University of Southern California Dissertations and Theses

LIGHT-ing up prostate cancer: remodeling the tumor microenvironment and enhancing therapeutic vaccine efficacy through forced LIGHT expression in prostate cancer

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LIGHT-ing up prostate cancer: remodeling the tumor microenvironment and enhancing therapeutic vaccine efficacy through forced LIGHT expression in prostate cancer

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Content i

LIGHT-ING UP PROSTATE CANCER:
REMODELING THE TUMOR MICROENVIRONMENT AND ENHANCING
THERAPEUTIC VACCINE EFFICACY THROUGH FORCED LIGHT
EXPRESSION IN PROSTATE CANCER
by
Lisa Yan

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the Requirements of the Degree
DOCTOR OF PHILOSOPHY
(GENETICS, MOLECULAR and CELLULAR BIOLOGY)

MAY 2015

Copyright 2014 Lisa Yan
ii

The moment you doubt whether you can fly, you cease for ever to be able to
do it.
- J.M. Barrie

iii

Dedication

This dissertation is dedicated to my parents, Mr. Yan King Man and Mrs. Yan Situ
HuiJuan, who supported me without a question and to Jason, Wilson, Bebe and OC, who
believe in me. Thank you for all the love and support you have given me through this
journey, because of you all, I am a better person.
iv

Acknowledgements

To W. Martin Kast , my mentor: You have been the most supportive adviser I have ever
come across, your knowledge, comfort and counseling has gotten me through successes
and failures in the past 5 years. I owe you a tremendous debt of gratitude and I will never
forget what you have done for me.
To Diane Da Silva, my friend and immunology guru: I remember interviewing at the
Kast lab and you were the last person I spoke with, I jokingly asked “Is the lab always
this quiet?” and you replied “Yes” with a stern face. I was nervous, but after joining the
lab and becoming part of the family, I have connected with you and trust you as a friend
and as my mentor. You have given me so much support on my projects and late-night
mouse experiments, no words can describe how much I appreciate you, your help and
your knowledge.
To the members of the Kast lab, my colleagues: Thank you all for being so kind,
encouraging and supportive through successes, failures, and frustrating moments in the
lab. Thank you Bhavna Verma, Brooke Nakumura, Heike Brand and Tania Porras
for believing in me.
To my committee members, Michael K. Wong and Omid Akbari: Thank you so much
for all the time and advice you have given me. Dr. Wong, thank you, you have been so
supportive throughout my doctoral degree, I cannot describe how thankful I am to have
you as my committee member. Dr. Akbari, you have always been so encouraging and I
appreciate the effort and time you have put into my future career, thank you for
contributing to the success of my future.
To my colleagues and friends: Emily Adler, Damian Wang, Ying Wu, Junko
Yaegashi, and Megan Yardley, I will not be here today without your utmost support and
encouragements. I appreciate and love you all dearly, thank you for everything.
To my family: Mom, Dad, Jason, Wilson, Bebe and Ong Choy. I could not have done it
without your support and love. Thank you for all the joy you guys have brought me
through the good and hard times.
To Alvin Lee: No words can describe how thankful I am for the support you have given
me through my journey. Thank you so much for being my friend, support and partner, I
appreciate all the attention you have given me during good and bad times.

v

Table of Contents

Dedication .......................................................................................................................... iii
Acknowledgements ............................................................................................................ iv
List of Tables .................................................................................................................... vii
List of Figures .................................................................................................................. viii
Thesis Narrative .................................................................................................................. 1
Chapter 1 Introduction ....................................................................................................... 2
1.1 The prostate, prostate cancer and current treatments ........................................... 2
1.2 Tumor associated antigens and prostate cancer immunotherapy ......................... 5
1.3 Immune activation: Roles of T cells and antigen presenting cells ..................... 11
1.4 Immune suppression: Regulatory T cells ........................................................... 13
1.5 LIGHT (Lymphotoxin-like inducible protein that competes with glycoprotein D
for HVEM on T cells) ................................................................................................... 14
1.6 Prostate Stem Cell Antigen TriVax: An immune stimulating treatment against
prostate cancer ............................................................................................................... 18
1.7 Hypothesis and Working model: LIGHT’s role in remodeling the tumor
microenvironment ......................................................................................................... 19
Chapter 2 Forced LIGHT expression in prostate tumors overcome Treg mediated
immunosuppression and synergizes with a prostate cancer therapeutic vaccine by
recruiting effector T lymphocytes. .................................................................................... 23
2.1 Abstract .............................................................................................................. 23
2.2 Introduction ........................................................................................................ 25
2.3 Methods and Materials ....................................................................................... 28
2.4 Results ................................................................................................................ 39
2.4.1 Combined Ad-LIGHT and PSCA TriVax therapies synergize to reduce tumor
burden ........................................................................................................................ 39
2.4.2 Ad-LIGHT synergizes with PSCA TriVax by increasing TIL numbers. ......... 42
vi

