eBioscience 17-0251
CD25 7D4 FITC BD Bioscience 553071
CD25 PC61.5 FITC eBioscience 11-0251
CD44 IM7 V500 BD Bioscience 560780
CD44 IM7 FITC eBioscience 11-0441
FoxP3 FJK-16s PerCP-Cy 5.5 eBioscience 45-5773
FoxP3 FJK-16s PE eBioscience 12-5773
CD80 (B7-1) 16-10A1 PE eBioscience 12-0801
CD11c N418 APC eBioscience 17-0114
MHC II M5/114.15.2 eFlour450 eBioscience 48-5321
CD45R/B220 RA3-6B2 PerCP-Cy 5.5 eBioscience 45-0452
Live/dead - eFlour780 eBioscience 65-0865
Live/dead - DAPI BioLegend 422801
45
2.5.5 Intracellular Fox P3 and Ki-67 staining
Samples that require intra-cellular staining were prepared by washing the cell
pellet in 500 µl of permeabilisation buffer once and resuspending the cell pellet in
permeabilisation/fixative buffer, which was then incubated for 1 hour at 4°C. Cells were
washed once with permeabilisation buffer and resuspended in 50 µl of master mix
containing FoxP3 and Ki-67 in the recommended concentrations (diluted in
permeabilisation buffer) and incubated at 4°C for 30 minutes. The cells were washed
with permeabilisation buffer and resuspended in FACS buffer for analysis by flow
cytometry.
2.5.6 Preparation of compensation controls for flow cytometry
Single stain controls are required for compensating intrinsic spectral overlaps
that can occur when using multi-colour flow cytometric analyses. If left uncorrected
without using compensation controls, this can lead to misinterpretation of data with
false-positives and artifactual populations when analysing contour plots (Baumgarth and
Roederer, 2000). Compensation beads (positive and negative beads) are thereby used to
set the gates for what proportion of cells is positive and thereby help with interpreting
data (Maecker and Trotter, 2006). Compensation beads were used to create different
single stained controls for all the fluorochromes (Table 2.2) used in the flow cytometry
experiments. Depending on the number of fluorochrome conjugates used in the
experiment, individual tubes were labelled as such for the conjugates. A drop
(approximately 60µl) of positive and negative compensation beads were dispensed into
each tube; following manufacturer’s instructions (BD Bioscience BDTM CompBead cat#
552843). 0.5 µl of the respective fluorochromes (irrespective of the antibody bound to
them) was added and incubated at 4°C for 30 minutes. The controls were then washed
twice with FACS wash buffer before being resuspended in 500 µl of FACS wash buffer,
ready to be used for the compensation step before analysing the samples.
46
2.5.7 Flow cytometric analysis of samples
Flow cytometry was performed using FACS CantoII bench-top flow cytometer
using the FACSDiva software and the data collected was analysed using the FlowJo
software from Treestar Inc.
2.5.7.1 Analysis of T cells
The gating strategy for live T cells (CD4+ and CD8+) is shown in Figure 2.2.
2.5.7.2 Analysis of Regulatory T cells
Regulatory T cells (Tregs) were defined as CD4+CD25+FoxP3+ T cells and the
gating strategy for Tregs are shown as a percentage of CD4+CD25+ T cells that are also
positive for FoxP3. The gating strategy used for determining Tregs in the TDLNs and
tumours is shown in Figures 2.3A and B.
2.5.7.3 Analysis of DC cells
Dendritic cells (DCs) were defined as CD11c+MHCII+ and their gating is as
shown in Figure 2.4.
2.5.7.4 Determination of relative expression of CD80
Relative expression of CD80 by DCs was determined using Geometric Mean
Fluorescence Intensity (MFI) of the CD11c+MHCII+ cells co-stained with CD80-PE as
shown in Figure 2.4.
47
48
Figure 2. 2 Gating strategy for analysing CD4+ and CD8+ T cells. Total lymphocytes population was firstly selected based on their size (forward scatter FSC) and their granularity (side scatter SSC) respectively. Doublets and triplets are eliminated by plotting the size of the lymphocytes based on their area versus height and singlets are selected. The viability dye ef780 (live/dead) was then used to select for live cells that did not fluoresce. Out of these live cells, the population of cells that were positive for CD8 and CD4 T cells were then determined.
49
Figure 2. 3 Gating strategy for Treg estimation in TDLNs and tumours. CD4+ T cells are selected by plotting against viability dye for TDLNs (A) or against granularity (SSC) when examining within tumours (B). The percentage of cells within this CD4+ population that also express CD25 are then determined. The expression of FoxP3 by the CD4+CD25+ T cells is then plotted as a histogram.
50
Figure 2. 4 Gating strategy for analysing dendritic cells. For determining total dendritic cell (DC) population, the total live cell population were plotted as MHCII versus CD11c. The gates for positive and negative expression of CD80 were set using the single stained compensation controls. The expression of CD80 by the DCs was then analysed by determining the mean fluorescence intensity (MFI) of the fluorochrome (PE) used for identifying CD80 expression in these cells.
CD80
51
2.5.7.5 Determination of cell numbers
The number of cells per samples analysed was determined using TruCount tubes
(BD Bioscience) according to the instructions from the manufacturers.
2.5.8 Isolation of immune cells by Fluorescence-activated cell sorting (FACS)
Spleen cells to be sorted by FACS were prepared as mentioned in 2.5.2.
However, instead of the FACS wash, sterile PBS enriched with 5% FCS was used. The
extra-cellular staining of the single cell suspension was the same as the above-
mentioned methodology in 2.5.4. With the one exception that 5% FCS enriched PBS is
used instead of FACS wash in all the steps. Before cells were sorted, they were
resuspended at 5 × 106 cells/ml in 5% FCS enriched PBS.
The FACS sorting was conducted at The Centre for Microscopy,
Characterisation and Analysis (CMCA), UWA, by the CMCA staff using BD Influx
flow cytometer with an 86 µm nozzle and a pressure of 33 psi. The live/dead cell stain
(DAPI at 1µg/ml concentration) was added to the samples prior to sorting and mixed
well, and sorting was initiated. The sorted cells were collected in 50 ml Falcon tubes
containing 5 ml of sterile, cold FCS to maintain the viability of these cells.
2.5.9 Adoptive transfer of immune cells into mice
The immune cells of interest collected in the 50 ml Falcon tubes were centrifuged
at 1500 × g for 3 minutes, and the supernatant was discarded. The pellets were then
resuspended in sterile PBS and counted, reconstituted with sterile cold PBS to the
appropriate concentrations and were injected intra-venously into the recipient mice (tail-
vein injections) using 0.5 ml insulin syringe fitted with 29g needle. The recipient mice
were warmed in an approved warming cabinet before the i.v injection and were
monitored for any sign of overheating which included increased rapid respiration or
panting, red extremities, decreased activity and excessive salivation.
52
2.6 Tumour specific Immunoglobulin detection analysis
Immunoglobulin G (IgG) in the serum of mice that react specifically against
tumours was determined by enzyme linked immunosorbent assay (ELISA).
2.6.1 Preparation of serum
Blood was collected by cardiac puncture into uncoated blood tubes and was
allowed to clot overnight at 4°C. The serum was transferred into a new microfuge tube
and stored at -20°C. Multiple cycles of freeze/thaw were avoided for serum samples.
2.6.2 Preparation of cell lysate coated ELISA immunosorbent plates
Tumour cells were harvested as mentioned in section 2.2.4 and splenocytes
(from naïve mice) were prepared as single cell suspensions as mentioned in 2.5.2 and
the respective cells were counted and resuspended to a concentration of 2 × 107 cells per
10 ml of sterile PBS. The cells were subjected to 3 cycles of freeze-thaw (liquid
nitrogen followed by thawing at 37°C, 5 minutes each). The resulting lysed cell
suspension was centrifuged at 300×g for 10 mins and the supernatant collected. 100 µl
of the supernatant was added to each well of an immunosorbent 96 well ELISA plate
and incubated overnight at 4°C without shaking. The following day, the supernatant was
removed, and the wells washed thrice with 200 µl of 0.01% Tween-20 in PBS. The
plates were then used immediately or stored at -20°C for later use.
2.6.3 Detection of tumour cell lysate specific IgG in serum
The cell lysate coated wells prepared as mentioned in 3.7.1 were blocked with
200 µl of 5% FCS in PBS for 1 hour at room temperature before running an ELISA.
The blocking solution was removed, and the wells were washed thrice with 200 µl of
0.01% Tween-20 in PBS. After the 3 washes, 100 μl pooled sera diluted in ELISA assay
diluent were added to the plate and incubated without shaking at room temperature for 2
hrs. The wells were washed thrice with 200 µl of 0.01% Tween-20 in PBS once the
53
serum was removed. Following three washes, 100 µl of biotin conjugated anti-mouse rat
IgG antibody diluted in assay diluent (final concentration of 2 µg/ml) was added and
incubated for 1 hr at room temperature. The wells were washed three times in washing
buffer followed by the addition of 100 µl avidin-horseradish peroxidase (avidin-HRP,
diluted 1:1000 times with assay diluent) and incubated for 30 min at room temperature
and the plate was washed thrice with 200 µl of 0.01% Tween-20 in PBS.
2.6.4 Detection of IgG in serum specific to live tumour cells
For the modified ELISA, tumour cells and splenocytes (from naïve mice) were
harvested as described in sections 2.2.4 and 2.5.2 respectively and used instead of cell
lysates. The cells were counted and diluted, such that there were 2 × 104 cells/100 µl.
The assay was performed in 1.5 ml microcentrifuge tubes with each tube containing 300
µl of the suspended live cells. Washes were performed with sterile PBS and involved
centrifugation and supernatant removal. After incubation with avidin-HRP, the cells
were resuspended in PBS and transferred into pre-blocked (5% FCS in PBS) 96 well
plates (ELISA plate wells were blocked overnight at 4°C with 200 µl of 5% FCS in PBS
and washed twice with 200 µl of 0.01% Tween-20 in PBS before using the plates).
2.6.5 Measurement of optical density of serum IgG specificity
100 µl of TMB substrate solution (substrate for HRP) was added to the
appropriate wells of the 96 well immunosorbent plates and the colour reaction that
develops after 10 minutes; was stopped by the addition of 50 µl of 1M Phosphoric acid
to each well. The optical density was then measured using POLARStar Optima plate
reader at 450nm.
Appropriate controls used with every ELISA run were:
a. NO LYSATE: Wells which were not coated with tumour cell lysates, but
incubated with serum and the rest of the reagents that followed;
54
b. NO SERUM: Tumour cell lysate coated wells to which no serum was added and
received the subsequent detection antibodies (to detect cross-reactivity); and
c. NO BIOTIN CONJUGATE: Tumour lysate coated wells that were incubated
with serum and all other reagents excluding anti-mouse rat IgG-Biotin, and
d. ONLY LIVE CELLS: An additional control of only live tumour cells (with
nothing else added to them) was used for the whole live cell ELISA analysis to
account for auto-fluorescence of live cells.
2.7 Statistical analysis
All immune cell data collected by flow cytometry are presented as the mean ± SD.
For all tumour size measurements, mean tumour size (± SEM) in mm2 was calculated.
Statistical significance set at p<0.05 were used when only two groups were compared
and were determined by performing student’s two-tailed t-tests. One-way analysis of
variance (ANOVA) with Tukey’s multiple comparison post-hoc tests and linear trend
analysis set at p<0.05 were used for determining differences in immune cell populations
between different groups. All calculations and statistical tests were carried out using
Microsoft Excel (2011) and GraphPad Prism version 5.04 for Windows (GraphPad
Software, San Diego, CA, USA). The Kaplan-Meier survival curves including median
survival calculations were generated using GraphPad Prism version 5.04.
2.8 Materials and Reagents
2.8.1 Mice
Female C57BL/6J and BALB/c mice aged 6-8 weeks were obtained from the
Animal Resource Centre (Perth, Western Australia) and maintained under standard
housing conditions in the M block Animal Care Facility at The University of Western
Australia. The B cell deficient mutant mice (Kitamura et al., 1991) on the BALB/c
background (C.129S- Igh-6tm1Cgn/J; referred to as BKO) aged 4-10 months were bred
55
in-house at the University of Western Australia animal holding facility and were
provided by Animal Care Services (Perth, Western Australia). All animal work was
carried out in accordance with the guidelines of the National Health and Medical
Research Council of Australia and approval of The University of Western Australia
Animal Ethics committee (RA/3/100/1184).
2.8.2 Reagents
Benzyl Penicillin CSL, Australia
Deoxyribonuclease (DNase) I Sigma, USA
ELISA assay diluent (5x concentrate) eBioscience, USA
Ethanol (96%) Hurst scientific, Australia
Fixation/Permeabilisation concentrate eBioscience, USA
Fixation/Permeabilisation diluent eBioscience, USA
Foetal Calf Serum (FCS) Gibco BRL, USA
Gentamicin Deltawest, Australia
L-glutamine Sigma, USA
Liberase Blendzyme III Roche, USA
Methoxyflurane Medical Developments Australia,
Australia
Permeabilisation buffer eBioscience, USA
Phosphate buffered saline tablets Oxoid, Australia
Phosphoric acid BDH Chemical, Australia
56
Protease inhibitor cocktail Sigma, USA
RPMI 1640 media Gibco BRL, USA
Sodium azide Sigma Aldrich, USA
Stabilisation fixative (3x concentrate) BD Bioscience, USA
Tris Gibco BRL, USA
Tris-Hydrochloride Sigma, USA
Triton X-100 BDH Chemicals, Australia
Trypan Blue Hopkin and Williams, England
Trypsin/EDTA Sigma, USA
Tween-20 Promega, USA
2-Mercaptoethanol BDH Chemicals, Australia
2.8.3 ELISA reagents
Avidin-HRP 250x concentrate eBioscience, USA
Rat anti-mouse IgG-Biotin eBioscience, USA
3’,3’,5’,5’-Tertramethylbenzidine (TMB) substrate eBioscience, USA
2.9 Equipment
Acrodisc Micropore Syringe Filters Gelman Sciences, USA
BD Influx Becton Dickinson, USA
Blood collection vials
- Non-coated MICROTAINER 2ml Becton Dickinson, USA
57
Cell strainer (40 µm nylon mesh) BD Falcon, USA
Centrifuge and Eppendorf tubes
- 15 ml and 50 ml Falcon tubes Becton Dickinson Falcon, USA
- 1.5 ml and 2 ml Eppendorf tubes Eppendorf, Germany
Centrifuges
- Eppendorf microfuge 5424 Eppendorf, Germany
- BECKMAN Model TJ-6 centrifuge Beckman Coulter, USA
ELISA plates
- Immunosorbent 96 well plates BD Falcon, USA
FACSCanto II Becton Dickinson, USA
FACS tubes
- Polystyrene round bottom tubes BD Falcon, USA
- Polypropylene round bottom tubes BD Falcon, USA
Flow Cytometry software
- FACSDiva BD Biosciences, USA
- FlowJO Treestar Inc, San Carlos, CA
Incubator
- Forma Series II Water Jacket Thermo Fischer Scientific, USA
Neubauer chamber Hawksley, England
Nunc cryovials 1.8 ml Nalge Nunc International, Denmark
58
POLARStar Optima BMG Labtech
Rotating wheel Chiltern Scientific, England
Syringes
- 0.5 ml 31g insulin syringe BD Biosciences, USA
- MICROLITRE 100 μl Hamilton, USA
- 50ul syringe (50R-GT) SGE, Australia
Tissue Culture flasks
- 75 cm2 Nalge Nunc International, Denmark
- 225 cm2 Nalge Nunc International, Denmark
Tissue culture hood model BH180 Gelman Sciences
TruCount tubes BD Biosciences, USA
Vernier microcalliper Kaiser, Australia
2.10 Buffers, media and solutions
Antibiotics (4mg/ml Gentamicin and 6mg/ml Benzylpenicillin) (100ml)
Five ampules of Gentamicin and 600mg (1 ampule) of Benzylpenicillin was
dissolved in 100ml of ddH2O, filter sterilised using 0.2 µm filter and stored at -20°C in 5
ml aliquots.
DNAse I (2.3ml)
23mg of DNase I was dissolved in 1.3ml of 10mM Calcium chloride and 1 ml of
ddH2O and filter sterilised through 0.2 µm filter and stored at -20°C in 100 µl aliquots.
59
ELISA assay diluent
ELISA assay diluent (5X) was diluted in PBS
FACS wash
0.352g of sodium azide (0.005mM NaN3) was dissolved in 100 ml of PBS,
autoclaved and stored at room temperature. Prior to use, 5 ml of FCS was added and
stored thereafter at 4°C.
FCS (Foetal Calf Serum)
Frozen FCS was thawed in water bath at 37°C and heat inactivated for 30
minutes in a 60°C water bath and cooled at room temperature. The heat inactivated FCS
was then aliquoted into 50 ml falcon tubes and stored at -20°C.
Freezing media
10 ml of DMSO was added to 90 ml of FCS and mixed well.
L-glutamine (250ml)
7.3 g of L-glutamine was dissolved in 250 ml of ddH2O, filter sterilised through
0.2 µm filter and stored at -20°C in 5 ml aliquots.
Liberase Blendzyme III
7 mg of Liberase Blendzyme III was dissolved in 2 ml of sterile ddH2O, then
incubated on ice for 30 minutes with intermittent mixing to ensure complete dissolution.
The solution was then filter sterilised using a 0.2 µm filter and stored at -20°C in 100 µl
aliquots.
60
2-mercaptoethanol (2-ME)
1.74 ml of 2-mercaptoethanol was added to 50 ml of ddH2O and filter sterilised
using a 0.2 µm filter and stored in 500 µl aliquots at -20°C.
Phosphate buffered saline (PBS) (100 ml)
One phosphate buffered saline tablet was dissolved in 100 ml of ddH2O,
autoclaved and stored at room temperature. Typical concentration formula for PBS is
8.0 g/L Sodium chloride, 0.2 g/L Potassium chloride, 1.15 g/L di-sodium hydrogen
phosphate and 0.2 g/L Potassium di-hydrogen phosphate.
Red cell lysis buffer
8.3 g of Ammonium chloride (NH4Cl) was dissolved in 1L of ddH2O to make a
0.83% solution. 450 ml of the 0.83% solution was added to 50 ml of Tris solution
(2.059 g of Tris dissolved in 10 ml of ddH2O and pH adjusted to 7.6) and filter
sterilised.
Supplemented RPMI 1650 media
5 ml of L-glutamine, 5 ml of media antibiotics, 50 ml of FCS and 40 µl of 2-
mercaptoethanol was added to 500 ml of RPMI 1640 media and stored at 4°C for up to a
month.
61
CHAPTER 3 Development and characterisation of an effective triple
immunotherapy in a TGF-β secreting murine mesothelioma model (AE17 model)
62
63
3.1 Introduction
A greater understanding of the interactions between the immune system and
developing tumours has led to immune system modulation being a promising approach
to reducing the morbidity and mortality of cancers. To date, clinical immunotherapy
protocols have predominantly involved only a single agent (O'Brien et al., 2003;
Gerena-Lewis et al., 2009; Hodi et al., 2010; Kantoff et al., 2010; Wolchok et al., 2010;
Wolchok et al., 2013). Such monotherapies have had promising results in animal
models and clinical settings, but their effectiveness has often been limited by peripheral
tolerance induced by tumour growth.
Peripheral tolerance can be induced by regulatory T cells (Tregs) (Sakaguchi,
2004). During tumour growth, increases in Treg numbers have been observed in the
periphery and also within different tumours, including murine and human
mesotheliomas, thereby maintaining T cell anergy and inhibiting the cytotoxicity of
CD8+ T cells and NK cells (Chen et al., 2005; Needham et al., 2006; Smyth et al., 2006;
Zou, 2006; Rabinovich et al., 2007; Hegmans et al., 2010; Whiteside, 2013; 2014).
Danull et al (2005) have shown that elimination of Tregs using Denileukin Diftitox also
enhanced the activity of a DC vaccine that was administered to patients with renal cell
carcinoma (RCC) (Dannull et al., 2005). Elimination of Tregs with corresponding
increase in cytotoxic capacity of CD8+ T cells have also been reported in other human
clinical trials against non-Hodgkin lymphoma, T-cell lymphoma and non-small cell
lung cancer (Olsen et al., 2001; Dang et al., 2007; Gerena-Lewis et al., 2009).
When Treg depletion is carried out systemically, it can lead to undesirable
effects. Work carried out in BALB/c mice by Sakaguchi et al (2006) showed that
adoptive transfer of spleen cells depleted of Tregs into athymic nude BALB/c mice
caused severe autoimmune diseases in multiple organs in these recipient mice
64
(Sakaguchi et al., 2006). To avoid such systemic problems, we have shown that intra-
tumoural injection of Treg depleting antibodies (as opposed to systemic delivery) can
avoid undesirable autoimmune effects (Needham et al., 2006).