2.4.3 LIGHT treatment alters the tumor microenvironment from an
immunosuppressive to an immunostimulatory milieu. .............................................. 45
2.4.4 LIGHT prevents the maturation of Tregs from naïve CD4+ T cells and
compromises the suppressive function of existing Tregs. ......................................... 49
2.4.5 PSCA TriVAX induces memory T cells in TRAMP-C2 re-challenge study ... 52
2.4.6 Induction of pro-inflammatory responses improves the efficacy of PSCA
TriVax in the TRAMP model .................................................................................... 53
2.5 Discussion .......................................................................................................... 55
2.6 Conclusion .......................................................................................................... 63
2.8 Acknowledgement .............................................................................................. 64
Chapter 3 Working Model, Pitfalls and Future Directions .............................................. 65
3.1 Working model: Current view of : LIGHT’s role in remodeling the tumor
microenvironment ......................................................................................................... 65
3.2 Pitfall: Delivery and intratumoral expression of Ad-LIGHT ............................. 68
3.3 Future direction: Targeted therapies: Development of Bi-specific Fusion
Proteins with LIGHT ..................................................................................................... 69
3.5 Research Summary ............................................................................................. 71
Chapter 4 Additional Projects ......................................................................................... 72
4.1 Publications by the author ....................................................................................... 72
References ......................................................................................................................... 73

vii

List of Tables

1.1 Expression of LIGHT, LT βR and HVEM. 15
2.1 Number of Mice undergoing tumor challenge, tumor resection and 52
tumor re-challenge.

viii

List of Figures

1.1 Prostate gland 2
1.2 BCR-ABL chromosome 9 and 22 translocation 7
1.3 Sipuleucel-T processing and assumed mechanism 10
1.4 Immune response and immune tolerance via DC and T cell activation 12
1.5 Inhibitory mechanisms of Tregs 14
1.6 Binding partners of LIGHT and HVEM 16
1.7 LIGHT’s Role in various tumor models 17
1.8 Immune modulating properties of forced LIGHT expression 20
1.9 Working model 22
2.1 Vaccination scheme with LIGHT injections alone 29
2.2 Tumor excision and rechallenge study 34
2.3 Surgical procedure of intraprostatic injections of Ad-LIGHT into 37
TRAMP mice
2.4 Membrane bound LIGHT expression in TRAMP-C2 cells peaks within 40
the first 48 hours of infection
2.5 Ad-LIGHT and PSCA TriVax therapeutic vaccination delays 41
TRAMP-C2 tumor growth
2.6 Tumor growth curve of untreated, Ad-Control and Ad-LIGHT treated 42
tumor bearing mice
ix

2.7 Intratumoral LIGHT treatment in combination with PSCA TriVax shows 43
an increased number of CD4+ and CD8+ in a prostate cancer tumor model.
2.8 Combined Ad-LIGHT and PSCA TriVax vaccination shows an 44
increased trend in TAA specific T cells
2.9 TAA specific T cells after untreated, Ad-Control, or Ad-LIGHT treatment. 45
2.10 Phenotype of tumor infiltrating lymphocytes. 46
2.11 Ad-LIGHT treatment modulates the tumor microenvironment to an 48
immunostimulatory environment
2.12 Intratumoral expression of LIGHT reduces the frequency of induced 50
Tregs and causes existing Tregs to lose their suppressive capacity.
2.13 Untreated and Naïve B6 mice display functional Tregs. 51
2.14 Prostate tumor weights of TRAMP mice at 20 weeks of age 53
3.1 Revised working model 66
3.2 Differentiation of Naïve CD4+ T cells to effector cell types. 67
3.3 Delivery of Ad-LIGHT via intratumoral injections. 68

1

Thesis Narrative

Prostate cancer affects approximately 200,000 men and account for 30,000 deaths
annually in the United States alone. This major health problem cost Americans $255.6
million dollars in health care and even then, current treatments for prostate cancer
including surgery, chemotherapy and radiotherapy, may lead to devastating side effects
such as impotence and incontinence. Prostate cancer immunotherapy offers an advantage
to patients, avoiding these potential side effects, where a patient’s own immune system is
stimulated and taught to eradicate the existing tumor. The goal of this research is to
improve current therapeutic vaccines by boosting the immune system with a new
treatment, LIGHT, in combination with a prostate cancer therapeutic vaccine. This study
will assess the efficacy of combination immunotherapy treatment and measure survival in
a prostate cancer mouse model that mimics prostate cancer progression in a subset of
patients. With this translational project, we will generate a synergistic combination
therapy that will push the field of prostate cancer immunotherapy forward, providing a
curative option for patients of this disease.