In the work conducted previously in our laboratory on the AE17 murine
mesothelioma (C57BL/6J) model, intra-tumoural (i.t) depletion of Tregs with low dose
anti-CD25mAb (PC61) effectively inhibited growth of tumours for approximately 10
days (Needham et al., 2006). As opposed to high dose anti-CD25mAb therapy which
can in fact enhance tumour growth (Jackaman et al., 2009), we demonstrated that low
dose and local intra-tumoural Tregs depletion using an anti-CD25mAb resulted in
transient tumour growth inhibition and activation of CD4+ helper T cells, CD8+
cytotoxic T cells and dendritic cells (Kissick et al., 2010). A limitation of low dose anti-
CD25mAb therapy, even when administered directly into the tumour, is the transient
nature of tumour growth inhibition, as Treg cells re-accumulated in the tumour
microenvironment ten days post treatment.
We, therefore, hypothesised that multiple mechanisms of peripheral tolerance
are involved in immune suppression within the tumour microenvironment and that;
combinatorial therapies targeting multiple mechanisms will improve anti-tumour
immune responses. This has become a major international research focus; with animal
studies confirming that targeting multiple mechanisms of tumour immunosuppression
result in stronger immune responses against multiple tumour types (van Elsas et al.,
1999; Uno et al., 2006; Kissick et al., 2009; Curran et al., 2010; Jackaman et al., 2012;
vom Berg et al., 2013).
The AE17 model of murine mesothelioma is an excellent model for assessing
pre-clinical efficacy of immune modulation therapies.It is also known to secrete
transforming growth factor-β (TGF-β) (Fitzpatrick et al., 1994; Ireland et al., 2012), an
65
important immunosuppressive cytokine that has been shown to actively promote
accumulation of Tregs and also assist Tregs in inhibiting cytotoxic capacity of CD8+ T
cells as well as NK cells within the tumour microenvironment (Ghiringhelli et al., 2005;
Smyth et al., 2006). Further investigation of the transient response to Treg cell depletion
in this model found that more than 50% of TGF-β still remained in the tumour area post
Treg depletion; thereby promoting Treg cell re-accumulation or conversion (Kissick et al.,
2010). In order to overcome the immunosuppressive effect of TGF-β, we developed a
combined regime of Treg depletion and timed TGF-β neutralisation. The idea behind this
double immunotherapy regimen was to target two mechanisms of immune suppression
that would potentially overcome Treg based immunosuppression. In this double
immunotherapy (DI) experiment, mice were treated with a single intra-tumoural
injection of anti-CD25mAb when tumours reached 9mm2 followed by daily intra-
tumoural injections of TGF-β soluble receptor (SR) for 7 days. This resulted in around
50% of the tumours being reduced in size such that they were barely palpable; with
significant inhibition of tumour growth in the remaining mice and a significant increase
in the time to death (TTD) (defined as tumours reaching 100mm2) (Kissick et al.,
2009). This combined treatment also resulted in increased intra-tumoural IFN-γ
concentrations- suggesting an increase in activated cytotoxic T cells, when compared
with either treatments administered as monotherapies (Kissick et al., 2010). Though the
DI treatment was clearly an improvement over the anti-CD25mAb monotherapy, long-
term cures of the tumours was not achieved i.e. all the mice eventually grew end-point
tumours.
In order to determine whether the efficacy of this immune modulating therapy
could be further enhanced towards complete eradication of tumours in the murine
model; the promising new anti-CTLA-4mAb therapy was added (Callahan et al., 2010).
Cytotoxic T lymphocyte antigen (CTLA)-4 is a second receptor on T cells for B7 and a
66
negative receptor involved in immune-regulation (immune check-point). Under normal
circumstances, CTLA-4 is an antagonist for activated T cell proliferation, which is
essential for maintaining self-tolerance and minimising collateral tissue damage
(Korman et al., 2006). However, in the presence of a growing tumour, CTLA-4 binds
with higher affinity to CD80 and CD86 on antigen-presenting cells (APC), out-
competing CD28. A subsequent inhibition of the proliferation of T cells occurs and
ergo, aids in maintaining T cell anergy (Walunas et al., 1994; Krummel and Allison,
1995; Greenwald et al., 2005). CTLA-4 blockade using anti-CTLA-4mAb allows the
interaction between CD28 on T cells with CD80 and CD86 present on dendritic cells,
thereby potentiating T cell activation and consequently assisting in tumour destruction
(Grosso and Jure-Kunkel, 2013). A number of animal studies have been carried out and
have demonstrated enhanced anti-tumour activity following CTLA-4 blockade;
especially when used in conjunction with a tumour vaccine (van Elsas et al., 1999;
Greenwald et al., 2005). The fully humanised monoclonal antibody, Ipilimumab
(Yervoy, Bristol-Myers Squibb, New York, NY, USA) has been approved by the FDA
for treating patients with advanced melanoma and has been found to enhance overall
survival (Hodi, 2010; Robert et al., 2011).
Therefore, the aim of this study was to examine for improved efficacy of the
combination of CTLA-4 blockade with the previously published Treg depletion/TGF-β
neutralisation (double immunotherapy) in the AE17 murine mesothelioma model. We
hypothesised that this triple immunotherapy treatment regime would result in complete
tumour clearance, associated with a sustained depletion of intra-tumoural Treg cells and
activation of cytotoxic T cells. This overall objective of inducing complete tumour
eradication was achieved.
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3.2 Results
3.2.1 Intra-tumoural administration of anti-CD25mAb together with intra-
peritoneal anti-CTLA-4mAb and sequential anti-TGF-βmAb, completely
eradicates established AE17 murine mesothelioma tumours
The efficacy of the triple immunotherapy regime by the addition of anti-
CTLA4mAb to the existing double immunotherapy (DI) was examined. A dose of
100µg of anti-CTLA-4mAb delivered intra-peritoneally (i.p) was added to the DI. This
dosage and administration route was based on a previously published protocol (Hurwitz
et al., 1998). This timed triple immunotherapy (TTI) therefore consisted of intra-
tumoural depletion of Tregs using anti-CD24mAb and systemic blocking of CTLA-4 at
treatment initiation (day0), followed by 7 consecutive days of TGF-β neutralisation
within the tumours with anti-TGF-βmAb.
DI or TTI treatments were initiated in sub-cutaneous AE17 tumour bearing mice
when the tumours reached 9mm2. Figure 3.1 shows that 46% of the mice treated with
the TTI (13/28 from 3 independent experiments) underwent complete tumour
regression. Mice receiving the DI of anti-CD25mAb and anti-TGF-βmAb alone had no
complete cures (median survival 24 days compared to untreated mice with a median
survival of 13.5 days, p<0.0001). The remaining 50% of the mice did not completely
respond to the TTI treatment (partial responders), and underwent delayed tumour
growth, reaching end-points later than untreated controls (median survival 25 days;
p<0.0001). Ergo, the TTI treatment was a substantial improvement over previous
treatments tested in our laboratory, where long-term regression of tumours was never
achieved (Kissick et al., 2012).
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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). C57BL/6J mice were implanted s.c with 1x107 AE17 cells. When the tumours reached an established size of 9mm2 (14±1 days), treatments were initiated. DI regime (■) consisted of one 2 µg i.t dose of anti-CD25mAb (given on day 0) followed by 7 daily i.t injections of 1 µg anti-TGF-βmAb. TTI regime (▲) consisted of one 2 µg i.t dose of anti-CD25mAb and one i.p 100 µg dose of anti-CTLA-4mAb concurrently (given on day 0) followed by 7 daily i.t injections of 1 µg anti-TGF-βmAb. Untreated tumours (●) were used as tumour growth controls for the double and triple treatments. Mice were monitored daily and Kaplan-Meier survival curves were plotted using tumour sizes of 100mm2 as end point (n=10 for untreated mice, n=17 for DI treated mice and n=22 for TTI; from three independent experiments).
69
3.2.2 Administration of timed triple immunotherapy into established AE17 tumours
inhibits re-accumulation of Tregs in tumour draining lymph nodes
The first step towards understanding the anti-tumour immune response elicited by
the TTI was to examine the impact on the immunosuppressive Tregs. Tumours and
tumour draining lymph nodes (TDLNs: pooled brachial, axillary and inguinal) from
C57BL/6J mice with established 9mm2 AE17 tumours treated with either DI or TTI and
were analysed by flow cytometry on days 0, 1, 3 and 10 for changes in Treg
(CD4+CD25+FoxP3+) populations. Tumours and TDLNs collected for determining Treg
cells on day 0 were from untreated tumour bearing mice before the treatment was
initiated. The population of Tregs from these untreated mice (n=6 from two independent
experiments) were used to determine baseline numbers before treatment initiation. The
DI treatment was used as control to determine the added benefit of anti-CTLA-4mAb in
the TTI.
Figure 3.2A shows that Treg numbers within tumours were not significantly different
in mice that received either DI or TTI over a period of 10 days (p=0.45, post-hoc linear
trend analysis). Interestingly, analysis of the TDLNs showed (Figure 3.2B) significant
differences between the two treatment groups. Prior to treatment, the majority of CD4+
T cells present in the TDLNs are Tregs, making up 65% of the total CD4+ T cell
population (Figure 3.2B). On day 1 post treatment with anti-CD25mAb and anti-CTLA-
4mAb, a significant reduction in total Tregs in the TDLNs to 21,824 (±7,839) was
observed in the TTI treated mice as compared to pre-treatment levels of 55,127 (±1,388)
on day 0 (P<0.01, by post hoc Tukey’s multiple comparison testing). This was close to a
60% drop in the total Treg numbers in the TDLNs of TTI treated mice. Even by day 3,
the total Tregs 21,576 (±14,772) in the TTI treated group remained significantly lower
compared to day 0 (p<0.01) and this low Treg levels continued till day 10 with only
23,715 (±12,764) cells present (p< 0.001). In the DI treated mice, though a significant
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decrease in Treg numbers (>80% reduction to 8,675 (±3,209)) one day after treatment
initiation was observed (p<0.001), this level of depletion was not maintained. By day 3,
the Treg numbers had increased by 30% to 25,356 (±10,391) and by day 10, the TDLNs
of the DI treated mice were re-populated by Tregs and were close to the pre-treatment
levels (50,290 (±9,759), p=0.97).
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Figure 3. 2. Sustained depletion of Treg cells in TDLNs by TTI treatment even 3 days post treatment completion. Tumours (Figure A) and tumour-draining lymph nodes (pooled unilateral brachial, axillary and inguinal) (Figure B) were removed from mice on days 0, 1, 3 and 10 following treatment with either DI (■) or TTI (▲) and the population of Treg cells (CD4+CD25+FoxP3+) were determined by flow cytometry. Treg numbers from untreated tumour-bearing mice were used as baseline numbers on the day of treatment initiation (day 0). (n=6 for untreated only on day 0 from two experiments, n=3 on days 1 and 3 from one experiment and n=8 for day 10 for both DI and TTI treated mice from two experiments. The errors bars represent standard deviations of the mean). Significant differences in Treg cell numbers on day 10 between DI and TTI were determined by unpaired t-test indicated by** p<0.01. Significant differences between all groups were determined by One-way ANOVA.
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3.2.2.1 TTI treatment results in greater maturation of DCs and subsequent activation
of effector T cells in the TDLNs
The effect of the TTI treatment on dendritic cells (DCs) in the TDLNs was assessed
by measuring CD80 expression. DCs were defined as CD11c+MHCII+ cells with the
CD80 expression measured in terms of fold changes in mean fluorescence intensity
(MFI) compared to untreated levels on day 0. One day post treatment initiation when
most of the Tregs had been depleted in the TDLNs; a significant (1.70 (± 0.24) fold)
increase in MFI of CD80 expression was observed in the TTI treated mice (p<0.01) as
shown in Figure 3.3 A. TTI treated mice were also found to have a 2 fold higher MFI of
CD80 expression than the DI treated mice on day 1 (p=0.002). By day 3, the level of
CD80 expression in the TTI treatment group slowly decreased (1.23 (±0.07)) and was
close to pre-treatment levels by day 6 (1.02 (±0.12)) (p=0.12). The fold changes in
CD80 expression remained constant on days 1, 3 and 6 and not significantly different to
the pre-treatment levels in the DI treatment mice (p=0.23). It was only 3 days post
treatment completion (day 10), that fold increase in CD80 expression almost doubled in
both DI (1.99 (±0.11)) and TTI (1.80 (±0.18)) treatment groups compared to the pre-
treatment levels on day 0 (1 (±0.21)) (p=0.001).
The number of CD4+ cells and CD8+ T cells in the TDLNs were also examined.
Activated helper T cell (CD4+CD25+FoxP3-) numbers were determined 1, 3, 6 and
10 days following TTI treatment in the TDLNs. With the sustained depletion of Tregs
in the TTI treated mice; Figure 3.3B shows that one day post Treg depletion, the
number of helper T cells was significantly lower than the pre-treatment levels of
73,778 (±19,256) in both DI (12,158 (±4,965)) and TTI (23,231 (±8,939)) treated
mice (p<0.001 and p<0.01, respectively). This number of helper T cells in the DI
treated mice remained significantly lower throughout the treatment period than
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untreated levels (p=0.03, post-hoc linear trend analysis) and TTI treated mice
(p=0.001).
However, in the TTI treated mice, the number of helper T cells began to increase
by day 6 to 146,329 (±73,083) to remain higher than that seen in the DI treatment mice
(38,917 (±7645)) on day 6 (p=0.02, post-hoc un-paired student t-test). A similar trend
was observed in the CD8+ T-cell population with a 2.5 fold increase in numbers on day
6 (1,007,951 (±344,234)) compared to untreated controls (438,381 (±162,170))
(p<0.01), figure 3.3C. This increase in CD8+ T cells was more enhanced in the TTI
treated mice when compared to DI over the time-period that they were analysed
(p=0.01, post-hoc linear trend analysis).
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Figure 3. 3 Enhanced CD80 expression by dendritic cells and increased effector T cell levels in mice treated with TTI. C57B/6J mice were implanted s.c with 1×107 AE17 cells and the treatment with either DI (■) or TTI (■) was initiated at a tumour size of 9mm2. Fold increases in expression of CD80 mean fluorescence intensity over untreated controls of CD11c+MHCII+ cells (A), number of CD4+CD25+FoxP3- helper T cells (B) and CD8+ T cells (C) present in the TDLNs (pooled unilateral brachial, axillary and inguinal) were removed from mice on days 1, 3, 6 and 10 post treatment and analysed by flow cytometry. Data collected from untreated tumour-bearing mice on day 0 were used as baselines for the representative populations. Error bars represent standard deviation of the mean. (n=6 for untreated only on day 0 from two experiments, n=3 on days 1 and 3 from one experiment and n=8 for day10 for both DI and TTI treated mice from two experiments) Significant differences between all groups were determined by one-way ANOVA. Significant differences between DI and TTI at the same time-points analyzed by un-paired student t-test: *p<0.05, ** p≤0.01 respectively; n.s-not significant.
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3.2.3 A specific anti-tumour memory response results from TTI treatment
Mice cured with TTI treatment remained tumour free for up to three months
post-treatment. The next step was to investigate their resistance to tumour re-challenge.
Cured mice re-challenged with a second AE17 inoculum at the original inoculation site
were found to be resistant to tumour re-challenge for a further two month period of
follow-up (Table 3.1). All re-challenged animals were observed to undergo
inflammation at the inoculation site which resolved within 2-3 days of re-challenge and
the mice remained tumour-free. This memory response was systemic as three cured
mice re-challenged on the contralateral flank also resisted tumour re-challenge. Naïve
control mice inoculated at the time of the re-challenge with the same batch of AE17
cells all grew tumours that reached end-points within the expected time frame (median
survival 17 days).
To determine if immunological memory was tumour specific, TTI cured mice
were re-challenged with B16 melanoma cells (syngeneic tumour type). Cured mice were
either re-challenged at the original inoculum site or their distal flank with B16
melanoma cells, and the tumour growths were compared to naïve C57BL/6J mice
challenged at the same time (Table 3.1). In contrast to the AE17 re-challenge, all cured
mice re-challenged with B16 cells developed end-point tumours. The B16 tumours
inoculated in the naïve mice grew within the expected time frame (median survival was
13 days). Compared to naïve mice, the TTI treated cured mice re-challenged on the
same and opposite flanks grew the B16 tumours significantly slower (median survival
of 24 (p= 0.0046) and 31 days (p=0.01) respectively), as summarised in table 3.1.
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n-number of mice challenged; Avg-average and SD- Standard deviation.
Naïve mice challenged TTI cured mice re-challenged with original AE17 cells Original site Contralateral site
n Avg tumour size (mm2) ± SD at day 20
n Avg tumour size (mm2) ± SD at day 20
n Avg tumour size (mm2) ± SD at day 20
9 57.84 ± 12.72 17 0 3 0
Naïve mice challenged TTI cured mice re-challenged with B16 melanoma Original site Contralateral site
n Avg tumour size (mm2) ± SD at day 14
n Avg tumour size (mm2) ± SD at day 14
n Avg tumour size (mm2) ± SD at day 14
3 99 ± 9 5 27.66 ± 25.08 3 18.6 ± 0.96
Table 3. 1 TTI cured mice are resistant to re-challenge with original inoculum (AE17) and partially resistant to syngeneic B16 melanoma re-challenge. AE17 tumour-bearing C57BL/6J mice cured by the TTI treatment were re-challenged 1-3 months post treatment completion with either the original AE17 (1 × 107 cells) inoculum or with B16 melanoma (5 × 105 cells) at the same inoculum site or the contralateral site. Naïve mice were challenged with the same batch of AE17 and B16 cells as controls.
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3.2.3.1 Sustained depletion of Tregs in the TDLNs of re-challenged AE17 cured mice
In response to the tumour re-challenge, accumulation of Tregs within the TDLNs
of these cured mice was investigated. The TDLNs of cured mice that have been re-
challenged with AE17 cells were analysed for Tregs (CD4+CD25+FoxP3+) by flow
cytometry 3 days post challenge. The naïve mice challenged with the same inoculum 3
days prior to the analysis were used as controls. Compared to the naïve mice with
percentage of Tregs at 50.2 ± 1.6 %, the cured mice were significantly lower (43.7 ± 2.8
%, p<0.007 by post hoc student t-test) as shown in figure 3.6. This sustained depletion
of Tregs induced by the TTI during treatment (as observed earlier in figure 3.2) is
maintained in the TDLNs of cured mice, even 1-3 months post treatment completion.
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Figure 3. 4 Sustained depletion of Tregs in the TDLNs of re-challenged cured mice. Cured mice (■) that remained tumour free 1-3 months post treatment completion were re-challenged with 1 × 107 AE17 cells. Naïve untreated mice (●) challenged with the same inoculum were used as controls. Tumour-draining lymph nodes (pooled unilateral brachial, axillary and inguinal) were removed from these mice 3 days post tumour challenge and percentage of CD4+CD25+ T cells that were also Foxp3+, were analysed by flow cytometry. (n=3 for naïve challenged and n=7 for cured re-challenged from one experiment. The errors bars represent standard deviations of the mean). Significant differences in Treg percentages between naïve and cured were determined by unpaired t-test indicated by** p<0.01.
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3.2.3.2 Induction of memory T cells by the TTI treatment
A significant increase in expression of the T cell memory marker CD44 was
observed in the cured mice (15.6 ± 4.5%) (figure 3.7A). This increase in CD44+
expression was close to double the percentage of CD44+CD4+ T cells observed in naïve
mice challenged for the first time with AE17 tumour cells (9.09 ± 4.5%, p=0.01
significance determined by post-hoc unpaired t-test). A similar doubling in percentage
of CD8 T cells co-expressing CD44 marker was also found in the cured mice (19.8 ±
6.5%); compared to the naïve challenged mice (10.17 ± 3.9%, p=0.004), as shown in
figure 3.7B. This activation of the memory response in both the CD4+ and CD8+ T cell
compartments determined 3 days post tumour re-challenge was long-lasting and
tumour-specific, as confirmed by the re-challenge experiments carried out and
summarised in table 3.2.
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Figure 3. 5 Induction of memory T cells in mice cured of AE17 murine mesothelioma by TTI treatment. Mice cured of AE17 tumours with TTI were re-challenged at the same anatomical site with AE17 cells (1 × 107 cells) 1-3 months post treatment completion. Naïve fully immune-competent C57BL/6J mice were challenged with the same batch of tumour cells and were used as controls (●). 3 days post challenge, the TDLNs (pooled unilateral brachial, axillary and inguinal) were removed from mice and analysed for the expression of the memory CD44 marker within A) CD4+ T cell (▲) and B) CD8+ T cell (♦) compartments (n=6 for naïve challenged mice and n=12 for cured re-challenged mice from two experiments respectively). Significant differences in CD44 marker expression between naïve and cured were determined by unpaired t-test indicated by** p<0.01.