2

Chapter 1

Introduction

1.1 The prostate, prostate cancer and current treatments
The healthy human prostate is typically a small walnut-sized organ, part of the male
reproductive system that is responsible for sexual response and secretion of prostatic
fluids during sexual activity (1). This organ is directly located below the bladder,
surrounds the urethra and is connected to the seminal vesicles and vas deferens (2). The
prostate is made up of 4 different lobes: anterior, median, lateral and posterior lobes (3).
These lobes play an important role in diagnosis and the development of enlarged
prostates which may be due to benign prostatic hyperplasia, prostatitis or prostate cancer.
Enlargement of the prostate may cause urinary and sexual discomfort due to its close
proximity to the urethra (Figure 1.1).

Figure 1.1 Prostate gland. The prostate is located below the bladder, surrounding the
urethra (4). The prostate gland is sectioned into 4 different zones.
3

The health of the prostate is evaluated through various screening tools that are
recommended for men over the age of 40, an annual exam may include digital rectal
exam (DRE), prostate-specific antigen (PSA) blood test and/or a transrectal ultrasound
(TRUS) (5-7). Various screening tools are readily accessible for patients, especially in
first world countries where screening is more prevalent. However, there are many
controversial discussions about the accuracy of these screening tools. A simple blood test
evaluating the serum PSA levels may determine the risk of prostate cancer, a value above
4 ng/ml may yield red flags, yet false-positives or false-negatives may easily be
influenced by hormones and sexual activity prior to testing (8,9). There are many areas of
uncertainty with the PSA blood test due to the lack of randomized early-detection trials of
prostate cancer and the varying levels of PSA in the blood serum. The most routine
examination for prostate cancer is the DRE (10), where physicians evaluate abnormalities
of the posterior prostate based on size and rigidity, this tool provides an accurate screen
for diagnosing and clinical staging of prostate cancer. However, patients complain about
discomfort and physical pain during a DRE, which is the leading barrier to prostate
examinations (11-13). These challenges have led to many clinical trials that have
attempts to develop improved screening tools and reevaluate current methods to diagnose
and stage prostate cancer.
Prostate cancer is the second most common cause of cancer related death in men in the
United States and is most commonly diagnosed in men over the age of 68 (14). Elderly
patients who are diagnosed with prostate cancer usually do not die from the tumor itself
but of other causes, which is why physicians recommend “active surveillance” to early-
4

stage prostate cancer patients who do not wish to undergo surgery or radiation (15).
Active surveillance was previously termed as “watchful waiting,” however due to
negative controversy of the “stagnate” terminology; the term active surveillance took
over and encourages routine screening to maintain observation of the tumor stage (16).
Patients with localized disease may opt to do various treatments or combination
treatments including cryotherapy, radiation therapy, hormone therapy and/or radical
prostatectomy. Cryotherapy delays tumor cell growth and reproduction through low
temperature treatment in the localized organ, the mechanism of this therapy is to freeze
the cytosol of tumor cells, decrease inflammation, and constrict tumor blood vessels
(17,18). Radiation therapy eradicates tumor cells via high-energy rays that are aimed
towards the disease area, which may include the prostate gland or areas of bone
metastasis (19). Hormone therapy is primarily used in patients where prostate cancer
spreads beyond the prostate, since prostate cancer cells thrive off of testosterone, cutting
this supply will delay the progression of the diseases (20). Radical prostatectomy is a
procedure where the entire prostate gland is surgically removed (21). This procedure may
be performed through various anatomic approaches , including radical retropubic (lower
abdomen incision), radical perineal prostatectomy (incision between the rectum and
scrotum), suprapubic transvesical (incision made in the bladder), and lastly, the
computer-assisted laparoscopic radical prostatectomy (several abdomen incisions
performed by a surgeon controlled robotic instrument) (22). For localized disease, these
potential curative treatments relieve patients of prostate cancer but possible permanent
side effects exist, which may include impotence and incontinence.
5

For patients with high-risk, advanced or castrate-resistant metastatic disease, the
treatment options include chemotherapy, alternative hormone therapy or immunotherapy
(23-25). The lack of curative treatments for these high-risk patients calls for the
development of novel therapies, improved treatments options and encourages men with
advanced disease to participate in clinical trials.
Immunotherapy is a preferred mode of treatment for cancer because it offers low toxicity
and may synergize with current treatments. It is a treatment option that manipulates the
patient’s immune system to identify tumor associated antigens (TAAs) that results in
targeted therapy directed towards the tumor.
1.2 Tumor associated antigens and prostate cancer immunotherapy
The prostate cancer are thought to derived from mutated prostate cells that originate from
normal prostate epithelium, therefore these transformed prostate cells may possess a high
degree of similarity with normal ‘self’ characteristic. These differences may not be
resolvable at the level of the immune system thus immunotherapy lacks a distinguishable
target resulting in a high immunological threshold, requiring a high cellular activation to
recognize the modification between self and cancer. Exceptions include virus-induced
cancer or cancer specific mutations, like HPV-associated cervical cancer or BRCA
mutation in breast cancer. In these situations, these unique tumor specific antigens may
play crucial role in generating an immune response and break self-tolerance (26). These
TAAs are important tumor targets for the field of cancer immunotherapy.
6