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3.3 Discussion
Tumour eradication was achieved in close to 50% of mice bearing established
AE17 mesothelioma tumours treated with the TTI. This was a significant improvement
over any of the single or double immunotherapy treatments previously trialled in our
laboratory. Depletion of Tregs alone by a single anti-CD25mAb treatment only delayed
the growth of AE17 tumours, though the treatment did not confer any significant
improvement in tumour growth inhibition. Monotherapies exclusively involving just
TGF-β neutralisation or CTLA-blockade, both resulted in only a marginal improvement
in survival time (median survival of 17 and 20 days respectively) but ultimately, all the
mice grew end-point tumours (Kissick et al., 2012). Complete tumour eradication or
long-term survival of mice bearing murine mesotheliomas (AC29 and AB12) have been
previously achieved by treatment with anti-CD25mAb by other groups. However, it is
important to note that, these experiments involved systemic depletion of Tregs with anti-
CD25mAb before tumour challenge (Anraku et al., 2010). Though single treatment
approaches are ideal for certain diseases, the factors involved in immune suppression in
the presence of a growing tumour are multivariate and more complex. This supports our
hypothesis that combinatorial immunotherapy approaches improved the efficacy to
overcome tumour immune evasion.
Different double immunotherapy combinations trialled in our laboratory on
C57BL/6J mice bearing AE17 tumours showed that the increased survival time due to
significant tumour growth inhibition was greatest with the combination of TGF-β
neutralisation and Treg depletion (median survival improved to 24 days). Neither the
combinations TGF-β neutralisation/CTLA-4 blockade nor Treg depletion/CTLA-4
blockade conferred any improvement in survival (median survival of 19 days and 21
days respectively) when compared to mice that received CTLA blockade alone (median
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survival 20 days). Ultimately, long-term regression of tumours was never achieved
(Kissick et al., 2012).
Combining all three components (anti-CD25mAb, anti-TGF-βmAb and anti-
CTLA-4mAb) conferred a significant survival advantage over the best DI combination
of anti-CD25mAb and anti-TGF-βmAb. Most importantly, for the first time in our
laboratory, complete eradication of tumours in 46% of mice bearing established AE17
tumours was achieved with the novel TTI treatment (Figure 3.1). The remaining 50% of
mice that did not completely respond to the treatment (partial responders) did undergo
delayed tumour growth with increased survival time when compared to untreated mice.
This question of partial responders to the TTI could be a result of a sub-optimal
response to the treatment, and perhaps experiments involving recovery of these mice
with additional top-ups or by titrating the treatment components could enhance the cure
rate (see chapter 4 section 4.2.4).
From the previous work conducted in our lab, we knew that the DI treatment
only achieved transient tumour regression. This observation was in conjunction with the
re-accumulation of Tregs 7-10 days post depletion within the tumour microenvironment
(Kissick et al., 2010). Despite the addition of anti-CTLA-4mAb to the combination
therapy, Treg numbers were not significantly different between the DI and TTI treated
mice in the tumours as analysed by flow cytometry (Figure 3.2A). This could be
because, the treatment components that are injected intra-tumourally (anti-CD25mAB
and anti-TGF-βmAb) are not different between the DI and TTI (anti-CTLA-4mAb in
the TTI treatment is administered i.p). However, it was found that even 3 days post TTI
treatment completion; the Treg levels remained significantly lower in the TDLNs when
compared to Treg levels reaching pre-treatment levels in the TDLNs of mice that
received the DI (Figure 3.2B). This was a significant finding, as this suggested that the
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TTI treatment had a more profound impact on inhibiting the re-accumulation of Tregs
within the TDLNs (Kissick et al., 2012). Selective recruitment and expansion of Tregs in
the periphery and within other tumours has been reported in different types of cancers
(Woo et al., 2001; Liyanage et al., 2002; Wolf et al., 2003; Curiel et al., 2004). It is,
therefore, possible that the TTI approach developed here may be useful in treating other
tumours (see chapter 6).
Although it is well recognised that Treg depleting therapies can inhibit tumour
development in several murine models and human cancers; a common cause for concern
is the long-term side effects of autoimmune diseases observed with the administration
of Treg cell depletions systemically or for prolonged periods of time (Takeuchi et al.,
2004; Ruddle and Prince, 2007). Another cause for concern is the production of
neutralizing antibodies by the patient, as observed by Sterman et al (2010) with their
phase I trial of repeated IFN-γ gene transfer treatment for treating mesothelioma and
metastatic pleural effusions (Sterman et al., 2010). In relation to the novel TTI
treatment, the following observations can be made: a) by employing intra-tumoural
administration, the risk of systemic depletion of Tregs is substantially reduced. Indeed, no
auto-immune based ill-effects (such as weight loss, lethargy, reduced appetite, bloating
or diarrhoea) were observed in any of the treated mice, b) the complete cures without
the need for repeated administration suggests that neutralising antibody problems will
be similarly reduced, and c) the anti-CTLA-4mAb was systemically administered in
these experiments and hence could cause auto-immune issues. It would be appropriate
to examine the possibility of incorporating this reagent into the intra-tumoural
administration (see chapter 4).
Tregs are known to regulate the proliferation and maturation of DCs resulting in a
shift of the DC phenotype in an IL-10/TGF-β dependent manner (Kim et al., 2006;
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Onishi et al., 2008). Concurrent to the maintenance of low Tregs in the TDLNs over the
time-period of treatment with TTI, increased expression of CD80 by DCs was also
found in the TDLNs of TTI treated mice (Figure 3.3A). The increased CD80 expression
observed on day 1 in the TTI treated mice could suggest the removal of brakes on the
proliferation of DCs. This activation of DCs correlated with the expansion of helper T
cells (CD4+CD25+FoxP3-) and CD8+ T cells within the TDLNs by day 6 (Figures 3.3 B
and C respectively). The decrease in helper CD4+ T cells and CD8+ T cells observed on
day 10 could indicate the possible migration of DC primed CD4+ and CD8+ T cells into
the tumour microenvironment, as the TTI treatment induced tumour clearance in the
tumour-bearing mice. Flow cytometric analysis for the detection of these subsets of T
cells within the tumours would aid in confirming the migration of these anti-tumour
effector cells into growing tumours. Also, it is important to note that CD80 is just one of
many markers that are expressed on DCs. Future experiments that examine other
maturation and proliferation markers such as CD86, CD83 and CD40 would further
strengthen the phenomenon observed. Another option is to analyse concentrations of IL-
12 that are secreted by DCs and are necessary for the differentiation of activated T cells
into T helper cells.
The TTI treated mice that underwent complete tumour regression were kept for
between 1-3 months with no re-emergence of tumours. Development of immunological
memory in these mice was substantiated with these long-term cured mice failing to
develop tumours when re-challenged with the same inoculum of AE17 tumour cells
(Table 3.1). These re-challenged mice remained tumour free for a further two months
post re-challenge, irrespective of site of inoculum, thereby suggesting systemic immune
memory. This emergence of resistance to re-challenge in the TTI treated long-term
cured mice denotes the first reversal of dominant tolerance within the tumour
microenvironment.
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In response to tumour re-challenge, a significant doubling in the percentage of
CD4+ and CD8+ T cells co-expressing the memory CD44 marker was seen in the
TDLNs of TTI-cured mice, just 3 days after tumour re-challenge, when compared to
untreated, naive mice challenged for the first time with a single inoculum of AE17
tumour cells (Figure 3.7 A and B). Preliminary analysis of the tumour implantation site
of three of the TTI cured and re-challenged mice showed a rapid accumulation of CD4+
T cells at the re-challenge site just 3 days after tumour challenge. This was not observed
in the untreated naive mice implanted for the first time with tumour cells (data not
shown).
The systemic immunity elicited in the cured mice was found to be tumour-
specific, as re-challenge with B16 melanoma (syngeneic tumour model for the
C57BL/6J) resulted in tumour growth. Though the B16 tumours grew in these AE17
cured mice, their growth to end-point was significantly slower compared to their growth
in untreated naïve mice. This could be indicative of some partial immunity generated in
the cured mice towards the melanoma. Melanoma and mesothelioma are both cancers
arising from squamous epithelial cells, and perhaps share common tumour antigens
(Rivera et al., 2012). This could be the reason for the slow growth of melanoma
tumours in the cured mice. Perhaps re-challenge with a different syngeneic tumour cell
line such as EO771 breast carcinoma could help in understanding this notion of partial
immunity in the future.
Together, these data support the hypothesis that targeting multiple mechanisms
of immune suppression induces a superior anti-tumour immune response. Data from
these experiments have provided evidence that long-term tumour eradication can be
achieved, with continued resistance to tumour re-challenge when various aspects of
immune suppressive mechanisms are targeted. More importantly, all treatments used in
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this triple combination approach have human equivalents, that have been approved for
treating cancer patients. Denileukin Diftitox (DD), a recombinant fusion protein that
consists of peptide sequences of diphtheria toxin and human IL-2, is a FDA approved
drug for depleting Tregs in different cancers (Dannull et al., 2005; Dang et al., 2007;
Mahnke et al., 2007; Gerena-Lewis et al., 2009). Moreover, as mentioned earlier,
Ipilimumab (anti-CTLA-4mAb) has been approved for treating patients with advanced
melanoma (Hodi, 2010; Robert et al., 2011).
The concept of targeting multiple mechanisms of immune suppression is
evidenced in the literature, with combination therapies involving more than two
components being tested in various other laboratories. For example, combination
therapy involving 4-1BB (CD137) activation with anti-CTLA-4mAb treatment given as
a triple therapy in the setting of radiation therapy was found to improve survival of mice
with glioblastomas (Belcaid et al., 2014). More recently, a phase I clinical trial was
reported, that involved administering castration-resistant prostate cancer patients with
Ipilimumab, vaccine containing transgenes for prostate-specific antigen (PSA) and a
triad of co-stimulatory molecules (PROSTVAC). 30 patients were trialled, and these
patients were found to have increased overall survival (OS) (Jochems et al., 2014).
Combinatorial therapies are leading the way into the future of cancer treatments.
Coupled with the fact that current mesothelioma treatments are of low efficacy and
provide minimal improvements in survival rates, makes the TTI a promising new
development. Testing the efficacy of the TTI in a different murine mesothelioma model
would assist in translation of this novel TTI for treating human patients with
mesothelioma. Based on this, the following chapter will investigate the efficacy of the
TTI in the AB1 (BALB/c) murine mesothelioma model.
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CHAPTER 4 Improved efficacy of the triple immunotherapy in the non-TGF-β
secreting murine mesothelioma model (AB1 model)
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4.1 Introduction
The previous research in the AE17 murine mesothelioma model showed that the
timed triple immunotherapy (TTI) completely eradicated tumours in close to 50% of the
treated C57BL/6J mice. This was a significant improvement, as tumour eradication has
never been achieved with single or double immunotherapies (DI) trialled in our
laboratory. Long-term survival of treated mice was achieved with tumours failing to re-
grow post cessation of treatment. Development of immunological memory was
confirmed by flow cytometric analyses of memory T cells in TDLNs, and with the
continued resistance of the cured mice to AE17 tumour re-challenge. This denotes a
reversal of the dominant tolerogenic immune-surveillance present in the tumour
microenvironment and is particularly impressive, given the ability of the AE17 tumour
cells to secrete TGF-β that is known to recruit and/or convert Tregs (Chen et al., 2003).
In this chapter the TTI approach was applied to the BALB/c murine mesothelioma
(MM) tumour model, AB1. AB1 tumours were derived from the peritoneal cavity of H-
2d BALB/c mice injected with crocidolite asbestos fibres. Ascites, solid tumours or
peritoneal flushing were collected from the mice and grown in-vitro, where they were
serially passaged to derive the MM tumour cell line (Davis et al., 1992).
Immunohistological staining of intra-peritoneal AB1 tumour biopsies have already
provided phenotypic evidence for the increased accumulation of regulatory T cells
(CD4+CD25+FoxP3+) within growing tumours (Veltman et al., 2010). The AB1 MM
has also shown sensitivity to immunotherapy (Caminschi et al., 1998; Hegmans et al.,
2005; Mahaweni et al., 2013). Moreover, the tumour cells themselves are known to not
secrete the immunosuppressive cytokine TGF-β (Fitzpatrick et al., 1994). It was,
therefore, hypothesised that the TTI approach would induce significantly greater
percentage of AB1 tumour bearing mice to undergo complete tumour clearance as these
tumours are non-TGF-β secreting.
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Experiments described in this chapter aimed a) to study the efficacy of the triple
immunotherapy treatment in a murine mesothelioma that does not secrete TGF-β (AB1)
b) to examine the importance of timing in the delivery of the three immunotherapeutic
agents and c) to improve the 50% cure rate achieved in the AE17 murine mesothelioma
model.
4.2 Results
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
AB1 mesothelioma cells were subcutaneously injected into BALB/c mice and
the TTI treatment consisting of anti-CD25mAb, anti-TGF-βmAb and anti-CTLA-4mAb
was initiated when tumours reached 9mm2; as described for AE17 tumours in the
previous chapter and in Kissick et al, 2012. Immediately post treatment, a period of
local inflammation at the tumour site was observed with tumours swelling to a
maximum size of 15.4 (±4.8) mm2 in the TTI treated mice on day 3 (Figure 4.1). By
day 10 (3 days post treatment completion), no palpable tumour could be felt at the
inoculation site of the TTI treated mice. This was significantly different to the tumours
growing in the untreated controls which had an average size of 28.6 (±11.6) mm2
(p=0.001 significance determined by un-paired student t-test). These TTI treated mice
remained free of tumours for at least 3 months post-treatment completion.
This timed treatment of established AB1 mesotheliomas was trialled in 16 mice,
and figure 4.2 shows that 100% of animals (16/16, from three independent experiments)
underwent complete tumour clearance, compared to untreated controls (median
untreated survival 19 days; p<0.0001).
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Figure 4.1 Complete tumour eradication in 100% of AB1 tumour bearing BALB/c mice treated with TTI. BALB/c mice were implanted s.c with 1 × 106 AB1 tumour cells. TTI was initiated at a tumour size of 9mm2 (7 ± 1 days). TTI regime (▲) consisted of one 2 µg i.t dose of anti-CD25mAb and one i.p 100 µg dose of anti-CTLA-4mAb concurrently (given on day 0) followed by 7 daily i.t injections of 1 µg anti-TGF-βmAb. Untreated tumours (●) were used as tumour growth controls. (n=3 for untreated mice and n=5 for TTI; from one experiment). The data are shown as mean ± SD.
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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
Clinically, a single immunotherapy treatment regime would be easier to
implement than a timed daily dose regimen. A combined triple immunotherapy cocktail
(TIC) delivered directly into the established 9mm2 tumours via a single injection was,
therefore, tested. The TIC for intra-tumoural administration combined the anti-
CD25mAb at the same total concentration employed in TTI, the anti-TGF-βmAb at 2
µg instead of 1 µg and the anti-CTLA-4mAb at 2 µg reduced from the 100 µg
administered intra-peritoneally. The TIC was developed to test the importance of timed
component administration relative to the simplicity of a single injection. Figures 4.2A
and B show that a single intra-tumoural injection of the TIC was as efficient as the TTI
(p=0.1636), with complete tumour clearance seen in 88% of treated mice (15/17 mice
over three independent experiments) compared to untreated controls (p<0.0001)
(Krishnan et al., 2014).
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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). Mice were implanted s.c. with 1 × 106 AB1 tumour cells. The triple immunotherapy, either TTI (▲) or TIC (■), was initiated at a tumour size of 9 mm² with tumour growth compared to untreated mice (●). Mice were monitored daily for (A) survival and plotted as Kaplan-Meier survival curve with 100mm2 tumour as endpoint and (B) tumour growth (mean ± SD) was calculated with measurements made using microcalipers. The pooled data are from three independent experiments (n=16 for untreated mice and TTI treated mice and n=17 for TIC treated mice).
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4.2.3 Induction of systemic immune response in cured mice
Mice treated successfully with TTI were kept 1-3 months post treatment
completion, and all tumours failed to regrow. These long-term cured mice also failed to
re-grow tumours when re-challenged with the same inoculum of AB1 cells at either the
original or contralateral inoculation sites (Table 4.1). In comparison, 100% of control
naïve BALB/c mice inoculated with the same batch of AB1 cells grew tumours to
endpoint (100mm2) (Table 4.1).
Similar to mice cured by the TTI; Table 4.1 shows that BALB/c mice cured by
the TIC failed to regrow tumours when challenged on the same flank with AB1 tumour
cells one month after complete tumour clearance. Naïve BALB/c mice were inoculated
with AB1 cells at the same time as the re-challenge, and all grew end-point tumours.
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n-number of mice challenged; Avg-average; SD- Standard deviation; and ND-not done.
Naïve mice challenged TTI cured mice re-challenged Original site Contralateral site
n Avg tumour size (mm2) ± SD at day 20
n Avg tumour size (mm2) ± SD at day 20
n Avg tumour size (mm2) ± SD at day 20
9 57.84 ± 12.72 11 0 3 0
Naïve mice challenged TIC cured mice re-challenged Original site Contralateral site
n Avg tumour size (mm2) ± SD at day 20
n Avg tumour size (mm2) ± SD at day 20
n Avg tumour size (mm2) ± SD at day 20
6 54.54 ± 6.91 5 0 ND ND
Table 4. 1 BALB/c mice cured of established AB1 tumours with TTI or TIC are resistant to re-challenge. AB1 tumour-bearing mice cured by TTI or TIC treatment were re-challenged 1-3 months after complete tumour clearance with 1 × 106 AB1 cells at the same inoculum site or the contralateral site. Naïve mice were challenged with the same batch of AB1 cells as controls.
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4.2.3.1 Susceptibility of cured mice to re-challenge with syngeneic alternate tumour
type-4T1 breast carcinoma
To establish whether this immunological memory in the cured mice was tumour
specific, mice cured of AB1 tumours by TIC treatment were re-challenged with
syngeneic 4T1 breast carcinoma cells. Similar to the re-challenge experiment described
above, the TIC cured mice were either re-challenged at the original inoculum site or the
contralateral site with 4T1 breast carcinoma cells. Naïve BALB/c mice were also
inoculated at the same time as the re-challenge (Table 4.2). All TIC cured mice re-
challenged with syngeneic 4T1 cells, developed tumours irrespective of whether they
were challenged on the original or contralateral flanks. 20 days post 4T1 tumour cell
inoculation; compared with tumour growth in naïve BALB/c mice challenged with 4T1
cell, no significant difference in tumour growth was observed between mice injected on
the original site or contralateral sites (p=0.23 and p=0.208, respectively).
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n-number of mice challenged; Avg-average; SD- Standard deviation.
Naïve mice challenged TIC cured mice re-challenged with 4T1 cells
Original site Contralateral site
n Avg tumour size (mm2) ± SD at day 20
n Avg tumour size (mm2) ± SD at day
20
n Avg tumour size (mm2) ± SD at
day 20
3
40.33 ± 9.60
3
62.66 ± 26.02
2
52.50 ± 4.94
Table 4. 2 Immunological memory generated in BALB/c mice cured of established AB1 tumours with TIC are tumour specific. AB1 tumour-bearing mice cured by TIC treatment were re-challenged 1 month after complete tumour clearance with 1 × 105 4T1 cells at the same inoculum site or the contralateral site. Naïve mice were challenged with the same batch of 4T1 cells as controls.
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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.
Compared to only 50% survival in the TGF-β secreting AE17 mesothelioma
model (Chapter 3 and Kissick et al., 2012), complete tumour clearance in 100% of mice
treated with TTI in the AB1 murine mesothelioma model was achieved (Krishnan et al.,
2014). The improved outcome seemed most likely associated with the lack of TGF-β
secretion from the AB1 murine mesothelioma cells (Fitzpatrick et al., 1994). This
formed the basis for the next set of experiments; that aimed at improving the 50% cure
rate of AE17 tumour bearing C57BL/6J mice treated with TTI. The 50% of TTI treated
mice that grew end-point tumours were called partial responders, as they had a median
survival of 25 days and had a significantly delayed time to death (TTD) when compared
to UT controls (median survival 13.5 days, p<0.0001)).
The time-point at which TTI treated mice, based on the response to the TTI
treatment, differentiated into responders and partial responders, was investigated. Once
the treatment regime of 8 days was completed, due to the local inflammation at the
tumour site, tumours swelled to a maximum size of 8 (±2) mm2 in the TTI treated mice
on day 7. By day 10, the swelling continued and reached a maximum of 10 (±3) mm2 as
shown in figure 4.3A. At this point, the tumour sizes in both the TTI and DI treated
mice were similar and were significantly different to the untreated controls (p=0.001
and p=0.01 respectively, significance determined by un-paired student t-test,
respectively).