There are five classifications to different types of TAA: viral antigens, overexpressed
antigens, mutated cell surface glycoprotein antigens, oncofetal antigens and cell type-
specific differentiation antigens (27,28). Viral antigens are one of the most
immunogenic prospects for cancer vaccines since they are only expressed in infected
cells. The human papillomavirus (HPV) has been causally linked to the development of
cervical cancer, where the viral genome may randomly integrate into the cellular DNA of
keratinocytes and the expression of early (E) oncoproteins, E6 and E7, interfere with
tumor suppressor genes p53 and pRB (26,29). The expression of these viral antigens is
suggested to be an excellent target for cancer therapeutics. High levels of cellular
metabolism or biosynthesis are characteristics of tumor cell lines, resulting in the
overexpression of antigens such as eukaryotic elongation factor 2 (eEF2) found in
breast, prostate and lung cancer and the tyrosinase enzyme found in melanoma, all which
may trigger an immune response due to the abundant expression of these antigens
(30,31). Oncofetal antigens are proteins that are normally found during embryonic
development, prior to the development of immune-mediated self-tolerance. The re-
expression of these antigens in some adult cancers can activate an immune response since
self-tolerance to these immunogens are lacking. Mutated antigens may be tumor specific
due to point mutations, chromosome deletions, translocations or insertions in ubiquitous
proteins leading to unique tumor specific antigens. An example of a mutated tumor
antigen is the Philadelphia chromosome, BCR-ABL gene, where a genetic translocation
of chromosome 9 and 22 exchange places and drives the development of chronic
myelogenous leukemia (CML) (Figure 1.2) (32,33).
7

Figure 1.2 BCR-ABL chromosome 9 and 22 translocation. An elongated chromosome
9 (9+) and a truncated chromosome 22 (22-) is generated when the Abl1 gene on
chromosome 9 is juxtapositioned onto the BCR gene on chromosome 22. This oncogenic
truncated mutation translates into a membrane-bound protein, tyrosine kinase that is
constitutively active, resulting in unregulated cellular division. Figure taken from
creativecommons.org.

Lastly, cell type-specific differentiation antigens are expressed in tumor cells but are
not normally expressed in healthy tissues, examples of these are the cancer/testis antigens
(CTA). CTAs are normally only expressed in the testis and the prostate but can be found
in melanoma, bladder, and moderately expressed in breast and prostate cancer (34), the
abnormal expression in these organs activates the immune system resulting in T cell
specific targeting.
There are two forms of vaccine-based immunotherapy, prophylactic vaccines and
therapeutic vaccines (35). Prophylactic treatments are used on a preventative bases while
therapeutic vaccines are used after the diseases have manifested. There are currently 3
approved prophylactic treatments against cancers, Gardasil that protects against HPV 6,
11, 16 and 18 and Cervarix that protects against HPV 16 and 18, viruses that may cause
8

cervical cancer (36,37). These two preventative vaccines are aimed to prevent the
development of cervical cancer by blocking the transmission of HPV from partner to
partner. The other FDA approved vaccine is against the Hepatitis B virus which may lead
to the development of liver cancer (38). These treatments only work in healthy patients
whom have never been infected by these viruses. These vaccines operate by inoculating a
healthy uninfected patient with inactivated viral antigens that trigger an immune response
against these foreign particles, further preventing any future infections by these
pathogens.
The ultimate goal of cancer immunotherapy is to stimulate the immune system to
eradicate existing cancer. There are three types of immunotherapies: cell-based therapy,
antibody therapy and cytokine therapy. One of the most common responses effective cell-
based therapeutic vaccines elicit is tumor specific T cells against tumor associated
antigens (TAA) (39,40). Antibody therapies have had a long history of success
beginning in 2001 with Alemtuzumab against chronic lymphocytic leukemia and multiple
sclerosis (41,42). These treatments exert their cancer-specificity through the engineering
of the antibody’s targeting domain so that it recognizes tumor promoting receptors or
ligands. One of the recent triumphs in immunotherapy are the 2 FDA approved antibodies
ipilimumab (Yervoy
TM
) (43), an anti-CLTA 4 monoclonal antibody inhibitor, and
pembrolizumab (Keytruda
®
) an anti-PD1 inhibitor (44), both approved for unresectable
metastatic melanoma, which is known to be an aggressive disease with unmet medical
treatment. Interferon and interleukin cytokines are the two main cytokine
9

immunotherapies that promote an anti-tumoral effect through activation of effector T
cells and direct lysis of tumor cells (45,46).
Cancer immunotherapy has long been implicated to be a promising but has had many
hurdles translating from the bench to bedside. The top challenges include the limitation of
animal models in predicting immune response in human trials, delayed initiation of
clinical trials, and lastly, immune escape due to the complexity of heterogeneous tumors
(47). Despite these hurdles, the first cell-based immunotherapy had success in 2010 with
the first FDA approved prostate cancer immunotherapy, Sipuleucel-T (Provenge
®
).
Sipuleucel-T is a dendritic cell (DC) based vaccine, where patients monocytes are
isolated and activated with a TAA, prostatic acid phosphatase (PAP), and DC stimulator,
GM-CSF-PAP (Figure 1.3) (48,49). The cells are then re-infused back to the patient
where these DC’s will activate naïve T cells to eliminate cells expressing PAP (50).
Provenge has been shown to increase survival by 4 months in prostate cancer patients as
compared to standard of care (50).

10

Figure 1.3 Sipuleucel-T processing and assumed mechanism. Leukocytes are first harvested from asymptomatic or
minimally symptomatic prostate cancer patients via leukopheresis. Upon a high density-gradient centrifugation, monocytes are
isolated and co-cultured with GM-CSF PAP fusion protein for 2-3 days before being infused back into the patient. Once the
infused monocytes are circulating the system, these monocytes mature into activated DC mounted with PAP that may activate
PAP specific T cells. These activated T cells then migrate to the site of infection and eradicate PAP expressing tumor cells
(50).
11

1.3 Immune activation: Roles of T cells and antigen presenting cells
Therapeutic immunotherapy manipulates the patient’s immune system by activating DCs
and T cells against TAA expressing cells so that they can eradicate tumors. However,
immune tolerance poses a great challenge in prostate cancer patients (51). In an instance
where foreign antigen or viral vectors infect a cell, the immune system may easily mount
an immune response against infected cells and induce T cell killing mechanisms.
However, the immune system has a difficult time in mounting an immune response
against prostate cancer, where TAA present on prostate cancer cells are also present on
the healthy prostate cells. This phenomenon leads to immune tolerance against self-
antigens and therefore, the lack of an immune response (Figure 1.4).
DCs are specialized antigen presenting cells that survey the peripheral tissue for foreign
antigens. DC have two important receptors that facilitate antigen presentation to naïve T
cells, major histocompatibility complex (MHC) molecules and CD86 costimulatory
molecules (52). MHC molecules come in two classes, MHC Class I and MHC Class II,
the former mediates cellular immunity against viruses and bacteria and interacts with the
T cell receptors (TCR) on CD8+ T cells while the latter mediates humoral immunity and
interacts with the TCR on CD4+ T cells (52-54).
12

Figure 1.4 Immune response and immune tolerance via DC and T cell activation.
During an immune response, DCs mount viral antigenic peptides onto its MHC class
molecule and further educates naïve T cells against the foreign antigen in the lymph
nodes. T cells then proliferate and differentiate into mature T cells that may directly lyse
cells that are infected. Immune tolerance on the other hand, occurs when DC mount a
self-antigenic peptide and presents it to naïve T cells.

CD4+ T helper cells and CD8+ cytotoxic T cells play a crucial role in cell-based vaccine
that activates DCs. The latter may directly induce lysis of virally infected cells or cells
expressing TAA, via the release of inflammatory cytokines (55). CD4+ T helper cells aid
the activity of B cell antibody and maturation and differentiation of CD8+ cytotoxic T
13

cells (56). Therefore, it is important to provide proper stimulation and activation signals
to CD4+ and CD8+ T cells to break immunological tolerance. This will induce T cell
mediated killing mechanisms against infections, viruses, or cellular mutations that lead to
cancer.
1.4 Immune suppression: Regulatory T cells
Tumor escape mechanisms contribute to vaccine failure in the prostate cancer setting.
These mechanisms include tolerogenic DC, myeloid derived suppressor cells, defective
antigen presentation, production of immunosuppressive cytokines and regulatory T cell
(Treg) mediated immunosuppression. Regulatory T cell (CD4+CD25+FoxP3+) mediated
suppression is a well-studied mechanisms because it is a critical barrier to the success of
vaccines. The transcription of FOXP3 helps maintain immunological tolerance against
self antigens via the release of suppressive cytokines, TGF- β and IL-10. It also mediates
inhibitory interactions with DCs and T cells (Figure 1.5) (57).
Tregs may either be thymus or periphery derived and can be found in abundance in many
solid tumors (58). Their presence there is thought to be a consequence of chemo-
attractants released by tumor infiltrating macrophages. Movement into the tumor
microenvironment and exposure to self-antigens further activates and induces
proliferation of these cells. The ratio of Tregs versus cytotoxic T cells is important
because an overabundance of the former within malignant tumors is associated with poor
prognosis (59,60). One explanation for this is the ability of Tregs to suppress the activity
and proliferation of CD8+ cytotoxic T lymphocyte primed to recognized TAAs (61).
14

Therefore, the development of improved or novel immunotherapy treatments should be
geared towards targeting the inhibitory aspect of the tumor microenvironment while
activating inflammatory pathways that will induce antigen specific T cells.