The threshold, wherein the separation within the TTI treated mice into a)
responders that undergo complete tumour clearance and b) the partial responders, took
place 3 days post-treatment completion (day 10). It is at this point in the TTI treated
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mice, that the tumours either gradually decreased in size (3/5, 60%) and no palpable
tumours were detectable (responders); or increased in size (2/5, 40%) and eventually
reached end-point tumours (partial-responders) as shown in figure 4.3B. Though the
partial-responders reached end-points significantly slower than untreated controls as
mentioned earlier, their TTD was not significantly different from DI treated mice
(median survival 25 days, p=0.27 significance determined by log-rank test).
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Figure 4. 3 Tumour growth of partial responders to the TTI treatment is not significantly different from the DI treated mice. C57BL/6J mice were inoculated s.c with 1 × 107 AE17 cells and treatments were initiated when the tumours reached 9mm2 (14 ±1 days). A) Growth of AE17 tumours in TTI (■) (n=5) and DI (▲) (n=5) treated mice post treatment initiation compared to UT controls (●) (n=3). B) Growth of AE17 tumours in TTI diverged into responders (▼) (n=3) and TTI partial responders (♦) (n=2) compared to DI and UT controls. Treatment with DI consisted of one i.t dose of anti-CD25mAb (given on day 0) followed by 7 daily i.t injections of anti-TGF-βmAb. TTI regime consisted of one i.t dose of anti-CD25mAb and one i.p dose of anti-CTLA-4mAb concurrently (given on day 0) followed by 7 daily i.t injections of anti-TGF-βmAb. Tumour growths were monitored daily with a tumour size of 100 mm2 considered as end-point. The data are shown as mean ± SD.
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In a separate experiment, the immunomodulatory environment within the
TDLNs of partial responders (average tumour size of 28 (±8) mm2) was investigated, in
comparison to the untreated end-point tumour bearing controls (average tumour size of
68 (±21) mm2). The immune responses elicited in the partial responders were not
significantly different from the untreated controls (Table 3.1). These results demonstrate
that despite the increased TTD in the partial responders, the activation of immune cells
in these mice is nevertheless sub-optimal.
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Immune cells in TDLNs Total number of cells (Avg ± SD) Significance
p<0.05 Partial
responders
(n=3)
UT controls (n=3)
CD4+CD25+ T cells 38,242 ± 7,458
28,773 ± 5, 938 0.16 (n.s)
CD8+ T cells 263,913±
144,896
387,378 ± 46,135 0.27 (n.s)
CD11c+MHCII+ cells 64,745 ± 29,761 89,386 ± 95,675 0.69 (n.s)
n, number of mice analysed; Avg, average; SD, Standard deviation; n.s-not significant
Table 4. 3 No significant improvement in immune cell numbers in partial responders. TDLNs (pooled unilateral brachial, axillary and inguinal) were removed from TTI treated partial responders and total number of CD4+CD25+ T cells, CD8+ T cells and CD11c+MHCII+ dendritic cells were analysed by flow cytometry and compared with the respective populations present in untreated end-point tumour bearing controls. Significant differences in immune cell numbers between partial responders and untreated controls were determined by t-test.
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4.2.4.1 Sub-optimal response observed in partial responders despite attempts at
recovering them with additional top-up of immunotherapy treatment
With the TTD of partial responders being similar to DI treated mice, the idea
that perhaps this was due to a sub-optimal treatment needed to be investigated. In order
to do that, an experimental protocol involving treatment of tumour bearing mice at
9mm2 with the normal TTI treatment followed by either a single i.p shot of anti-CTLA-
4mAb on day 10 (figure 3.5A) or secondary regimen of i.p anti-CTLA-4mAb and
sequential TGF-βmAb dosages (i.t) starting from day 10 (figure 3.5B) was assessed. A
second dose of anti-CD25mAb was not included in the secondary treatment protocol; as
previous work in our laboratory has shown that excess depletion with anti-CD25mAb
within the tumour microenvironment accelerated tumour growth (Kissick et al., 2009).
Figures 3.5A and C show that the mice that received the additional top-up of a
single i.p injection of anti-CTLA-4mAb had a median survival of 33 days which was
significantly longer than UT controls (median survival 17 days, p=0.01). Though their
survival was longer (by 16 days) than the untreated mice, only 50% (3/6 from one
experiment) of the treated mice underwent complete tumour regression.
A similar trend was observed in the mice treated with TTI coupled to the
secondary regimen of single i.p treatment with anti-CTLA-4mAb on day 10 followed by
7 daily i.t injections of anti-TGF-βmAb. Though their median survival (40.5 days) was
significantly longer than vehicle (median survival 16 days, p<0.01) or UT controls
(p<0.01); only 50% of the treated mice (3/6 from one experiment) underwent complete
tumour regression (Figures 3.5 B and C). Also, the survival of the secondary
administration group was not significantly different from the group that received just a
single additional top-up dose of anti-CTLA-4mAb (p=0.82). With regards to the vehicle
control group, the additional i.t injections of PBS within the tumours made no
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difference to tumour growth as their median survival was not significantly different
from UT controls (p=0.68).
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109
Figure 4. 4 Failed recovery of partial responders despite attempts with secondary round of immunotherapy. C57BL/6J mice were inoculated s.c with 1 × 107 AE17 cells and primary treatments were initiated when the tumours reached 9mm2 (14 ±1 days). (A) TTI treated mice received secondary single i.p dose of anti-CTLA-4mAb alone (▲) (n=6) on day 10 and their tumour growths were compared with UT controls (●) (n=3). (B) TTI treated mice received secondary regime consisting of one i.p dose of anti-CTLA-4mAb (given on day 10) followed by 7 daily i.t injections of anti-TGF-βmAb (▲) (n=6). Their tumour growths were compared with UT controls and vehicle control mice (■) (n=3). Vehicle control mice received 8 100 µl i.t injections of PBS from day 0-day 8, followed by only 5 100 µl i.t injections of PBS from day10-14 as the tumour sites were too inflamed to complete the remaining 3 i.t injections. C) Tumour growth was monitored daily and Kaplan-Meier survival curves were plotted using tumours with an end-point of 100mm2. The data are from one experiment and are shown as mean ± SD. Unpaired t-test were used to determine significant differences in tumour growth on day 18 with *p<0.05 between UT controls and TTI+1 anti-CTLA-4mAb treated group and * p<0.05 between vehicle controls and TTI +1 anti-CTLA-4mAb+ 7 anti-TGF-βmAb treated group.
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4.2.4.2 Increased anti-TGF-βmAb dosage in the original TTI results in complete
tumour eradication in AE17 tumour bearing mice
Based on the conclusions of the previous experiment, it was hypothesised that
targeting the TGF-β early with double the dosage could have a more profound effect on
neutralisation than a second round of late treatments.
C57BL/6J mice with established 9mm2 AE17 tumours were treated with the
modified TTI (mTTI); that consisted of anti-CD25mAb and anti-CTLA-4mAb at the
same total concentrations as before, but anti-TGF-βmAb was increased from 1 µg to 2
µg over the initial 7 day treatment period. After treatment initiation, tumours swelled to
a maximum size of 17.50 (±5.58) mm2 in the mTTI treated mice on day 4 (Figure 4.5).
By day 10 (3 days post treatment completion), tumours had reduced to an average size
of 4 (±0.70) mm2 and were barely palpable at the inoculation site of the treated mice.
This was found to be significantly different to the untreated controls which had an
average tumour size of 97.2 (±4.5) mm2 (end-point tumour size) (p<0.0001 significance
determined by un-paired student t-test).
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Figure 4. 5 Complete tumour eradication in 100% of AE17 tumour bearing C57BL/6J mice treated with mTTI. C57BL/6J mice were implanted s.c with 1 × 107 AE17 tumour cells. Modified TTI (mTTI) was initiated at a tumour size of 9mm2. mTTI regime (▲) consisted of one 2 µg i.t dose of anti-CD25mAb and one i.p 100 µg dose of anti-CTLA-4mAb concurrently (given on day 0) followed by 7 daily i.t injections of 2 µg anti-TGF-βmAb. Mice with untreated tumours (●) were used as tumour growth controls. (n=7 for untreated mice and n=11 for mTTI; from two experiment). The data are shown as mean ± SD.
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4.2.4.2.1 Induction of systemic immune response in the mice cured using mTTI
Mice that were cured successfully of AE17 tumours with mTTI were kept for
four weeks post-treatment completion, and all mice failed to regrow tumours. These
cured mice also failed to re-grow tumours when re-challenged with the same inoculum
of AE17 cells at either the original or contralateral inoculation sites (Table 4.4). In
comparison, 100% of control naïve C57B/6J mice inoculated with the same batch of
AE17 cells grew tumours to endpoint (100mm2) (Table 4.4).
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n-number of mice challenged; Avg-average; SD- Standard deviation.
Naïve mice challenged mTTI cured mice re-challenged with AE17 cells
Original site Contralateral site
n Avg tumour size (mm2) ± SD at day 9
n Avg tumour size (mm2) ± SD at
day 9
n Avg tumour size (mm2) ± SD at
day 9
6
79.70 ± 13.90
8
0
3
0
Table 4. 4 Mice cured of established AE17 tumours with mTTI are resistant to re-challenge. AE17 tumour-bearing C57BL/6J mice cured by mTTI treatment were re-challenged 4 weeks after complete tumour clearance with 1 × 107 AE17 cells at the same inoculum site or the contralateral site. Naïve mice were challenged with the same batch of AE17 cells as controls.
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4.3 Discussion
The work described in this chapter has demonstrated complete tumour clearance in
100% of mice treated with the TTI in the non-TGF-β secreting AB1 murine
mesothelioma model (Figure 4.1), compared to the 50% cures observed in the TGF-β
secreting AE17 mesothelioma model (Chapter 3 and Kissick et al., 2012). This supports
the hypothesis, as the TTI approach induced significantly greater percentage of tumour
bearing BALB/c mice to undergo complete tumour clearance in the non-TGF-β
secreting AB1 mesothelioma model. The improved outcome is most likely associated
with the lack of TGF-β secretion from the AB1 murine mesothelioma cells (Fitzpatrick
et al., 1994). Increasing the anti-TGF-βmAb dosage used in the mTTI (modified TTI)
improved the outcome in the AE17 model to 100% (Figure 4.5). The reduced levels of
TGF-β in the AB1 tumour microenvironment probably also resulted in lower levels of
Treg accumulation within these tumours (see Chapter 6). This may also be a factor
contributing to the 100% tumour clearance rate when using far less anti-TGF-βmAb in
the TTI.
It was initially thought that the efficacy of the TTI was dependent on the precise
timing of the TGF-β neutralisation injections, which consisted of 7 separate injections
given consecutively for 7 days. However, with the lack of TGF-β secretions by the
AB1 tumours themselves, the TIC treatment was devised where all three components
were administered directly into the AB1 MM tumours as a single injection. The TIC
was as effective as the TTI in the AB1 model with 88% of mice undergoing complete
tumour eradication (Figures 4.2 A and B). This suggested that timing based
administration of immunotherapy was not critical in the AB1 MM model and that, this
single intra-tumoural administration of the TIC was sufficient for overcoming immune
suppression and reactivating anti-tumour immunity within the tumour
microenvironment (Krishnan et al., 2014). It should be noted that the high effectiveness
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of TIC in the AB1 MM model resulted in no control studies. Specifically, it cannot be
excluded that a double immunotherapy cocktail might not be as effective as the triple
immunotherapy cocktail.
In order to develop the triple immunotherapy cocktail (TIC), anti-TGF-βmAb
dosage was increased to allow for the single intra-tumoural administration and their
sustained presence within the tumour microenvironment. The concentration of anti-
CTLA-4mAb for the TIC was reduced significantly (as it was modified from systemic
to local delivery) from the dosage used previously in the timed triple immunotherapy
(TTI) (Kissick et al., 2012). Changing the route of administration of anti-CTLA-4mAb
from i.p to i.t was to overcome the systemic toxicities that have been reported and
particularly associated with anti-CTLA-4mAb treatment in humans. Severe systemic
toxicities associated with the intravenous administrations that have been reported
include neutropenia, diarrhoea, rash, colitis and transaminitis in over 60% of the
melanoma patients in different clinical trials (Hodi et al., 2010; Graziani et al., 2012;
Madan et al., 2012). A single intra-tumoural administration of this mAb, either alone or
in combination with other immune modifiers as shown here, may reduce the risk of
these systemic side effects. The mice were routinely monitored for side effects to the
therapy including weight loss, lethargy, decreased appetite, bloating and diarrhoea. To
date, no ill-effects to either the TTI or TIC have been observed in the treated mice.
Intra-tumoural administration of a combined immunotherapy has also been recently
shown in a mouse brain tumour model (vom Berg et al., 2013). In this work, the serious
adverse events seen with systemic IL-12 delivery were avoided by intra-tumoural
delivery. Furthermore, when combined with systemic anti-CTLA-4mAb, the glioma
rejection was so efficient, that the rapid translation to human clinical trials was called
for. Clinical combined immunotherapies for cancer are, however, in their infancy. The
single published example at the time of writing (Wolchok et al., 2013) used fully
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humanised antibodies against PD-1 and CTLA-4. These antibodies have complementary
roles, in that, anti-PD-1mAb contributes to T cells avoiding programmed cell death and
anti-CTLA-4mAb blocks attenuation of effector T cell activity. Rapid and substantial
tumour regression was seen in advanced melanoma patients, and these responses were
deemed superior to either agent used in monotherapy.
Long-term clearance of AB1 tumours was also achieved in the mice that were
treated with either the TTI or TIC treatments. Cured mice were also protected from
further re-challenges with the original inoculum (Table 4.1). Notably, although these
cured mice were resistant to tumour relapse or re-challenge with the same tumour, they
were found to be susceptible to challenges with a different syngeneic tumour type, as
demonstrated with the growth of 4T1 breast carcinoma tumours (Table 4.2). This
indicates specificity of the immunological memory. Previous work by Twitty et al
(2011) reported that mice vaccinated with short-lived tumour-specific antigens elicited
an effective cross-protection against sarcoma tumour challenge in these mice (Twitty et
al., 2011). However, in this study, the TIC treatment could not induce effective cross-
protection against 4T1 tumour challenge in the AB1 cured mice. This absence of cross-
protection could be explained by the fact that mammary carcinomas arise from
glandular epithelial cells, whereas mesotheliomas arise from squamous epithelial cells
and the tumour associated antigens (TAA) could be distinct between the two tumour
types. This question of whether there are any common TAAs between the two groups
can be confirmed by detecting tumour antigen-specific antibodies in the TTI or TIC
cured mice sera by ELISA (see chapter 5, section 5.2.4.1).
Despite the depletion of Tregs by anti-CD25mAb, neutralization of existing TGF-β
by anti-TGF-βmAb and the boost to T cell activity by anti-CTLA-4mAb, the TTI
treatment only effectively eradicated tumours in 50% of the TGF-β secreting AE17
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mesothelioma model at the initial anti-TGF-βmAb concentration. Day 10 (3 days post
first round of TTI treatment) formed a threshold at which point the differentiation
between mice responding to the treatment and partial responders occurred (Figure 4.3
B). The recovery of these partial responders was not successful even with the additional
supplementation of top-up treatments at day 10 (Figure 4.4 A and B). This research
demonstrated that it was hard to recover the mice that did not respond to the treatment,
once the tumours grew past the threshold. A preliminary analysis was conducted on
mice bearing AE17 tumours, where the treatment was initiated at a tumour size of
25mm2 and at this size, only 20% survival was achieved (1/5 from one experiment)
(data not shown). This inability to reject larger tumour burden (tumour size of 25mm2)
further supports the idea that once the tumours grow beyond a threshold point, it is
harder to achieve tumour eradication. The inability to reject significant tumour burden is
supported by work undertaken by other groups, including pre-clinical trials on B16-
OVA melanomas lung metastases, CMS5 fibrosarcomas and AE17 mesotheliomas
model. Therapies and/or adoptive transfer studies conducted on the above mentioned
tumour models were found to be less effective against large tumour burdens
(Dobrzanski et al., 2000; Hanson et al., 2000; Jackaman et al., 2008).
This led to testing the efficacy of the TTI by doubling the dosage of anti-TGF-
βmAb for sustained presence within the tumour microenvironment. This would also
help avoid the second round of consecutive injections as translation of such a
complicated treatment protocol into the clinic will be difficult. Also, the prolonged
treatment could likewise lead to the production of neutralising antibodies, which could
make the treatment ineffective. One such example, for the production of neutralizing
antibodies against repeated treatments, making them less potent, was reported in the
phase I trial of IFN-γ gene transfer therapy for patients with mesothelioma and
metastatic pleural effusions(Sterman et al., 2010).
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Treatment of established AE17 tumour-bearing mice with the modified TTI (mTTI)
with double the anti-TGF-βmAb dosage proved to be highly effective, with 100% of the
mice undergoing complete tumour eradication. This data suggested that the increased
dosage of anti-TGF-βmAb in the mTTI treatment aided in sustained neutralisation of
TGF-β within the tumour microenvironment. This also suggests that the rejection of
MM tumours is anti-TGF-βmAb dose dependent, and ergo, complete tumour
eradication of established AE17 tumours was achieved.
Patients presenting to the clinic would be in various stages of the disease
progression with varying tumour mass. With the ability of TTI, TIC and mTTI to treat
9mm2 tumours raises questions such as a) what about larger tumours, b) the TGF-β
present within these larger tumours and c) ability of the injected material to diffuse
through a larger tumour? This leads to future experiments that could involve titration
studies of anti-TGF-βmAb in larger tumours, increase in treatment volume which could
aid in better diffusion of the treatment within the large tumours and perhaps even
multiple injections at different angles for the efficient delivery of the treatment intra-
tumourally.
Another option for the future testing of the triple immunotherapy include testing
their efficacy in a more clinically relevant model. There are multiple studies involving
sub-cutaneous injections of the murine mesothelioma tumour cells, including the work
performed in our laboratory (Jackaman et al., 2003; Needham et al., 2006; Jackaman
and Nelson, 2012; Kissick et al., 2012). This is advantageous because sub-cutaneous
growth allows the direct monitoring of tumour size and easier delivery of intra-tumoural
therapy treatments. However, this does not approximate the patterns of tumour growth
or sensitivity to the treatments of intra-thoracic tumours (Nakataki et al., 2006). Using a
mouse model that mimics natural anatomical location and a tumour microenvironment
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similar to the human disease would allow for better translation of the triple
immunotherapy for human clinical trials. All the experiments that were performed on
the s.c model of mesothelioma can also be conducted on the MM tumour cell lines that
are grown i.p (intra-peritoneally) in the same mice backgrounds. The i.p model is
regarded as being closer to the human pleural cavity area of mesotheliomas and several
such studies have been done on the i.p MM AE17 murine models previously (van
Bruggen et al., 2005; Andujar et al., 2007). Intra-peritoneal tumours produce ascites with
local growth and invasion without metastases, and are associated with cachexia reminiscent
of human MM disease progression, which can commonly occur in the peritoneum
(Bielefeldt-Ohmann et al., 1994). The i.p model of murine mesothelioma allows for the
systemic study of the efficacy of new therapies in a model with more nodular and diffused
tumours reminiscent of the clinically defined disease. One such i.p model that can be used is
the AB1-DsRed MM model. In this i.p model, the AB1 tumour cell line is transfected with
pDsRed2-1 (Clontech) which encodes for DsRed2. DsRed is a variant engineered for faster
maturation, lower non-specific aggregation and higher expression in mammalian cells. An
investigation of efficacy of the triple immunotherapy in this murine i.p model of
mesothelioma (a more clinically relevant model) could aid in the clinical translatability of
this preclinical research.
Chapter 3 detailed the role of T cells (both CD4+ and CD8+) in the tumour
eradication mediated by TTI treatment in the AE17 murine mesothelioma model. The
role of B cells in tumour eradication has not been previously investigated in our
laboratory. A small amount of literature suggests that B cells positively enhance cellular
immune responses by serving as APCs, which can lead to tumour-specific cytotoxic T
cell activation and aid in the subsequent production of antibodies that contribute
modestly to anti-tumour immunity (Manson, 1994; Crawford et al., 2006; DiLillo et al.,
2010b; Jackaman et al., 2010). Antibodies have been reported (in both animal tumour
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models and in cancer patients) to recognise tumour antigens present on the surface of
cells, as well as intracellular antigens (Canevari et al., 1996; Disis et al., 1997). More
specifically, previous findings by Jackaman et al (2010) suggested a role for B cells in a
partially successful CD40 based immunotherapy against the two MM models used in
the present study (AE17 and AB1 murine mesotheliomas) (Jackaman et al., 2010). The
next step as described in the subsequent chapter dealt with the analysis of tumour
specific IgG antibody responses investigated in both the AE17 and AB1 tumour models
and the importance of these IgG antibodies during tumour eradication. The involvement
of B cells in tumour eradication as previously reported by Jackaman et al (2010) was
also investigated in the AB1 tumour model.