Figure 1.5 Inhibitory mechanisms of Tregs. Under stimulating conditions with TGF β,
IL-2, CTLA-4 or PD-1, naïve T cells differentiate into suppressive regulatory T cells.
These induced Tregs (iTregs) survey inflamed tissue and lymph nodes where they may
interact with DC’s and T cells via direct receptor-ligand binding or through cytokine
release.

1.5 LIGHT (Lymphotoxin-like inducible protein that competes with
glycoprotein D for HVEM on T cells)
LIGHT molecule is a ligand that is highly expressed during lymphogenesis and aids in
the recruitment of T cells and immune cells into the lymph node (62,63). This ligand is a
15

costimulatory functional homotrimer that has various binding partners, which may
activate the immune system. LIGHT binds (i) soluble decoy receptor 3 (DcR3) which
neutralizes the effects of LIGHT, (ii) lymphotoxin β receptor (LT βR) which releases T
cells chemokines and (iii) HVEM that is capable of activating immune cells. The
expression of these molecules vary amongst immune cells (Table 1.1) (64).

T cells B cells DC stroma
LIGHT + + ++ -
LTβR - - + ++
HVEM + + + -

Table 1.1 Expression of LIGHT, LT βR and HVEM. LIGHT expression is usually
present on T cells and B cells but predominately on DCs. LT βR which is responsible for
the recruitment of T cells once engaged with LIGHT is present on DC and stroma, while
HVEM is present on T cells, B cells and DC.

LIGHT can function as a transmembrane or soluble protein that may act as an
independent co-stimulatory molecule that is distinct from CD28/B7 signaling. LIGHT
can replace the role of CD28/B7 as signal 2 upon DC and T cell engagement.
HVEM has been coined as the “molecular switch” as it plays a crucial role in
inflammatory and inhibitory response. HVEM may become activated via LIGHT binding
or become inactivated upon binding to B and T lymphocyte attenuator (BTLA) (64). The
engagement of HVEM on immune cells with BTLA on neighboring Tregs activates and
enhances the suppressive capacity of Tregs, therefore creating an environment that
supports immune tolerance.

16

Figure 1.6 Binding partners of LIGHT and HVEM. LIGHT binds to two cellular
receptors HVEM and LTR which leads to costimulation and activation of various gene
pathways that aids in the recruitment of T cells and boost immune response against
pathogens on primed T cells. BTLA competes with LIGHT for HVEM binding, however
due to a higher avidity and affinity of LIGHT-HVEM, LIGHT may dislodge BLTA from
HVEM to induce a positive co-stimulation. LIGHT is also seen as an independent
costimulatory molecule.

In various cancer settings including cervical cancer and colon cancer, forced expression
of LIGHT molecule on the tumor cell surface can costimulate T cells, recruit T cells into
and remodel the tumor microenvironment and activate DCs (Figure 1.7) (65-67). LIGHT
has never been used in a prostate cancer thus the net effect of its expression is unknown,
and is the subject of study of this thesis project.

17

Figure 1.7 LIGHT’s Role in various tumor models. LIGHT is capable of stimulating T
cells independent of CD28/B7 signaling. LIGHT is capable of recruiting effector T cells
and naïve T cells via the release of chemokine, CCL21 through LTβR and LIGHT
engagement. Lymph nodes may become enlarged due to restructuring of the tumor
microenvironment after forced expression of LIGHT. Lastly, LIGHT may aid in the
activation and recruitment of DC.

18

1.6 Prostate Stem Cell Antigen TriVax: An immune stimulating treatment
against prostate cancer
Prostate cancer vaccine is the subject of extensive development over the past decade.
Strategies include (i) viruses as delivery devices for TAAs and adjuvants (PROSTVAC-
F) (68), (ii) an anti-CTLA4 inhibitor (ProstAtak) (69), (iii) a Trojan horse method to
destroy prostate cancer cells, (iv) antibody directed to the cancer cells (anti-OX40) (70),
and antibody based checkpoint inhibitors to CTLA-4 (nivolumab) (71), or to PD-1
antibody. Peptides have also served as prostate cancer vaccines. These strategy consist of
long or short peptides to mimic TAAs and frequently involve ab adjuvant to stimulate
DCs. These vaccines suffer from HLA restriction and the literature only shows modest
tumor burden reductions (72). Recent strategies such as the addition of Toll-Like
Receptor (TLR) agonist or anti-PD-1 antibodies appear to have increased peptide vaccine
efficacy.
Prostate Stem Cell Antigen (PSCA) TriVax consists of three components; (i) a peptide
vaccine targeting the TAA PSCA, sequence 83-91, (ii) a TLR3 agonist, Poly-ICLC, and
(iii) a DC activator, α-CD40 antibody (ab). PSCA is an overexpressed antigen present in
prostate cancer cells. Poly-ICLC is a double stranded RNA pathogen-associated
molecular pattern that acts as a danger signal on immune cells. Poly-ICLC is known as an
adjuvant because its engagement with TLR3 activates an antitumoral response resulting
in the release of the inflammatory cytokine IFN, chemokines and costimulatory factors
(73-75). Poly-ICLC has been used with partial success in various clinical trials as its
19

optimum dosing, schedule and mode of administration remains in flux (76,77) (78). α-
CD40 ab induces a maturation signals on DCs (79,80) and promotes the anti-tumoral
response of cytotoxic T cells (81). Given these attributes, Poly-ICLC and α-CD40 ab are
known as effective vaccine adjuvants in mounting a DC-driven immune response.
1.7 Hypothesis and Working model: LIGHT’s role in remodeling the
tumor microenvironment
The developments of new vaccines and vaccine combinations have given new
momentum to the field of immunotherapy. Despite this, the mortality rates of prostate
cancer have remained unchanged for the past decade (82-84), and a pressing need
remains to improve current treatments. Critical roadblocks to therapeutic success include
tumor heterogeneity, the immunosuppressive peritumor environment and immune
tolerance.
In the section below, we present the case that the advantages of using LIGHT in
combination with a peptide vaccine possess advantages that solve many of the issues we
raise here. We hypothesize that the forced expression of LIGHT in prostate cancer cells,
in the presences of an administered prostate-specific peptide vaccine will overcome Treg
mediated immunosuppression, increase the recruitment of effector T cells and induce
TAA specific T cells (Figure 1.8). A working model of this hypothesis is described in
Figure 1.9.
20

Figure 1.8 Immune modulating properties of forced LIGHT expression in prostate
cancer cells. In the absence of LIGHT, Tregs release suppressive cytokines, survey the
tumor microenvironment and inactivate local effector T cells from attacking self. In the
presence of LIGHT, Tregs are inactivated upon engagement with LIGHT, effector T cells
are recruited to the tumor microenvironment and are subsequently costimulated upon
engagement with LIGHT.

In this model, LIGHT is expressed on the tumor cell surface via the delivery of an
adenovirus encoding membrane bound LIGHT. LIGHT subsequently interacts with
resident LT βR on stoma, causing a cytokine release that attract naïve and effector T cells
into this microenvironment. T cells are then be primed by the tumor cell surface with
21

MHC molecules and receive costimulation from LIGHT molecules. This leads to the
release of IFN- γ, which is a known T cell mediated killing mechanisms. The subsequent
apoptosis of tumor cells leads to the release of TAAs which in turn begin the desired
cascade of phagocytosis and T cell presentation by local DC. Intratumoral LIGHT has the
additional advantage of blunting the immunosuppressive capacity of Tregs, further
contributing to an environment permissive for immunotherapy.

22

Figure 1.9 Working Model. (1) LIGHT is delivered to the prostate cancer cell via an
adenovirus vector coding for membrane bound LIGHT. (2) Once membrane LIGHT is
expressed, LIGHT engages with LT βR on stroma cells resulting in the release of
chemokine CCL21 that attracts T cells. (3) Membrane bound LIGHT on prostate cancer
cells costimulates T cells independently of B7/CD28 interactions leading to the release of
(4) inflammatory cytokines that induces tumor cell apoptosis. (5) The release of tumor
antigens primes DCs and leads to the migration of maturated DC to the lymph nodes
where DCs may present antigens to effector T cells (6). (7) Membrane bound LIGHT
inactivates suppressive T cells.

23

Chapter 2

Forced LIGHT expression in prostate tumors overcome Treg
mediated immunosuppression and synergizes with a prostate
cancer therapeutic vaccine by recruiting effector T
lymphocytes.

2.1 Abstract

Background: LIGHT, a ligand for lymphotoxin-  receptor (LT R) and herpes virus
entry mediator, is predominantly expressed on activated immune cells and LT R
signaling leads to the recruitment of lymphocytes. The interaction between LIGHT and
LT R has been previously shown to activate immune cells and result in tumor regression
in a virally-induced tumor model, but the role of LIGHT in tumor immunosuppression or
in a prostate cancer setting, where self antigens exist, has not been explored. We
hypothesized that forced expression of LIGHT in prostate tumors would shift the pattern
of immune cell infiltration toward an anti-tumoral milieu, would inhibit T regulatory cells
(Tregs) and would induce prostate cancer tumor associated antigen (TAA) specific T
cells that would eradicate tumors.
Methods: Real Time PCR was used to evaluate expression of forced LIGHT and other
immunoregulatory genes in prostate tumors samples. For in vivo studies, adenovirus
encoding murine LIGHT was injected intratumorally into TRAMP-C2 prostate cancer
cell tumor bearing mice. Chemokine and cytokine concentrations were determined by
multiplex ELISA. Flow cytometry was used to phenotype tumor infiltrating lymphocytes
24

and expression of LIGHT on the tumor cell surface. Tumor-specific lymphocytes were
quantified via ELISpot assay. Treg induction and Treg suppression assays determined
Treg functionality after LIGHT treatment.
Results: LIGHT in combination with a therapeutic vaccine, PSCA TriVax, reduced
tumor burden. LIGHT expression peaked within 48 hours of infection, recruited effector
T cells that recognized mouse prostate stem cell antigen (PSCA) into the tumor
microenvironment, and inhibited infiltration of Tregs. Tregs isolated from tumor draining
lymph nodes had impaired suppressive capability after LIGHT treatment.
Conclusion: Forced LIGHT treatment combined with PSCA TriVax therapeutic
vaccination delays prostate cancer progression in mice by recruiting effector T
lymphocytes to the tumor and inhibiting Treg mediated immunosuppression.
25