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Chapter 5 Evidence of B cell involvement in triple immunotherapy
122
123
5.1 Introduction
The role of B cells in anti-tumour activity is not well understood. B lymphocytes are
effector cells of the humoral immunity, a subsystem of the acquired immune response.
The primary functions of B cells include performing the role of antigen presenting cells
(APCs), production of cytokines, differentiation into antibody-secreting plasma cells or
developing into memory B cells post-antigen interaction (Mauri and Bosma, 2012).
Analysis of existing literature has shown conflicting data on the role of B cells in
tumour immunity.
Several studies have shown that B cells hamper the anti-tumour immune response.
Qin et al (1998) showed that the immunisation with irradiated tumour cells in B cell
deficient mice (µMT) provided protection against spontaneous mammary
adenocarcinoma development. Moreover, B cell deficiency in these µMT mice was
found to enhance T cell (CD4+ and CD8+) mediated tumour immunity (Qin et al.,
1998). Similarly, µMT mice were shown to have enhanced resistance to thymoma and
colon carcinoma tumour challenges when compared to their wild-type counterparts.
Delayed growth of melanoma tumours was also observed in these µMT mice in the
same study (Shah et al., 2005). Regulatory B cells (Bregs) have also been described in
some studies; whose functions have been found to be similar to Tregs, wherein these cells
negatively regulated the immune system and hence promoted tumour growth (Yanaba et
al., 2008; DiLillo et al., 2010a; Olkhanud et al., 2011). Antibody production has also
been implicated in enhancing tumour development due to chronic inflammation
(Houghton et al., 2005; de Visser et al., 2006).
In contrast to the preceding paragraph, a positive significance of B cells in anti-
tumour immunity has also been reported. Impaired T cell activation with enhanced
melanoma tumour growth have been demonstrated in mice that were depleted of mature
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B cells (DiLillo et al., 2010b). Adoptive transfer studies involving transfer of activated
or primed B cells into mice have shown a) to enhance anti-tumour T cell activity, b)
slow tumour growth and also c) production of high titres of tumour specific IgG
antibodies (Ritchie et al., 2004; Li et al., 2011). Jackaman et al. (2010) had suggested a
role for B cells in a partially successful CD40 based immunotherapy against AE17 and
AB1 murine mesotheliomas. Specifically, they found high levels of tumour reactive
IgG and IgM antibodies in the sera during agonist anti-CD40 Ab treatment, with
significantly high serum IgG levels detected by the 6th dose using enzyme-linked
immunosorbent assays (ELISAs). In the same study, sera from C57Bl6/J mice that had
been cured of their AE17 tumours by anti-CD40 Ab treatment were collected; adjusted
to contain 25 µg of IgG, and then were transferred into naïve mice (i.v) and these mice
were challenged 24 hours later with AE17 cells. 2/5 of these recipient mice that
received the IgG antibodies from the cured mice had significantly retarded tumour
growth (Jackaman et al., 2010).
The work in this chapter was designed to further investigate the immune responses
involved in the complete tumour eradication achieved by the triple immunotherapy, but
especially focuses on the involvement of B cells. In light of the significantly high IgG
levels observed in the AE17 mesothelioma model treated with the agonist anti-CD40
Ab treatment (Jackaman et al., 2010), experiments were designed to systematically
examine whether the successful tumour eradication achieved with the triple
immunotherapy (TTI and TIC) elicited such high IgG levels in the mice cured of AE17
and AB1 tumours respectively.
The primary aim of the studies described in this chapter; was to determine whether
high IgG levels could be detected in the TTI and TIC cured mice and also to determine
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whether B cells played a positive role in the anti-tumour immunity in our successful
triple immunotherapy for the treatment of murine mesothelioma.
5.2 Result
5.2.1 Detection of tumour specific IgG antibodies in the serum of AE17 tumour-
bearing mice by ELISA
In order to measure IgG antibodies specific to tumours, an ELISA that used
tumour cell lysates as the antigenic target (Dols et al., 2003) was developed in our
laboratory (see Chapter 2, section 2.6). Initial experiments to detect IgG antibodies were
carried out against the AE17 murine mesothelioma tumour cell lysates. Plate controls
were run on each experiment to account for any non-specific binding of antibodies, and
these included a) no serum control, b) no tumour lysate control and c) no IgG-Biotin
antibody conjugate control. Sera collected from mice with AE17 end-point (100mm2)
tumours were pooled and initially used to determine if reactivity to the tumour lysates
could be detected. The OD absorbance noted in the duplicate wells that contained the
neat sera pooled from untreated mice were significantly higher (2.68 (±0.34)) when
compared to the plate controls, with the absorbance being 24 fold higher than the no
serum control (0.115 (±0.02)), 55 fold higher than no tumour control (0.04 (±0.007))
and 51 fold higher than the no biotin control (0.05 (±0.0007)), as shown in Figure 5.1.
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Figure 5. 1 Elevated AE17 cell lysate specific IgG antibodies detected in the sera of AE17 end-point tumour bearing untreated mice. Pooled sera (neat) were collected from untreated end-point (100mm2) tumour bearing C57BL/6J mice (n=9) and were incubated, in duplicate, on an ELISA plate coated with AE17 cell lysates along with the appropriate plate controls. Neat sera from untreated mice were used for the no biotin and no tumour plate controls. No serum was added to the wells used as no serum control. Tumour specific IgG was detected using a biotinylated anti-mouse IgG antibody, measuring optical density at 450 nm. OD absorbances are shown as means across the duplicates ± standard deviations from one experiment. Significant differences in IgG levels are indicated: ** p≤0.01 as shown. OD indicates optical density.
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5.2.2 Elevated levels of tumour specific IgG antibodies in the sera of AE17 tumour-
bearing mice cured by TTI
With the high IgG levels observed in the sera from untreated AE17 end-point
tumour bearing mice, the next step was to investigate whether high IgG levels could be
detected in the mice cured of AE17 tumours by the TTI treatment. During the
standardisation step of ELISA tests; when neat sera from TTI cured mice were used,
maximal OD readings of 3.5 were noted (data not shown). This resulted in the dilution
of sera by 20 times (in assay diluent) to be used for all consecutive assays across the
different C57BL/6J groups.
IgG reactivity in the pooled sera collected from mice cured of AE17 tumours
more than 2 months post treatment with TTI, gave 4.1 fold and 3 fold higher absorbance
readings compared to pooled sera from naïve C57BL/6J mice and end-point AE17
tumour bearing untreated mice respectively (Figure 5.2). Though the IgG reactivity in
the sera of untreated tumour bearing mice was significantly higher than the reactivity
observed in sera from naïve C57BL/6J mice (p<0.003, significance determined by un-
paired student t-test), the fold increase was only marginal (1.3 fold).
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Figure 5. 2 Elevated levels of tumour specific IgG observed in sera of TTI cured mice compared to untreated controls. Pooled sera (1:20 dilution) were collected from naïve C57BL/6J mice ( ), untreated endpoint (100mm2) tumour bearing mice ( ) and TTI cured mice (■) (>2 months post treatment). Pooled sera from each group were incubated, in duplicate, on an ELISA plate coated with AE17 cell lysates along with the appropriate plate controls (■). Tumour specific IgG was detected using a biotinylated anti-mouse IgG antibody, measuring optical density at 450 nm. Mean absorbance are shown as means ± standard deviations for 5-9 mice per group over 3 independent experiments. Significant differences in IgG absorbance when comparing indicated columns by t-test are: **p<0.01 and ***p<0.001 respectively. OD indicates optical density.
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5.2.2.1 Partial cross-reactivity of IgG antibodies in TTI cured mice to B16 melanoma
cell lysates and spleen cell lysates
In order to determine the antibody specificity in the TTI cured mice, pooled sera
from the different groups were tested against a) syngeneic B16 melanoma cell lysates
and b) non-syngeneic AB1 murine mesothelioma tumour lysates and c) lysed self-cells
(splenocytes).
In terms of reactivity against AE17 cell lysates, the reactivity of IgG antibodies in
the pooled sera from TTI long-term cured mice were 5 fold and 3.6 fold higher than
reactivity found in naïve and untreated tumour bearing mice respectively (p<0.00001
and p<0.0001 respectively, Figure 5.3). In the case of reactivity to syngeneic B16
melanoma tumour cell lysates, a similar pattern was observed; wherein the reactivity of
the IgG antibodies was higher in the TTI cured sera compared to naïve sera and
untreated tumour controls (3 fold and 2.7 fold, respectively). When comparing the fold
change in IgG reactivity in the TTI cure mice to AE17 and B16 cell lysates (tumours
that are syngeneic to C57BL/6J mice), the reactivity against the non-syngeneic AB1
was the lowest (p=0.0003 and p=0.01, respectively).
In order to determine whether this IgG reactivity in the TTI cured mice was
auto-reactive, the pooled sera from the different C57BL/6J groups were incubated
against lysed splenocytes (self-cells) taken from naïve mice. Relative to the reactivity
against lysed splenocytes observed in the naïve group, no significantly different reaction
was seen in the untreated tumour group (0.30). However, the reactivity of IgG
antibodies against splenocytes were 2.4 fold and 2.13 fold higher in the TTI cured mice
group relative to both naïve and untreated groups (p=0.0001 and p=0.0003
respectively). Overall, though reactivity of the IgG from TTI cured mice group was
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present against lysed B16 tumour cells and naïve splenocytes, reactivity nevertheless
remained much higher against the AE17 tumour cell lysates.
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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. Pooled sera (1:20 dilution) were collected from naïve C57BL/6J mice ( ), untreated endpoint (100mm2) tumour bearing mice ( ) and TTI cured mice ( ) (>2 months post treatment). Pooled sera from each group were incubated, in duplicate, on an ELISA plate coated with cell lysates of AE17, B16, AB1 and naïve lysed splenocytes. Tumour specific IgG was detected using a biotinylated anti-mouse IgG antibody, measuring optical density at 450 nm. Fold increases are shown as means ± standard deviations for 5-9 mice per group over 2 independent experiments. Significant differences in fold increases of IgG absorbance when comparing indicated columns by t-test are: *p<0.05, ** p≤0.01 and ***p<0.001 respectively; n.s-not significant. OD indicates optical density.
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5.2.3 Development of a living whole cell ELISA for detecting serum IgG levels
against AE17 tumour cells
The next aim was to determine whether IgG reactivity could be detected in the
different groups against whole live in vitro cultured AE17 tumour cells. Technical
difficulties of losing cells during the washing steps in the 96 well plates were overcome
by performing the assay in 1.5ml microcentrifuge tubes as detailed in chapter 2 section
2.6.4.
The preliminary ELISA test conducted against whole live in vitro cultured AE17
cells: showed 7.9 fold and 6 fold higher IgG reactivity in sera from TTI cured mice and
untreated mice relative to naïve mice sera (p<0.001 and p<0.001, respectively). When
comparing the relative difference in reactivity between the untreated and TTI long-term
cured mice, the IgG levels were only 1.3 fold higher in the TTI than the untreated
control group (p<0.004). Overall, though the IgG reactivity in TTI cured mice against
whole live AE17 was only marginally higher than untreated tumour controls; both
reacted very strongly to live AE17 cells.
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Figure 5. 4 Detection of increased reactivity of serum IgG in TTI cured mice to whole live AE17 cells. Pooled sera (1:20 dilution) were collected from naïve C57BL/6J mice ( ), untreated endpoint (100mm2) tumour bearing mice ( ) and TTI cured mice ( ) (>2 months post treatment). Pooled sera from each group were incubated with live in vitro cultured AE17 cells along with the appropriate assay controls (■). Tumour specific IgG was detected using a biotinylated anti-mouse IgG antibody, measuring optical density at 450 nm. Mean absorbances are shown as means ± standard deviations for 4-8 mice per group from one experiment. Significant differences in IgG absorbance when comparing indicated columns by t-test are: **p<0.01 and ***p<0.001 respectively. OD indicates optical density.
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5.2.3.1 Increased reactivity of serum IgG to whole AE17 live cells in TTI long-term
cured mice
Compared to the IgG reactivity in the pooled serum collected from naïve and
untreated end-point tumour bearing mice, the IgG reactivity against whole live in vitro
cultured AE17 cells were 7.6 fold and 1.3 fold higher in the TTI cured mice sera
(p=0.003 and p=0.04 respectively, Figure 5.5). A similar pattern of reactivity was
observed against AE17 tumour cell lysates with IgG reactivity being 3.4 fold and 1.2
fold higher in the TTI cured mice pooled sera compared to pooled sera from naïve and
untreated groups (p<0.01 and p=0.003 respectively).
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Figure 5. 5 Increased reactivity of serum IgG to whole AE17 live cells in TTI cured mice compared to AE17 cell lysates. Pooled sera (1:20 dilution) were collected from naïve C57BL/6J mice ( ), untreated endpoint (100mm2) tumour bearing mice ( ) and TTI cured mice ( ) (>2 months post treatment). Pooled sera from each group were incubated, in duplicate, on an ELISA plate coated with AE17 cell lysates or incubated with live in vitro cultured AE17 cells. Tumour specific IgG was detected using a biotinylated anti-mouse IgG antibody, measuring optical density at 450 nm. The vertical axis shows the OD fold increases in tumour specific IgG over naïve mice sera. Fold increases are shown as means ± standard deviations for 4-8 mice per group from one experiment. Significant differences in fold increases of IgG absorbance when comparing indicated columns by t-test are: *p<0.05 and ** p≤0.01 respectively; n.s-not significant. OD indicates optical density.
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5.2.4 Increased levels of AB1 tumour specific IgG in sera of mice 95 days post
treatment with the TTI or TIC
The previous section determined that a higher IgG reactivity was observed in
C57BL/6J mice cured of AE17 tumours with TTI and these IgG antibodies had greater
reactivity to whole live AE17 cells than the tumour cell lysates. The next aim was to
determine if a similar high IgG reactivity could be detected in the sera from BALB/c
mice treated with TTI or the triple immunotherapy cocktail (TIC).
Serum IgG levels reactive with AB1 tumour cell lysates and whole live in vitro
cultured AB1 cells were compared in TTI and TIC treated mice; relative to IgG levels in
the sera of both naïve and untreated AB1 tumour bearing mice. Preliminary ELISA tests
conducted with sera diluted 20 times from TTI cured mice showed maximal OD
readings of 3.5 (data not shown). This resulted in the dilution of pooled sera by 50 times
(in assay diluent) being used for all consecutive analyses across all different BALB/c
groups.
IgG reactivity in the pooled serum collected from mice cured of AB1 tumours 95
days post treatment with the TTI or TIC gave 8.7 fold and 3.6 fold higher absorbance
readings respectively against whole live in vitro cultured AB1 cells, compared to pooled
sera from end-point tumour bearing untreated mice (p<0.001 and p<0.0001 respectively,
Figure 5.6). A similar pattern of reactivity was observed against AB1 tumour cell
lysates, though the IgG reactivity was only marginally higher (̴ 1.3 fold) in the pooled
sera from TTI or TIC cured mice compared to pooled sera from untreated mice with
end-point tumours (p<0.001 and p<0.001 respectively, Figure 5.6). Interestingly,
looking at the sera IgG reactivity within the TTI and TIC cured mice themselves,
though their reactivity to AB1 cell lysates are not significantly different from each other
(p=0.66); against whole live AB1 cells, the BALB/c mice that were cured with TTI had
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significantly higher reactivity (1.7 fold) than the TIC cured mice (p<0.001) (Krishnan et
al., 2014).
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Figure 5. 6 Elevated levels of tumour specific IgG antibodies detected in combined immunotherapy (TTI and TIC) treated mice. Pooled sera (1:50 dilution) were collected from naïve BALB/c mice ( ), untreated endpoint (100mm2) tumour bearing mice ( ), TTI cured ( ) and TIC cured mice ( ) (95 days post treatment). Pooled sera from each group were incubated, in triplicate, on an ELISA plate coated with AB1 cell lysates or incubated with live in vitro cultured AB1 cells. Tumour specific IgG was detected using a biotinylated anti-mouse IgG antibody, measuring optical density at 450 nm. The vertical axis shows the OD fold increases in tumour specific IgG over naïve mice sera. Fold increases are shown as means ± standard deviations for 5-8 mice per group over 3 independent experiments. Significant differences in fold increases of IgG absorbance when comparing indicated columns by t-test are: ***p<0.001; n.s-not significant. OD indicates optical density.
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5.2.4.1 Partial cross-reactivity of IgG antibodies in TTI and TIC cured mice to
syngeneic 4T1breast carcinoma cells
In order to determine the tumour specificity of the IgG antibodies, the next
experiment was designed to test IgG reactivity to other live tumour cells. The tumour
types used were, live in vitro cultured 4T1 BALB/c syngeneic breast carcinoma, the
allogeneic (specific to C57BL/6J) AE17 murine mesothelioma and B16 melanoma cells.
Partial cross-reactivity in the sera of TTI and TIC cured mice were higher against
their respective syngeneic live 4T1 breast carcinoma tumour cells (Figure 5.7). The IgG
reactivity in the TTI and TIC cured mice were 5.5 fold and 4.4 fold higher against 4T1
live tumour cells, relative to the fold increase in IgG reactivity observed in the untreated
end-point tumour bearing mice serum (p<0.0001 and p=0.002 respectively).
The IgG reactivity in TTI cured mice were not significantly different from naïve, or
untreated mice sera when incubated against live AE17 tumour cells (p=0.53 and p=0.75
respectively). The TIC cured mice sera were not tested against live AE17 tumour cells,
so no data is available for their IgG reactivity. In the case of allogeneic B16 live
tumours; there is some cross-reactivity in IgG present in the TTI and TIC long-term
cured mice compared to untreated controls (4.2 fold and 2.7 fold higher), though the
fold increases are not as high as the reactivity to syngeneic 4T1 tumour cells.
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Figure 5. 7 Partial cross-reactivity high against live syngeneic 4T1 tumour cells in TTI and TIC cured mice. Pooled sera (1:50 dilution) were collected from naïve BALB/c mice ( ), untreated endpoint (100mm2) tumour bearing mice ( ), TTI cured ( ) and TIC cured mice ( ) (95 days post treatment). Pooled sera from each group were incubated, in triplicate with live in vitro cultured AB1, 4T1, AE17 and B16 tumour cells. Tumour specific IgG was detected using a biotinylated anti-mouse IgG antibody, measuring optical density at 450 nm. The vertical axis shows the OD fold increases in tumour specific IgG over naïve mice sera. Fold increases are shown as means ± standard deviations for 5-8 mice per group over 3 independent experiments. Significant differences in fold increases of IgG absorbance when comparing indicated columns by t-test are: *p<0.05, ** p≤0.01 and ***p<0.001 respectively; n.s-not significant. OD indicates optical density.
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5.2.4.2 IgG antibodies present in the TTI and TIC cured BALB/c mice are tumour
specific and not auto-reactive
Compared to the high specificity of the reactivity of IgG antibodies to live AB1
tumour cells in both the TTI and TIC treated mice, marginal reactivity was observed
against live splenocytes taken from a naïve BALB/c mouse. Interestingly, though the
reactivity in TTI cured mice sera was higher than sera from the untreated group (1.5
fold higher, p=0.03), the reactivity in TIC cured mice sera was lower than the untreated
end-point tumour bearing control sera (p=0.03). Overall, this experiment determined
that the treatment with TTI or TIC did not elevate auto-reactive antibodies in these long-
term cured mice.
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Figure 5. 8 Low levels of auto-reactive antibodies in TTI and TIC cured mice. Pooled sera (1:50 dilution) were collected from naïve BALB/c mice ( ), untreated endpoint (100mm2) tumour bearing mice ( ), TTI cured ( ) and TIC cured mice ( ) (95 days post treatment). Pooled sera from each group were incubated, in triplicate with live in vitro cultured AB1 cells and live splenocytes from a naïve mouse. Tumour specific IgG was detected using a biotinylated anti-mouse IgG antibody, measuring optical density at 450 nm. The vertical axis shows the OD fold increases in tumour specific IgG over naïve mice sera. Fold increases are shown as means ± standard deviations for 5-8 mice per group from one experiment. Significant differences in fold increases of IgG absorbance when comparing indicated columns by t-test are: *p<0.05, ** p≤0.01 and ***p<0.001 respectively; n.s-not significant. OD indicates optical density.
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5.2.5 Tumour specific antibodies are not elevated during tumour eradication
The time to complete clearance of established 9mm2 AB1 tumours was between 10-
12 days post treatment with the TIC (Chapter 4). Sera collected on the day of TIC
injection (Day 0) and at time-points immediately post treatment were analysed by whole
live cell ELISA to determine the relative levels of anti-tumour IgG during the period of
tumour clearance.
Relative to IgG levels on day 0 (against whole AB1 cells), no significant increases
in IgG levels were observed within the period of maximal tumour regression (days 2- 8
post TIC treatment, Figure 5.9). The absorbance readings were also constant at days 16
and 20 post treatment, when no palpable tumours remained. Relative to pre-treatment
IgG levels measured on day 0, a significantly higher absorbance reading (5 fold
increase, p<0.001) was found for pooled sera collected on day 95 post TIC treatment
(Day 95, Figure 5.9) (Krishnan et al., 2014).
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Figure 5. 9 No significant change in serum IgG levels in TIC treated mice up to 20 days post treatment. BALB/c mice were implanted s.c with 1 × 106 AB1 tumour cells and treated intra-tumourally with TIC at a tumour size of 9 mm² (day 0). Pooled sera (1:50 dilution) were collected before TIC treatment on day 0 and at time-points post TIC treatment (days 1, 2, 4, 8, 12, 16, 20 and 95). Pooled sera from each time-point were incubated, in triplicate, with live in vitro cultured AB1 cells. Tumour specific IgG was detected using a biotinylated anti-mouse IgG antibody, measuring optical density at 450 nm. The left vertical axis shows the tumour areas (■) at each time-point (n=14) measured over three independent experiments. The right vertical axis shows the fold increases (■) in tumour specific IgG over day 0 sera. Fold increases are shown as means ± standard deviations for each time-point. OD indicates optical density.
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5.2.5.1 Elevated B cell numbers during TIC induced tumour eradication
In the same time course experiments described above, the percentages of B220+
cells within the tumour draining lymph nodes (TDLNs) and the spleens of TIC treated
mice were determined.
Flow cytometric analysis showed some minor fluctuations in B cell percentages on
days 1 and 2. However, the percentage of B cells in both spleen and TDLNs was
greatest on day 4 for the TIC treated mice compared to untreated mice at the same time
points (Figures 5.10 A and B). The percentage of B cells in the spleen increased from
46.4% on day 1 to 72.4 % on day 4. The percentage of B cells in TDLNs increased from
30.1% on day 1 to 42.8% on day 4. Day 4 post TIC treatment is central to the period of
maximal tumour shrinkage. By day 8, when the tumours were barely palpable under the
skin, the B cell percentages had reduced from the day 4 high in both the spleen and
TDLNs, but were still higher than the day 0 values (Krishnan et al., 2014).
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Figure 5. 10 Changes in B cell percentage post TIC treatment. Spleen (A) and tumour-draining lymph nodes (TDLNs: pooled unilateral brachial, axillary and inguinal) (B) were removed from mice on days 1, 2, 4, 8 and 12 days after treatment with TIC (■) and the population of B cells (B220+) was determined by flow cytometry and compared with cell populations in untreated mice (●) at the same time-points. Baseline percentages of B cells before treatment initiation were determined from untreated 9mm2 tumour bearing mice (day 0). (n=6 for untreated mice only on day 0 and n=2 for untreated and TIC treated mice for all other time-points, from one experiment. The error bars represent standard deviations of the mean). Significant differences in B cell percentages between untreated controls and TIC treated mice at different time-points determined by t-test are indicated: *p<0.05 and ** p=0.01 respectively.
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5.2.5.2 B cells are vital to the efficacy of the combined triple immunotherapy
The efficacy of the TIC in B-cell knock-out (BKO) mice were used to
substantiate the suggested role of B cells in the anti-tumour response. BKO mice
(C.129S- Igh-6tm1Cgn/J) on the BALB/c background were injected s.c with AB1 cells
and treated with the TIC when tumours reached 9 mm2. Figure 5.11 shows that BKO
mice left untreated grew tumours faster (median survival 16 days) than their wild-type
counterparts (median survival 19 days, p=0.0001 significance determined by log-rank
test). More importantly, Figure 5.11 also shows a complete failure of the TIC to
eradicate the AB1 tumours in these B-cell knock-out mice. This contrasts strongly with
the 88% clearance (15/17 mice over three independent experiments) seen with B cell
positive BALB/c wild-type mice. Note that 19/19 untreated BALB/c wild-type mice
developed tumours to end-point (Figure 5.11). The BKO treated mice instead had only a
partial response to the treatment with a transient delay in tumour growth compared to
the BKO mice left untreated (median survival 16 days, p=0.38) (Krishnan et al., 2014).
148
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Figure 5. 11 Successful tumour eradication with TIC requires B cells. B cell knock-out mice (BKO) and wild-type BALB/c mice were implanted s.c. with 1 × 106 AB1 tumour cells and TIC was administered intra-tumourally at a tumour size of 9 mm² (day 0). Mice were monitored daily for (A) survival and plotted as Kaplan-Meier survival curve with 100 mm2 tumour as endpoint and (B) tumour growth (mean ± standard deviation) was calculated with measurements made using microcalipers. (n=5 for BKO untreated (■) and n=5 for BKO TIC treated (◊). n=19 for wild-type BALB/c untreated (●) and n=17 for wild-type TIC treated controls (▼) have been included for reference).
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5.2.5.2.1 B cells are required for successful treatment of AB1 tumours by TIC
The previous section determined that in the absence of B cells in these BKO mice,
TIC was not successful in eradicating established AB1 tumours. Thus, the next
experiment was designed to assess the anti-tumour role of B cells at the time of intra-
tumoural treatment with TIC by re-constituting the B cells in these BKO mice. Long-
term cured wild-type (WT) BALB/c mice were re-challenged with the original 1 × 106
AB1 tumour cells, and their spleens were harvested 3 days later and made into single
cell suspensions as mentioned in chapter 2 section 2.5.2. These spleen cells were then
either (a) left unfractionated (from 3 re-challenged cured mice) or (b) positively selected
for B220+ B cells (from 2 re-challenged cured mice, purities of >90% were determined
by FACS) and were then transferred through the tail-veil into 9mm2 AB1 tumour-
bearing BKO mice simultaneous to TIC treatment given intra-tumourally.
Figure 5.12 shows that 25% (1/4, from one experiment) of BKO mice that received
the TIC treatment at the same time as positively selected B220+ B cells underwent
complete tumour clearance. This was a significant advantage over (a) the BKO mice
that received TIC plus unfractionated spleen cells (p<0.05) as well as (b) the BKO mice
left untreated (median survival 14 days, p=0.02). The BKO mice that received TIC plus
unfractionated spleen cells did not survive longer than either the untreated BKO
controls (median survival 16 days, p=0.11) or the WT BALB/c untreated control mice
(median survival 18 days, p=0.14) and all 100% of mice (4/4) grew AB1 tumours to
end-point.
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Figure 5. 12 Anti-tumour efficacy of TIC treatment is augmented by B cells. B cell knock-out mice (BKO) and wild-type BALB/c mice were implanted s.c. with 1 × 106 AB1 tumour cells and i.t TIC and i.v transfer of spleen cells were initiated at a tumour size of 9 mm² (day 0). Spleens were harvested from long-term cured mice (n=5) that were challenged with 1 × 106 AB1 tumour cells 3 days prior to day 0. The spleens from 3 mice were pooled and made into single cell suspensions and left unfractionated while the pooled splenocytes from the remaining 2 mice were sorted by FACS into B220+ B cells. On day 0, the BKO treatment groups along with TIC administered intra-tumourally, also received i.v transfers of either 1 × 107 unfractionated splenocytes (♦) or positively selected 1 × 106 B220+ B cells (▼). Tumour growth (mean ± standard deviation) was calculated with measurements made using microcalipers and the tumour growth in the treated mice were compared with AB1 tumours growing in untreated wild-type BALB/c mice (●) and BKO mice (■). (n=4 for the treated mice and n=2 for the untreated mice groups respectively).
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5.2.5.2.2 Overcoming the immunosuppressive tumour microenvironment is critical
to tumour clearance
The above data suggested that i.t TIC treatment was more effective post-B cell
reconstitution in BKO mice. However, this raised the question of what happens in WT
BALB/c mice that received only the primed immune cells from re-challenged cured
mice; in the absence of TIC treatment.
In the next experiment, WT BALB/c mice were inoculated s.c with AB1 tumour
cells and the tumours were allowed to grow to an established size of 9 mm2. Spleens
from long-term cured mice (re-challenged 3 days prior with 1 × 106 AB1 tumour cells)
were harvested and made into single cell suspensions, and positively selected for B220+
B cells or CD3+ T cells by FACS with purities > 98% for both T and B cells. These
positively selected immune cells were transferred through the tail vein into these 9mm2
AB1 tumour bearing BALB/c mice as either (a) B220+ B cells only, (b) CD3+ T cells
only or (c) a mixture of both B and T cells and the tumour growth in the respective
groups were monitored daily.
Figure 5.13 shows that irrespective of whether the naïve AB1 tumour bearing mice
received only B cells, T cells or both T and B cells, tumours grew in 100% of the mice
in all 3 groups (3/3, one experiment). Transfer of only B cells or mixture of T and B
cells into these tumour-bearing mice made no difference in delaying tumour growth, as
their median survival of 14 and 15 days were not significantly different to untreated
mice (median survival of 14 days, p=0.19 and p=0.45). Only the mice that received just
T cells survived longer than untreated controls (median survival 17 days, p=0.02).
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Figure 5. 13 Manipulation of tumour microenvironment is important for successful clearance of established tumours. Wild-type BALB/c mice were implanted s.c. with 1 × 106 AB1 tumour cells and antigen primed immune cells from re-challenged cured mice were transferred intra-venously at a tumour size of 9 mm² (day 0). Spleens were harvested from long-term cured mice (n=2) that were challenged with 1 × 106 AB1 tumour cells 3 days prior to day 0. The spleens were pooled and made into single cell suspensions and sorted by FACS into B220+ B cells and CD3+ T cells. These positively selected cells were then transferred either as (a) 1 × 106 B220+ B cells only (■) or (b) 1 × 106 CD3+ T cells only (▼) or as (c) mixture of 1 × 106 both T and B cells (♦). Tumour growth (mean ± standard deviation) was calculated with measurements made using microcalipers and the tumour growth in the treated mice were compared with AB1 tumours growing in untreated wild-type BALB/c mice (●). (n=3 for the both treated mice and untreated mice groups, from one experiment).
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5.3 Discussion
The work described in this chapter has demonstrated strong initial evidence that
rapid elimination of tumours caused by the TTI and the TIC treatments are B cell
dependent. There is some literature that indicated the role of B cells in positively
enhancing cellular immune responses by serving as antigen presenting cells (APCs).
The APC function of B cells subsequently led to the activation of tumour specific
cytotoxic T cells and production of antibodies (which may contribute modestly to anti-
tumour immunity) (Manson, 1994; Ritchie et al., 2004; Crawford et al., 2006; DiLillo et
al., 2010b; Jackaman et al., 2010; Li et al., 2011; Molnarfi et al., 2013).
More specifically, the interest in B cells was initiated with the publication of the
work conducted by Jackaman et al., 2010, wherein the involvement of B cells in the
same two- AE17 and AB1 murine mesothelioma (MM) cell lines was investigated using
intra-tumoural anti-CD40 Ab treatment. This paper was relevant at two levels: (a) the
treatment involved timed intra-tumoural administrations of their anti-CD40 Ab therapy
(similar to the TTI) and (b) tumour reactive IgG and IgM antibodies were determined in
the sera during agonist anti-CD40 Ab treatment, with significantly high serum IgG level
detected by the 6th dose (Jackaman et al., 2010).
Therefore, investigations of the IgG reactivity to AE17 tumour cell lysates in the
sera of C57BL/6J mice were initiated. The result from the first test using neat sera
showed that IgG reactivity against the AE17 tumour cell lysates could be detected in the
sera from untreated end-point tumour bearing mice (Figure 5.1). It was important to use
sera from untreated mice in the initial analysis, as these mice have had AE17 tumour
cells implanted in them sub-cutaneously, which grew to end-point. It was expected that
these mice would naturally have antibodies capable of recognising tumour antigens.
This initial experiment with neat sera was also crucial to show that the low absorbance
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reading noted in the plate controls account for the absence of cross-reactivity. Thereby,
the reactivity observed in the wells containing the neat pooled sera from untreated mice
was in fact, cross-reacting to the AE17 tumour cell lysates. It could be concluded from
this initial assay, that a) tumour reactive IgG antibodies were present in mice that have
been exposed to the AE17 tumour cells and also, b) reactivity to FCS (integral to the
RPMI media used for culturing the cells in vitro) is highly unlikely.
The following experiments showed that relative to these untreated mice, IgG
reactivity in the TTI long-term cured C57BL/6J mice were highest against AE17 cell
lysates (Figure 5.2). It is clear that the TTI treatment induces higher levels of circulating
IgG in these long-term (>2 months post TTI treatment) cured mice specific to the
antigens over-expressed by the MM tumours, with no adverse effects noted.
Numerous studies have identified common tumour antigens such as MUC1, CEA,
EMA and CSPG4 that are expressed on multiple human tumour types such as lung
cancer, breast cancer, mesothelioma, and melanoma among others (Pinkus and Kurtin,
1985; Pinto et al., 1986; Schumacher et al., 1993; Rivera et al., 2012). The work
conducted by Rivera et al., 2012 showed that Chondroitin Sulphate Proteoglycan 4
(CSPG4), a melanoma-associated antigen, was also found to be highly expressed in
malignant mesothelioma cells in humans (Rivera et al., 2012). This could explain the
partial cross-reactivity of the IgG present in the sera collected from the TTI cured mice
to the syngeneic B16 melanoma cell lysates (Figure 5.3). However, low reactivity to the
non-syngeneic AB1 (raised in BALB/c mice) cell lysates was noted. This raises the
possibility that perhaps tumour antigen recognition is mouse strain (on which the
tumours were raised against) specific, as the better reactivity to B16 tumour cell lysate
was due to AE17 and B16 both being raised against C57BL/6J mice. Moreover, the low
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reactivity to AB1cell lysates, despite it being another MM tumour model, could be due
to their background being the allogeneic BALB/c mice (Figure 5.3).
In the future, perhaps testing the C57BL/6J cured mice sera IgG specificity against
several different tumour cell lines could aid in further understanding the notion of cross-
reactivity. The syngeneic EO771 breast carcinoma cell line (in C57BL/6J mice) and the
non-syngeneic 4T1 breast carcinoma cell line (in BALB/c mice) would be ideal
candidates to cross-check IgG reactivity.
There is evidence in the literature that antibodies that recognise intracellular as well
as surface antigens have been reported in both animal cancer models and cancer patients
(Canevari et al., 1996; Disis et al., 1997; Dao et al., 2013; Grandjean et al., 2013; Wei
et al., 2013). The IgG reactivity in the sera from TTI cured mice was found to be
greatest against whole live AE17 tumour cells and lesser against AE17 cell lysates
(Figure 5.5). A similar reactivity was observed in the sera of BALB/c mice cured of
AB1 tumours by TTI and TIC treatments. Reactivity of the IgG antibodies in these
long-term (greater than 95 days post treatment) cured mice were greatest against whole
live AB1 tumour cells and lesser against AB1 tumour cell lysates (Figure 5.6 and
Krishnan et al, 2014) and very low against wild-type BALB/c whole live naïve
splenocytes (self-cells) suggesting tumour specificity (Figure 5.8). Similar to the cured
C57BL/6J mice, the IgG reactivity of the BALB/c mice cured mice was found to be
more cross-reactive against live syngeneic 4T1 breast carcinoma cells (that were also
raised in BALB/c mice) and very low against the other non-syngeneic AE17 MM cells
(Figure 5.7).
Antibody dependent cell-mediated cytotoxicity (ADCC) is an important mechanism
by which antibodies recognise tumour antigen receptors present on the target cells by
their Fab domains. The Fc portions of these antibodies bind to the corresponding Fc
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receptors present on immune cells such as monocytes, macrophages and natural killer T
(NKT) cells. This in turn leads to the activation and crosslinking of the Fc receptors and
ultimately, lysis of the target cells by the release of cytokines and formation of cytotoxic
granules that contain granzyme and perforin (Leibson, 1997; Lyubchenko et al., 2001).
ADCC has been reported as an important mechanism of action for monoclonal antibody
(mAb) mediated therapies for cancer, such as rituximab (anti-CD20mAb), cetuximab
(EGFR inhibitor) and trastuzumab (Her-2/neu inhibitor) (Carter et al., 1992; Reff et al.,
1994; Vermorken et al., 2008).
However, the time course experiments showed that the tumour specific IgG levels
remained stable and low during the tumour shrinkage post TIC treatment; compared to
the long-term cured mice (Krishnan et al., 2014). As elevated IgG levels were only
detected in long-term cured mice, it could mean that TIC mediated tumour eradication
was unlikely to involve IgG antibodies as low IgG levels were detected at this stage.
This suggested that perhaps tumour eradication in the AB1 BALB/c model by TIC is
not dependent on an ADCC mechanism as previously recorded for other tumour models
(Herlyn et al., 1980; Jasinska et al., 2003).
The high levels of IgG detected only in the long-term cured mice tended to indicate
that possibly an amplification of the adaptive immune response occurs in response to
endogenous adjuvants released during immunotherapy-induced tumour cell death.
Activation of dendritic cells by apoptotic cells and/or release of damage associated
molecular patterns have been previously shown to result in an increased tumour antigen
availability, which then leads to an increase in tumour antigen-specific antibodies (Shi
and Rock, 2002; Apetoh et al., 2007; Kono and Rock, 2008). It is also possible that high
levels of IgG antibodies in the cured mice played a more robust role in the prevention of
tumour relapse and are not actively involved in primary tumour eradication. Further
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studies to elucidate the exact function of antibodies, and their involvement in tumour
eradication could include immune-histochemical sectioning of receding tumours in
response to TIC from days 0 to day 12 for detection of IgG antibodies present in the
tumour microenvironment. Assessing the tumour microenvironment for NK cells,
macrophages and monocytes could also shed light on whether ADCC mediated tumour
lysis is prevalent in the triple mAb therapy. In the future, enzyme-linked immunospot
(ELISPOT) assays can also be used to study the secreted antibodies, but at a more
cellular level, as described previously (Sedgwick and Holt, 1983; Moody and Haynes,
2008).
Elevated numbers of B cells in the spleens and TDLNs of TIC treated mice at the
precise time of maximal tumour shrinkage (with low unchanged antibody levels)
suggested that B cells may be playing another role such as antigen presentation in
tumour immunity (Manson, 1994; Ritchie et al., 2004; Crawford et al., 2006; DiLillo et
al., 2010b; Jackaman et al., 2010; Li et al., 2011; Molnarfi et al., 2013). The minor
fluctuations in B cell percentages on days 1 and 2 may represent migration of B cells
but may also be within the sample size error range (Figure 5.10). Technical difficulties
of accurately determining B cell percentage in the rapidly shrinking tumours precluded
reliable, detailed analyses. Tumour-specific effector T cell percentages of both CD4+
and CD8+ remained unchanged in the spleens and TDLNs at day 4 of the treated mice
(data not shown) and this could be due to the de-repression of the existing effectors cells
being modulated by the TIC treatment. This observation, in conjunction with the
increase in B cells in both spleen and TDLNs of TIC treated mice on day 4, suggests
that B cells were rapidly stimulated by the agonist TIC treatment and perhaps B cells act
as antigen-presenting cells (APCs) and aid in optimal activation of CD4+ and CD8+
tumour immunity in the TIC induced tumour clearance. This anti-tumour activity
contributed by B cells, by playing the role of antigen presentation in tumour immunity
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is supported by the work conducted by other research groups (Manson, 1994; Crawford
et al., 2006; DiLillo et al., 2010b; Jackaman et al., 2012; Molnarfi et al., 2013). One
caveat on this interpretation is that the B220+ marker is not entirely specific to B cells.
Dendritic cells also can carry this marker and may be contributing to the day 4
elevations noted in the TDLNs and spleen. Additional work using extra lineage specific
antibodies is required to define the situation.
This notion of B cell involvement in tumour eradication was further supported by
the experiments in B cell knock-out mice. In the first experiment, it was noted that
untreated BKO mice grew tumours quicker than in BALB/c wild-type, and the TIC
failed to induce complete tumour clearance in any BKO mice treated, in comparison to
88% of BALB/c wild-type mice undergoing complete tumour eradication (Figure 5.11A
and B). This is supported by findings of impaired T cell activity in B cell deficient mice
reported with diminution in tumour specific effector T cell proliferation (Gordon et al.,
1982; Schultz et al., 1990; DiLillo et al., 2010b).
The preliminary data from the adoptive transfer experiment in the BKO mice was
also consistent with the hypothesised APC role of B cells for T cell-mediated tumour
eradication by TIC treatment (see Figure 5.12). The data from this experiment suggested
that i.t treatment with TIC was effective only in the presence of B cells (that was
adoptively transferred from cured mice) in the BKO mice, as BKO mice that received
only the TIC treatment alone, were not successful in eradicating AB1 tumours.
Though the simultaneous administration of TIC (i.t) and B220+ B cells (i.v) were
successful in tumour clearance in at least one (out of four) AB1 tumour-bearing BKO
mice, B220+ B cells from cured mice when administered in the absence of TIC- was not
sufficient to mount an effective anti-tumour immune response and clear established
AB1 tumours in WT mice (see Figure 5.13). In this adoptive transfer experiment, mice
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that received only T cells transferred through tail-vein from re-challenged mice showed
marginal improvement in survival compared to the other groups. The above mentioned
experiment showed that, though B cells involvement seemed to be critical in the TIC
mediated tumour clearance, in the absence of manipulation of the tumour micro-
environment, the tumours grew un-hindered.
Though the data from the adoptive transfer experiments mentioned above looks
promising; and supports the APC role of B cells, the factor that needs to be taken into
consideration is that, these were data from one experiment each. There is the possibility
of the adoptive transfers being sub-optimal as tail-vein transfers are difficult, and there
is always the chance that the transfers are not always 100% successful. One way to
confirm proper transfers would be to perhaps collect blood from the recipient mice one
hour post-transfer and look for the fluorescent tagged immune cell sub-sets. Also,
increasing the number of recipient mice could also aid with data validation. A repeat
examination of the adoptive transfer experiments would also help validate the results to
a greater extent. In the future, further differentiation of B cell sub-sets, such as memory
B cells (CD27+) and plasma cells (CD138+) and their adoptive transfers into tumour-
bearing mice could also help in determining which of the sub-sets are more important in
tumour eradication.
Although the exact mechanism of B cell involvement in a direct effector capacity is
not yet known; the work with the TIC in eradicating AB1 tumours; and the work on
BKO mice augment previous works that reported the requirement of B cells as APC for
T cell activation- in the context of tumour immunity. With the current mesothelioma
treatments being of low efficacy, the TIC looks particularly promising for clinical trials.
Also, the three components that constitute the triple therapy have been approved for
clinical use (as detailed in chapter 3).
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With more clinical trials at present focussing on combined therapy for treatment of
cancer in humans (Madan et al., 2012; Wolchok et al., 2013), we are on the right path
and testing the efficacy of this TIC treatment in other tumour models such as B16
melanoma, EO771 and 4T1 breast carcinoma; could perhaps facilitate their approval for
clinical trials. The next chapter details the preliminary work conducted on testing the
efficacy of the TIC against these non-mesothelioma tumour models.
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Chapter 6 Preliminary investigations of the intra-tumoural triple
immunotherapy in other tumour models and its effects on distal tumours
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6.1 Introduction
The effective triple immunotherapy (TIC) described in detail in chapter 4 and other
combination therapies examined in animal models; have resulted in stronger immune
responses against multiple tumour types (van Elsas et al., 1999; Uno et al., 2006;
Kissick et al., 2009; Curran et al., 2010; Jackaman and Nelson, 2012; vom Berg et al.,
2013). Clinical combined immunotherapies for cancer are, however, in their infancy.
The published example (Wolchok et al., 2013) used fully humanised antibodies against
PD-1 (Nivolumab) and CTLA-4 (Iipilimumab) for the treatment of advanced
melanoma. These antibodies have complementary roles, in that, anti-PD1mAb
contributes to T cells avoiding programmed cell death and anti-CTLA-4mAb blocks the
attenuation of effector T cell activity. Rapid and substantial tumour regression was seen
in 80% of the patients with advanced melanoma. Though adverse events related to the
systemic delivery of this treatment were observed, they were generally reversible and
this improvement in tumour reduction with the combined immunotherapy was greater
than when the treatments were administered as monotherapies (Wolchok et al., 2013).
More recently, different permutations of anti-CTLA-4mAb, anti-PD-L1mAb and
IDO inhibitor INCB23843 were tested in the murine B16-SIY melanoma model
(C57BL/6J mice). B16-SIY are engineered to express the antigen SIYRYYGL (SIY), a
green fluorescent fusion protein (GFP) (Blank et al., 2004). The B16-SIY is a useful
cell line, as it aids in monitoring T cell responses that are specific to the SIY antigen
(Kline et al., 2012). All three components that were used in the aforementioned study,
target the immunosuppressive mechanisms that operate within the tumour
microenvironment. In this murine study, the tumours were allowed to grow to an
established size and timed i.p injections of anti-CTLA-4mAb and anti-PD-L1mAb were
administered alternatively for 12 days and the IDO (indoleamine -2,3-dioxygenase)
inhibitor was given by oral gavage. For the combination of anti-CTLA-4/anti-PD-
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L1mAb, 55% of mice were shown to undergo complete tumour rejection and a lower
percentage of tumour clearance was observed with anti-CTLA-4mAb/IDOi (18%) and
anti-PD-L1mAb/IDOi (13%) and the triple combination. The doublet combinations
were also tested on the parent cell line B16-F10 tumours, but the authors mention that
though the tumour growth was significantly delayed, tumour clearance was never
achieved with the parent B16-F10 cell line (Spranger et al., 2014).
One of the main features of the TIC treatment is the depletion of Tregs by anti-
CD25mAb within the tumour microenvironment. Anti-TGF-βmAb is also partly
responsible for the depletion of Tregs, as is anti-CTLA-4mAb. From literature, it is
known that Treg associated immune suppression and maintenance of T cell anergy have
been previously determined in several murine and human tumours (Liyanage et al.,
2002; Curiel et al., 2004; Needham et al., 2006; Zhou et al., 2006; Zou, 2006; Hegmans
et al., 2010; Özdemir et al., 2014; Tang et al., 2014; Weiss et al., 2014). Most recently,
recruitment of Tregs and the mRNA expression of FoxP3 was found to be significantly
higher within the tumours than in the normal skin of patients suffering from oral and
cutaneous squamous cell carcinoma (SCC) (Schipmann et al., 2014). This led to the
hypothesis, that the newly developed TIC treatment that targets multiple mechanisms of
immune suppression would also be effective in eradicating other non-mesothelioma
murine tumours such as B16 melanoma, EO771 and 4T1 breast carcinomas.
The main aims of the experiments presented in this chapter was to a) firstly
determine the levels of Tregs within these alternate tumours to give an indication that
they may (at least theoretically) be sensitive to TIC and b) secondly, test the efficacy of
the TIC treatment i.t in the B16 melanoma, EO771 and 4T1 breast carcinoma tumour
types. Additionally, in a clinical setting, patients report to the clinic with varying
degrees of disease progression (from varied tumour sizes to wide-ranging levels of
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metastases). In order to replicate such conditions in the murine model, effectiveness of
the triple immunotherapy was also examined in BALB/c mice bearing large AB1
tumours (16-25mm2 tumour sizes). With metastases being another major factor to
decreasing survival time in patients, it was important to determine whether i.t treatment
of one tumour was sufficient to generate enough anti-tumour immune response to
eradicate distal tumours. Therefore, a final experiment to test the effectiveness of a
single treatment with TIC on a distal tumour was also undertaken.
6.2 Results
6.2.1 Detection of high levels of Tregs within the tumour microenvironment of non-
mesothelioma tumours by flow cytometry
The intra-tumoural (i.t) TIC treatment focuses directly (anti-CD25mAb) or
indirectly (anti-TGF-βmAb and anti-CLA-4mAb) on the importance of overcoming the
immune suppression maintained by Tregs. The presence of Tregs in these non-
mesothelioma tumours was, therefore, estimated prior to attempting TIC treatment.
The accumulation of Tregs within the growing tumours were analysed by flow
cytometry, and the tumours at the time of harvesting for the analysis were 26.5 (±6.75)
mm2 for AE17 tumours, 23 (±7.7) mm2 for B16 melanoma tumours and 15.7 (±5.23)
mm2 for EO771 breast carcinoma tumours in the C57BL/6J mice. For the tumours
examined in the BALB/c mice, the tumours sizes were 25.5 (±1.8) mm2 for AB1
mesothelioma and 22.4 (±7.9) mm2 for 4T1 breast carcinoma tumours respectively.
Figures 6.1A and B show that high levels of Tregs were detected in all the three
non-mesothelioma tumour models, with the highest percentage detected in the 4T1
breast tumours (52.86 (±5.73)), closely followed by the other breast model EO771 at
47.54 (±11.21) and B16 lastly with 36 (±6.72). However, when comparing the levels
within the tumours developed in C57BL/6J mice (Figure 6.1A), the Treg levels in B16
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and EO771 tumours were not significantly different from the Tregs percentage of total
CD4+ T cells examined within AE17 tumours (43.57 (±14.99), p=0.39 against B16 and
p=0.66 against EO771 tumours respectively, by post hoc student t-test). For the tumours
grown in BALB/c mice, the AB1 tumours had the lowest Treg percentage of total CD4+
T cells at 13.53 (±4.72) (Figure 6.1 B) but in contrast, the Treg percentage within 4T1
breast carcinoma tumours were ~4 fold higher than within AB1 tumours (p<0.0001).
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Figure 6. 1 Detection of Tregs within non-mesothelioma tumours grown in both C57BL/6J and BALB/c mice. C57BL/6J mice were inoculated s.c with either 1 × 107 AE17 mesothelioma cells (●), 5 × 105 B16 melanoma (■) or 5 × 105 EO771 breast carcinoma cells (▲). BALB/c mice were inoculated s.c with either 1 × 106 AB1 mesothelioma cells (●) or with 5 × 105 4T1 breast carcinoma cells (■). Tumour growth was monitored daily. Once the tumours sizes were between 16-25 mm2, the mice were humanely sacrificed and their tumours were harvested and the population of Treg cells was determined by flow cytometry. (n=4-5 for all tumour types, from one experiment). The errors bars represent standard deviations of the mean. Significant differences in fold increases of IgG absorbance when comparing indicated columns by t-test: ***p<0.001as shown; n.s-not significant.
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6.2.2 TIC treatment is not effective in eradicating B16 melanoma tumours in
C57BL/6J mice.
B16-F10 melanoma tumours are aggressive tumours with high metastatic abilities that
are syngeneic to the C57BL/6J mouse and are widely regarded as poorly immunogenic
in nature. B16 cells were originally derived from tumours that were chemically induced
in C57BL/6J mice, more than 60 years ago. A system of tissue culture and animal
transplantation was used to develop the B16-F10 cell line (Fidler, 1973). With the
detection of Tregs in the B16 melanoma tumours as shown in the previous section, the
next step was to investigate the efficacy of the TIC treatment in this tumour model.
B16 tumour cells were injected subcutaneously into C57BL/6J mice, and the TIC was
initiated when tumours reached 9mm2. When compared to the untreated mice (median
survival of 9.5 days), there was no significant difference in the tumour growth in the
TIC treated mice (median survival 10 days, p=0.96, significance determined by log-rank
test), as all 100% (9/9, from two independent experiments) grew tumours to end-point
size of 100mm2 (Figure 6.2).
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Figure 6. 2 Growth of melanoma tumours in C57BL/6J mice is unhindered by TIC treatment. C57BL/6J mice were inoculated s.c with 5 × 105 B16 cells and TIC treatment was initiated when the tumours reached 9mm2 (4 ± 1 days). TIC (■) consisted of anti-CD25mAb, anti-CTLA-4mAb and anti-TGF-βmAb given simultaneously at a dosage of 2 µg of all three components. Mice were monitored daily for tumour growth and tumour sizes were measured using microcalipers. Untreated mice (●) were used as tumour growth controls. (n=7 for untreated mice and n=9 for TIC; from two independent experiments). The data are shown as mean ± SD.
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6.2.2.1 Increased dosage of all three components of TIC has no effect on tumour
retardation.
Having established that the TIC treatment had no effect on melanoma growth,
let alone eradicating them entirely; in the next experiment, the dosage of all three
components of the TIC was doubled.
Mice were inoculated with B16 melanoma cells and the treatment with double
the dosage of all components of the TIC, called dTIC, was administered when the
tumours reached a size of 9mm2. Figures 6.3A and B show that despite the increased
dosage of all three cocktail components, the treatment had no effect on the melanoma
growth in the treated mice when compared to mice left untreated (p=0.78, significance
determined by log-rank test). Even 2 days post treatment with dTIC, the average
tumour size was 11.75 (±3.97) mm2 and this was found to be not significantly different
from the untreated controls with average tumour size of 11.83 (±3.68) mm2 (p=0.97
significance determined by un-paired student t-test). All dTIC treated mice reached end-
point tumour sizes of 100mm2 at the same time as the untreated tumour group (median
survival for both being 10 days).
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Figure 6. 3 Double the dosage of all three components in the TIC has no effect on melanoma growth in C57BL/6J mice. C57BL/6J mice were inoculated s.c with 5 × 105 B16 cells and dTIC treatment was initiated when the tumours reached 9mm2. dTIC (▲) consisted of anti-CD25mAb, anti-CTLA-4mAb and anti-TGF-βmAb given simultaneously at a dosage of 4 µg of all three components. Mice were monitored daily for (A) survival and plotted as Kaplan-Meier survival curve with 100 mm2 tumour as endpoint and (B) tumour growth (mean ± standard deviation) was calculated with measurements made using microcalipers. Untreated mice (●) were used as tumour growth controls. (n=3 for untreated mice and n=5 for dTIC; from one experiment).
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6.2.3 Transient period of EO771 breast carcinoma tumour growth retardation in
C57BL/6J mice treated with TIC.
Simultaneously, the efficacy of the TIC and dTIC were examined against EO771
breast carcinoma tumours that are also syngeneic to C57BL/6J mice. EO771 tumours
are adenocarcinoma of the mammary gland that are of spontaneous origin (Dunham and
Stewart, 1953). The tumour cells were inoculated s.c and treatment with either TIC or
dTIC was initiated when the tumours reached a size of 9mm2.
Figure 6.4 shows, compared to the tumours growing in the untreated control
group, a transient delay in tumour growth was observed in the TIC and dTIC treated
mice groups. Even by day 8, while the average tumour size of untreated controls was at
53 (±15.3) mm2, the tumour sizes were significantly smaller in the TIC treated with an
average of 22.5 (±6.4) mm2 and in the dTIC treated with a tumour size of 20.3 (±10.01)
respectively (p=0.01 and p=0.03 respectively, significance determined by un-paired
student t-test). The tumour sizes were not significantly different between the TIC and
dTIC treated groups (p=0.73). Despite the fact that the tumours grew slower in the
treatment groups, the time to end-point (median survival) was inconclusive, as the i.t
treatments lead to ulceration with exudation from within the tumours and the mice had
to be culled before end-point tumour sizes could be reached. 2/4 mice in the TIC group
and 2/3 mice in the dTIC group developed ulcers (a health concern under animal ethics
guidelines), and the mice had to be humanely culled.
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Figure 6. 4 Treatment with TIC or dTIC did not significantly improve survival of mice bearing EO771 tumours. C57BL/6J mice were inoculated s.c with 5 × 10
5 EO771 breast carcinoma cells and treatment with either TIC (■) or
dTIC (▲) was initiated when the tumours reached 9mm2 (5 ± 2 days). Mice were
monitored daily for tumour growth (mean ± standard deviation), calculated with measurements made using microcalipers. Untreated mice (●) were used as tumour growth controls. (n=3 for untreated and dTIC treated mice and n=4 for TIC treated groups, from one experiment). Significant differences in tumour growth between untreated controls and treated mice determined by t-test are indicated: *p<0.05 and ** p=0.01 respectively.
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6.2.4 Increased survival time in 4T1 tumour-bearing BALB/c mice treated with
either TIC or dTIC.
The efficacy of the TIC and the dTIC were then trialled in the BALB/c mice
bearing s.c 4T1 breast carcinoma tumours. The 4T1 breast carcinoma originates from
410.4 tumours that were in turn isolated from a spontaneously arising mammary tumour
of BALB/c mice (Dexter et al., 1978).
Figure 6.5 shows that TIC (median survival of 14 days) was as effective as dTIC
(median survival of 15 days, p=0.6073) in increasing the survival time of treated mice
compared to untreated controls (median survival of 11 days, p= 0.01 and p=0.0042,
respectively). Nevertheless, 100% of TIC (5/5) and dTIC (5/5) treated mice developed
tumours to end-point of 100 mm2. In the mice treated with dTIC, hollow depressions
were seen with some ulceration within the tumours, 6 ± 2 days post treatment. However,
these ulcerations were exudate free and by day 9, had healed completely as shown in
figure 6.6, leaving a centralised zone of tumour clearance, with raised periphery. The
tumours in the periphery eventually grew to end-point tumour sizes (Figure 6.6).
Overall, a significant improvement in survival with TIC or dTIC was only observed in
the 4T1 breast cancer cells syngeneic to BALB/c mice.
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Figure 6. 5 Improved survival with TIC or dTIC in BALB/c mice bearing 4T1 tumours, compared to mice left untreated. BALB/c mice were implanted sub-cutaneously with 5 × 105 4T1 breast carcinoma cells and treatment with either TIC (■) or dTIC (▲) was initiated when the tumours reached 9mm2 (4 ± 1 days). Mice were monitored daily and the pooled data from two experiments were plotted as Kaplan-Meier survival curve with 100 mm2 tumour as endpoint. Untreated mice (●) were used as tumour growth controls. (n=7 for untreated and n=5 for TIC and dTIC treatment groups).
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Figure 6. 6 Central zone of tumour clearance observed in mice post treatment with dTIC. BALB/c mice treated with the dTIC when the 4T1 tumours reached a size of 9mm2 were monitored daily and photographs were taken on day 6 (A), day 9 (B) and day 13 (C) post treatment initiation. The ulceration and the eventual development of central zone of hollow tumour clearance shown in the pictures were from the same mouse, and representative of all mice in the dTIC treatment group.
A B C
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6.2.5 Efficacy of the triple immunotherapy is diminished with an increase in tumour
burden.
Efficacy of immunotherapy against varying tumour burdens is clinically
relevant; as patients report to the clinic at varied stages of disease progression. The
effectiveness of the triple immunotherapy against a larger tumour size was, therefore,
trialled.
In this experiment, AB1 tumour cells inoculated sub-cutaneously were allowed
to grow to a size of 25mm2 before the treatment with TTI was started. Figure 6.7 shows
that compared to the untreated controls (median survival of 14 days), the mice with
large tumours, treated with the TTI had a significant improvement in their survival
(median survival 24 days, p=0.006). However, only 20% of mice (1/5, from one
experiment) receiving the TTI completely cleared the large AB1 tumours. This was a
significantly diminished efficacy in tumour eradication from the 100% achieved when
the TTI treatment was initiated when the tumours were 9mm2 (p<0.001).
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Figure 6. 7 Tumour eradication efficacy of TTI treatment lowered with increase in tumour burden in AB1 tumour bearing BALB/c mice. Mice were implanted s.c. with 1 × 106 AB1 tumour cells. Treatment with TTI (■) was initiated at a tumour size of 25 mm² (17 ± 3 days) with tumour growth compared to untreated mice (●). Mice were monitored daily and survival was plotted as Kaplan-Meier survival curve with 100 mm2 tumour as endpoint (n=3 for untreated mice and n=5 for TTI treated mice, from one experiment).
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6.2.5.1 TIC treatment was effective in generating partial concomitant immunity– a
preliminary study on the effect of TIC on distal tumours.
Mesothelioma is a metastatic tumour, and whilst they are slow growing and have
a latency period of 30-40 years, secondary metastases are always a feature of the
disease. In order to completely eradicate mesothelioma, an ideal immunotherapy would
be able to cause complete regression in not only the primary tumour that is directly
treated, but also initiate immune activation, to result in secondary tumour regression at
distal sites.
In this experiment, BALB/c mice were simultaneously co-challenged sub-
cutaneously with AB1 cells on both flanks. Tumour growths on both right and left
flanks were monitored daily, and the TIC treatment into the primary tumour (growing
on the right flank) was initiated when these tumours reached a size of 9mm2. The
growths of the primary and secondary tumours are split into two separate tumour
growth graphs (Figures 6.8A and 6.8B respectively) for better understanding of the
growth kinetics in these mice that were challenged on both flanks.
Figure 6.8A shows the tumour growth of the TIC treated primary tumours in
comparison to untreated controls. Growth of the primary tumours post TIC (the red line
in the graph) were split into cured and non-responders as only 40% of the mice (2/5,
from one experiment) that received the TIC treatment underwent complete tumour
regression of their primary tumours. The other 60% of the mice (3/5) did not respond to
the treatment (non-responders) and grew end-point tumours of 100 mm2. By day 18, the
average sizes of these primary tumours in the mice that did not respond to the TIC
treatment were 70.66 (±25.40) and were not significantly different to the untreated mice
that had an average end-point tumour size of 100.33 (±16.74) mm2 (p=0.16).
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Figure 6.8B shows the tumour growth of only the untreated secondary tumours
(on the contralateral flank) in the mice that were cured of the primary tumours and the
mice that did not respond to the TIC treatment. When it came to these secondary
tumours, all 100% of the tumours developed. However, the tumour growth of the
secondary tumours in the mice that were cured of their primary tumours (2/5) was
significantly retarded when compared to the sizes of secondary tumours in the mice that
did not respond to the TIC treatment. By day 20, the secondary tumour sizes of the TIC
non-responders were 83.08 (±13) mm2, while the secondary tumours in the TIC cured
mice were 32.5 (±10.60) mm2 (p=0.02). This shows that there is some partial
concomitant immunity generated in the mice that were cured of their primary tumours,
as the secondary tumours in these mice grew significantly slower. However, the
immunity was not sufficient to completely eradicate tumours.
Overall, though TIC treatment mediated tumour eradication was only 40% (2/5)
efficient in the mice with distal tumours, their secondary tumours continued to grow
progressively in the same mice, but at a much lower rate compared to the 60% that did
not respond to the treatment at all.
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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. BALB/c mice were implanted sub-cutaneously with 1 × 106 AB1 mesothelioma on both right and their left flanks and treatment with TIC was initiated when the tumours on the right flank reached 9mm2. Mice were monitored daily and tumour growths on (A) right flank and (B) left flank were calculated with measurements made using microcalipers. n=2 for the TIC mediated cured mice (♦) and n=3 for TIC non-responders (■). n=3 for untreated mice (●) that were only implanted with AB1 cells on their right flanks. Data are shown as mean ± standard deviation, from one experiment.
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6.3 Discussion
Despite the high level of Tregs detected within the non-mesothelioma tumours, the
TIC treatment was ineffective, as 100% of the mice that were treated with the TIC grew
end-point tumours; thereby rejecting the hypothesis. The primary work described in this
chapter focussed on testing the efficacy of the triple immunotherapy cocktail (TIC) in
different non-mesothelioma tumours that were syngeneic to both C57BL/6J and
BALB/c mice. All three components which constitute the triple immunotherapy directly
or indirectly involved removal of immune suppression within the tumour
microenvironment mediated by Tregs. In this study, the intention was to examine Treg
levels at the time of TIC treatment initiation (which was an established size of 9mm2).
However, the technical difficulties of losing cells during the preparation of these small
tumours for flow cytometry required the use of larger tumours. From the previous work
performed on AE17 tumours at various sizes by Needham et al, 2006, it is known that
percentage of Tregs accumulating within tumours do no significantly increase between
9mm2-30mm2 and it is only in tumours greater than 30mm2 that a difference is observed
(Needham et al., 2006). In the light of this historic data on Treg accumulation in AE17
tumours, these non-mesothelioma tumours were inoculated sub-cutaneously into their
respective mice strains and allowed to grow to sizes between 16 - 25mm2.
The role of Tregs in immune responses to growing tumours has been documented in
several studies that looked into depletion/inhibition of these cells by anti-CD25mAb;
which subsequently led to increased Teff activation and a potent antitumour immune
response. (Onizuka et al., 1999; Shimizu et al., 1999). The success of Treg depletion on
tumour eradication has been varied towards different tumour types raised against
different murine strain backgrounds (Ostrand-Rosenberg et al., 2002; Chaput et al.,
2007; Bergot et al., 2010; Kline et al., 2012); including 80-100% in the AB1 and 50-
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100% in the AE17 mesothelioma studies; detailed in this thesis (and published in
Kissick et al., 2012 and Krishnan et al., 2014).
However, in the experiments conducted on the non-mesothelioma tumour models,
despite the Treg populations being determined within these tumours, the TIC and dTIC
treatments did not predict the effectiveness in eradicating established tumours. Testing
the TIC at 4µg of all three components (dTIC) was based on the Treg contents
determined within these non-mesothelioma tumours. With the dTIC treatment,
improved survival of the tumour-bearing mice was found in the 4T1 breast model and a
transient delay in tumour growth with the EO771 breast model. In the low immunogenic
B16 melanoma, the dTIC treatment made no impact on tumour growth when compared
to the normal growth kinetics observed in the untreated control group (see Figures 6.3A
and B). In future, experiments testing the efficacy of the TIC at a much larger range of
concentrations (titration studies) can be carried out. These future studies can include (a)
dosage titration of anti-CD25mAb alone in the TIC, to better deplete all Tregs, or (b)
different dosages of all three components can be tested and/or (c) existence of other
immunosuppressive mechanisms operating within these tumours also need to be taken
into consideration for formulating future treatments. With such titration studies, there is
the possibility of spillage into the systemic immunomodulation at higher concentrations.
So these factors need to be taken into account, and the mice need to be monitored for
systemic auto-immune symptoms such as weight loss, lethargy, reduced appetite,
bloating and diarrhoea.
From the previous experiments detailed in chapter 4, it was known that TIC
treatment was effective in eradicating AB1 tumours-where less than 15% of the CD4+ T
cells present within the tumours were Tregs (Figure 6.1B). However, in the case of the
B16 melanoma tumours, the average percentage of CD4+ T cells that are FoxP3+ was
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~2.7 fold higher (40%) than the levels present in the AB1. Despite the high Treg content
within B16 tumours, targeting immune suppression by Tregs is clearly not enough in the
B16 melanoma model. The role of Tregs as the primary regulators of immune
suppression has been elucidated in previous studies carried out on B16 tumours (Turk et
al., 2004). Perhaps increasing the dosage of anti-CD25mAb (that depletes Tregs) alone in
the TIC could in fact, overcome the immune suppression and eradicate the B16 tumours
in tumour-bearing mice. So in the future, titration studies involving anti-CD25mAb in
the TIC could potentially enable the eradication of B16 tumours.
In the case of the EO771 breast carcinoma tumours, a transient period of growth
retardation was observed in both TIC and dTIC treatment groups (Figure 6.4). However,
2/4 in the TIC and 2/3 in the dTIC treatment groups had to be humanely culled due to
their development of ulceration with exudates at the tumour site and thereby, calculation
of median survival for the treatment groups was skewered (inconclusive). Also, it has to
be mentioned that the data presented was from only one experiment with small
treatment groups. It would have been beneficial to repeat the experiment with bigger
TIC and dTIC treatment groups. This would have also assisted in determining whether
the ulceration at the tumour site was (a) a rare incidence, or (b) if the intra-tumoural
delivery of all three components was highly effective in abrogating the immune
suppression, leading to increased tumour necrosis, which in turn led to ulceration at the
tumour site. In the case of the other breast cancer model, the 4T1 in BALB/c mice,
compared to untreated mice group, a significant increase in overall survival when
treated with TIC or the dTIC was observed.
Ulcerations were also observed in these 4T1 tumour bearing mice treated with the
dTIC (similar to EO771). However, in this case, the ulcerations were observed in all 5
mice that received the dTIC treatment (Figure 6.6A). Nevertheless, these ulcerations
187
healed quickly wherein the centre had collapsed onto itself and presented with a zone of
clearing in the centre of the tumours, with no exudates; leaving the impression of a ring
with hollowed out centre (Figure 6.6B). This eventually led to a central zone of
clearance with an raised edge, as the tumour cells in the periphery kept growing, and
these tumours with the hollowed out centre reached end-point sizes of 100mm2 (Figure
6.6C).
Treatment associated ulceration is not necessarily a negative response and one such
study that supports this concept was the adjuvant interferon (IFN) therapy used for the
treatment of melanoma by McMasters et al (2010). In their study, ulceration was found
to be a predictive marker for response in the stage III melanoma patients to IFN therapy
(McMasters et al., 2010). The authors also mentioned that perhaps the ulceration is
related to a greater inclination to vascular invasion of the tumours. A similar conclusion
of ulceration being a factor of good response to adoptive immunotherapy of tumour
infiltrating lymphocytes (TIL), was also reached with the treatment of melanoma stage
III patients (Peuvrel et al., 2011).
This active role of ulceration could be true in the ring-shaped tumour growth found
in the 4T1-dTIC treatment experiment. It is important to note that 4T1 breast carcinoma
tumours were found to have the highest Treg percentage (Figure 6.1B) amongst the non-
mesothelioma tumour models. This ring-shaped growth could mean that the single i.t
administration did have a response in these 4T1 tumours, with this zone of clearance
observed in the centre of the solid tumour. In the future, time course experiments,
wherein the analysis by flow cytometry or immunohistological sectioning of tumours at
various time-points immediately post treatment with TIC; would help in understanding
whether ulceration is beneficial to tumour eradication.
188
However, the central zone of clearance observed post dTIC treatment, also raises the
question that perhaps the volume (30µl) injected into the solid tumours was not
sufficient to permeate the treatment throughout the solid tumour, with the corresponding
inaccessibility of Teff cell infiltration, and this eventually leading to the ring-shaped
growth of the 4T1 tumours. In the future, varied volumes could be investigated, wherein
one treatment group receives 30µl as used in the earlier experiment, but the other
receiving 50µl (the volume that was originally used for i.t injections in the TTI) of the
dTIC intra-tumourally. This will be based on the hypothesis that increased volume of
the treatment would correspond with greater diffusion within the solid tumours, leading
to improved survival.
Results detailed in chapter 4 showed that a 100% and close to a 90% cure rate
were obtained with TTI and TIC treatment respectively in the AB1 BALB/c model.
However, this efficacy was achieved when the treatment was initiated at a tumour size
of 9mm2. Greenberg (1991) suggested that the effectiveness of any immunotherapy
could be hindered qualitatively and/or quantitatively by significant tumour cell burden
(Greenberg, 1991). The TTI treatment injected into 25mm2 AB1 tumours showed that
the tumour eradication was diminished to 20% (1/5 mice undergoing complete tumour
clearance) from the 100% against 9mm2 AB1 tumours. This is clear evidence that
bigger the tumour burden (125mm3, in terms of volume), lower the effectiveness of the
treatment (Figure 6.7). This is also supported by the work conducted by other groups on
different tumours models, wherein therapies and/or adoptive transfer studies using
effector T cells were not effective against large tumour burdens (Dobrzanski et al.,
2000; Hanson et al., 2000; Jackaman et al., 2008).
At the time of increase in tumour burden, several factors have been proposed to
account for the lowered effectiveness of the treatment. They are a) inaccessibility of
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tumour tissue as the tumour grows; b) decrease in antigen specific T cell priming and c)
induction of antigen specific tolerance (Hanson et al., 2000).
With regards to inaccessibility to tumour tissue; as the tumour increases in size, the
complexity of the tissue microenvironment also increases correspondingly. The tissue
microenvironment of a developing tumour consists of extracellular matrix (ECM)
proteins, fibroblasts, signalling molecules such as cytokines/chemokines, endothelial
cells and immune cells. Immune cells infiltrating the growing tumour, together with the
crosstalk with the surrounding tissue, fibroblasts and the extracellular matrix from the
scaffold, ultimately contributes to tumour growth expansion (Whiteside, 2008; Levental
et al., 2009). This could also help explain the need for greater volumes to be tested, for
greater diffusion of the treatments within the solid tumours.
In certain cases, a decrease in T cell priming is also known to occur, where tumour-
specific T cells are activated, however undergo anergy (unresponsiveness) and are
unable to affect tumour growth (Staveley-O’Carroll et al., 1998; Shrikant and Mescher,
1999). Immune tolerance to growing tumours are mediated by regulatory cells such as
Tregs, myeloid-derived suppressor cells (MDSC) and Th17 cells, and also by
cytokines/chemokines such as TGF-β, IL-6 and IL-10 just to name a few (Shankaran et
al., 2001; Curiel et al., 2004; Willimsky and Blankenstein, 2005; Lin and Karin, 2007;
Rabinovich et al., 2007; Gabrilovich and Nagaraj, 2009).
In the AE17 murine mesothelioma model, complete tumour eradication was
achieved due to the timed neutralization (for 7 consecutive days) of excess TGF-β
secreted by the growing tumour with the timed triple immunotherapy (chapter 3 and
Kissick et al, 2012). This 50% was improved to a 100% with the increased dosage of
anti-TGF-βmAb alone in the modified TTI, detailed in chapter 4. In the non TGF-β
secreting AB1 mesothelioma model, the TIC treatment formulation was developed
190
where a single treatment was sufficient to eradicate as effectively as the TTI (Krishnan
et al., 2014). With the focus now shifting more towards the neutralization of TGF-β in
the tumour microenvironment being pertinent to tumour eradication, perhaps the first
step for future studies on tumour burden should involve analysing TGF-β concentrations
as the tumours grow (by ELISA), and this could perhaps point towards dose titration
studies involving anti-TGF-βmAb and keep the other two components constant in the
TIC treatment protocol.
Removal of the immune suppression within the tumour microenvironment by i.t
treatments with avoidance of systemic toxicities have also been the focus of this thesis
so far. However, with the lowered effectiveness in the treatment of larger tumour
burdens, it is clear that the tumour burden is also an important factor that affects
efficacy of immunotherapy. This importance of both tumour burden and overcoming
intra-tumoural immune suppression were confirmed in the experiment where mice were
challenged on both flanks with AB1 cells and only one of the 9 mm2 (27 mm3 in terms
of volume) established tumours were treated. Intra-tumoural TIC treatment into the
primary tumours only aided in clearing these tumours in 40% (2/5) of the treated mice
(as opposed to the 5/5 cures usually noted) and no cures with the non-injected distal
tumour were observed, although this second tumour did grow significantly more slowly
(Figure 6.8B). This delayed growth of an already established secondary tumour could
be due to the partial activity of concomitant immunity. Concomitant immunity is a
process in which tumour-bearing hosts develop resistance to growth of secondary
inoculations of the same tumour. Tumour metastases (dissemination) are considered as
secondary tumours that develop concurrent to primary tumour growth and concomitant
immunity is considered as a means to control the development of these metastases
(Ruggiero et al., 1988).
191
The importance of eliciting concomitant immunity to combat secondary tumours
is further supported by a recent study conducted by Korrer and Routes (2014). In this
study, mice inoculated with MCA-205 fibrosarcomas transfected with ovalbumin
(OVA) on one flank, rejected a subsequent secondary inoculum of the MCA-205-OVA
tumours on the contralateral side in a OVA-specific T cell-mediated response (Korrer
and Routes, 2014). In another study, B-1 cells (subset of B lymphocytes) were
selectively obtained from BALB/c mice bearing Ehlrich tumours (derived from
mammary adenocarcinoma) developing in their footpad. These B-1 cells that were
adoptively transferred; were able to protect naïve BALB/c and BALB/c xid (B-1 cell
knockout) mice against primary tumour challenge with the same tumour type. This was
an example of concomitant tumour immunity being generated in a different mice post-
adoptive transfer of tumour primed B-1 cells (Azevedo et al., 2014).
Such examples are relevant to understanding the importance of concomitant
immunity. However, the preliminary distal tumour experiment detailed in this thesis is
significantly different, as the mice were co-challenged simultaneously and were not
challenged a few days apart. The tumour burden in this experiment, in toto, was 54 mm3
(2 × 27 mm3 tumours, based on volume). The tentative impression is that having two
tumours on a single mouse may be the same as a single 54 mm3 tumour, making the
overall treatment less effective. This analysis showed that the successful tumour
eradication and the corresponding anti-tumour immunity raised were not sufficient to
eradicate distal tumours due to an increase in tumour burden. Further investigation is
required, where mice with tumour on both flanks would be used as untreated controls
and the treatment groups that can be tested are:
a) dTIC treatment into only the right flank,
b) Target only right flank with dTIC at a higher volume (50 µ1),
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c) Target both right and left flank tumours with TIC at 9mm2,
d) Increase only the dosage of anti-TGF-β (preliminary study before testing in the
non-mesothelioma models) and
e) Administer i.t TIC into one tumour in conjunction with i.v lysed tumour
antigens (in vitro cultured AB1 cell lysates) to boost antigenic exposure of the
immune system.
These follow-up experiments could help in understanding the correlation between
tumour burden and the tolerance maintained within these growing tumours.
Furthermore, the data from the above experiment in addition to the TGF-β
concentrations assessed within the growing tumours (both mesothelioma and the non-
mesothelioma) could aid in making TIC as effective against the non-mesothelioma
tumours, as it is against mesothelioma tumours.
6.4 Conclusion and future perspectives
Current mesothelioma treatments are of low efficacy and provide minimal
improvement in survival rates. Immunotherapy is of particular interest for cancer
treatment, as it aids in overcoming immune evasion mechanisms employed by the
growing tumours and boosts the host’s anti-tumour immune response. The combined
triple immunotherapy combination using three agonist antibodies (anti-CD25mAb, anti-
TGF-βmAb and anti-CTLA-4mAb) produced complete tumour clearance in both the
C57BL/6J and BALB/c murine mesothelioma tumour models. The immunity generated
in these cured mice protected these mice against further re-challenges. All the treatment
components that constitute the triple immunotherapy have human equivalents and are
approved for use or are undergoing clinical trials. There is clinical potential for the
triple immunotherapy treatment to be used in clinical trials for treating mesothelioma
patients.
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6.4.1 Future of the triple immunotherapy
Future investigation into the efficacy of the triple immunotherapy in more highly
relevant pre-clinical models such as intra-thoracic mesothelioma model and mammary
carcinomas grown in the mammary pads of mice for the breast cancer models are
warranted. However, there are many factors that could affect the delivery of the
treatments intra-tumourally in these tumour models. Perhaps strategies such as CT,
endoscopy or ultrasound guided deliveries could enable the precisions with which the
treatments are delivered intra-tumourally. With the setback of treating larger tumours
with the triple immunotherapy, perhaps combination of chemotherapy with
immunotherapy can also be tested in the near future. While chemotherapy induces
lymphopenia in recipients, there is much research that indicates that a number of
chemotherapeutic agents provide some benefit to the anti-tumour function of the
immune system. Several mechanisms have been proposed for how this occurs, including
increase in antigen availability due to tumour cell death, with activation of dendritic
cells by apoptotic cells or the release of damage associated molecular patterns (Apetoh
et al., 2007), lymphablation of suppressive cells (North, 1982; Ghiringhelli et al., 2007)
and increased immunogenicity of tumour cells (Galetto et al., 2003). With this in mind,
certain chemotherapy combinations have been used with immunotherapy to good effect,
in both human and animal models, including gemcitabine/cisplatin and 5-
fluorouracil/cisplatin (Tanaka et al., 2002). Another possibility is a combination of
debulking surgery followed by immunotherapy. Surgery to remove mesothelioma
tumours, followed by radio- and/or chemotherapy forms part of the current treatment for
MM in the clinic (Sugarbaker et al., 1999). Resection of the tumour to decrease tumour
load with the corresponding increase in tumour antigen exposure will aid in effective
anti-tumour immunity being generated following immunotherapy.
194
6.4.1.1 Addition of more agonist antibodies to the TIC treatment to improve tumour
regression
In a recent article published in JEM, studies involving Treg depletion in mice
with chronic lymphocytic choriomeningitis virus (LCMV) showed that an up-regulation
of PD-L1 on T cells occurred, leading to inhibition of signals (Penaloza-MacMaster et
al., 2014). The authors also found that treatment combination of Treg depletion and PD-
L1 blockade resulted in a significant reduction in viral titres than when the treatments
were administered as monotherapies. Blockade of PD-1 by agonist antibody (BMS-
936558) have already been shown to have varying levels of objective responses in
patients with non-small-cell lung cancer, melanoma and renal-cell carcinomas (Topalian
et al., 2012).
In a more recent article, intra-tumoural treatment of 3mAb (CD137/CTLA-
4/PD-1) and 4mAb (CD137/PD-1/CTLA-4/CD19) combinations were tested by Dai et
al (2014) for treating sub-cutaneous murine models of B16 and SW1 melanoma and
TC1 lung carcinoma (Dai et al., 2014). The 4mAb combination treatment was found to
eradicate large tumours (~80mm2) in 50% of the treated mice. The treatment involved
injecting 0.25 mg of each of the monoclonal antibodies intra-tumourally into these large
tumours. Systemic toxicities associated with the aforementioned high dose intra-
tumoural treatment included hepatitis in liver, hair loss and depigmentation.
With the successful eradication of large tumours being achieved as described
above, perhaps the addition of other agonist antibodies such as anti-PD-1mAb could
further enhance the efficacy of the TIC. It is also important to note that the maximum
amount of the monoclonal antibodies that we have tested in our laboratory has been 4
µg. With mAb amounts that are 60 times more, being tested in other laboratories for
treating cancer, perhaps titrated dosages of 4mAb or even 5mAb combinations in the
195
future could be most ideal to eradicate not just large mesothelioma tumours, but other
non-mesothelioma tumours as well.
The significance of this work will aid in the development of a treatment
approach that will improve the poor outcomes for patients diagnosed with different
aggressive and incurable malignant cancers.
196
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