2.2 Introduction
Prostate cancer is the second most common cause of cancer related deaths in men in the
United States. Approximately 29,480 deaths are expected annually in the United States,
and 307,471 deaths are expected annually worldwide (82,85). The standard of care for
metastatic prostate cancer patients is androgen deprivation therapy, a hormone therapy
that reduces androgen levels that temporarily slows prostate tumor growth (86,87).
Alternatively, hormone manipulation can be avoided when treating asymptomatic or
minimally symptomatic prostate cancer with personalized therapeutic treatments such as
Sipuleucel-T (Provenge®), a dendritic cell based vaccine that is suspected to induce T-
cell mediated attack of cancer cells expressing tumor associated antigens (TAA) (88).
Sipuleucel-T extends median survival to 25.8 months as compared to 21.7 months with
standard of care. Although the true mechanism of action remains unclear, this treatment
is one of the many category 1 drugs recommended by the National Comprehensive
Cancer Network (89-91). Despite the development of new treatments such as this,
mortality rates remain unchanged (83,84,92). The efficacy of therapeutic vaccines have
been limited by tumor mediated immunosuppression and failure to elicit a TAA specific
immune response when used alone, but the discovery of adjuvants such as aluminum-
based mineral salts, toll-like receptor agonists, check point inhibitors and other immune
response stimulators have become key factors in vaccine development and have had some
success in overcoming these limitations (93). Therefore, identifying novel adjuvants or
checkpoint regulators to supplement the use of therapeutic vaccines is expected to
26

improve the overall efficacy of generating an immune response against prostate cancer,
ultimately increasing patient survival.
The aim of this study was to investigate the synergistic efficacy, anti-tumor immune
responses and immune modulatory mechanisms of adjuvant TNFSF14/LIGHT in
combination with a prostate cancer vaccine, prostate stem cell antigen (PSCA) TriVax.
LIGHT, lymphotoxin-like inducible protein that competes with glycoprotein D for
HVEM on T cells, is a membrane bound protein that is highly expressed during
lymphogenesis for the recruitment of lymphocytes via chemokine signaling of
lymphotoxin-  receptor (LT R) (94,95). LIGHT is a candidate adjuvant for therapeutic
cancer vaccines because it has been implicated in inducing activating co-stimulatory
signals on recipient cells in various cancer models (62,96). LIGHT has been shown to
recruit T cells into the tumor microenvironment in a melanoma and colorectal cancer
model, leading to a better prognosis (66,96). In addition, LIGHT may have a role in
inhibiting immune suppression by regulatory T cells (Treg), since Treg suppression is
enhanced when HVEM binds B and T Lymphocyte Attenuator (BTLA). This is pertinent
because LIGHT is also a binding partner to HVEM, an interaction that opposes the
effects of BTLA. Thus, we hypothesized that LIGHT expressed on tumor cells would
interact with HVEM on tumor infiltrating Treg cells, thereby inhibiting their ability to
suppress anti-tumor immune responses.
LIGHT has not been previously explored in the prostate cancer setting. Given the
multiple mechanisms of action of LIGHT, including homing of T cells to the tumor
27

microenvironment and induction of TAA-specific T cells in multiple tumor models, we
hypothesized that forced LIGHT expression in murine prostate cancer transgenic
adenocarcinoma of the mouse prostate (TRAMP-C2) tumor model would increase
prostate cancer survival by inducing prostate TAA specific T cells, would inhibit Tregs
and would synergize with a TAA therapeutic vaccine. For this study, we have chosen to
use PSCA TriVax, a vaccine that consists of PSCA
83-91
peptide and two dendritic cell
activators, anti-CD40 antibody (Ab) and Poly-ICLC. PSCA is an excellent prostate TAA
for the basis of a therapeutic vaccine since it is highly elevated in aggressive and
established prostate tumors (97). The results of this study indicate that addition of LIGHT
treatment to therapeutic cancer vaccines may improve their therapeutic effect.

28

2.3 Methods and Materials
Mice and cell lines.
Specific pathogen free C57BL/6 mice and C3H mice, 6 to 8 weeks of age, were
purchased from Taconic Farms (Germantown, NY). TRAMP-C2 (ATCC CRL-2731;
originally derived from the prostate tumor of a TRAMP mouse on the C57BL/6
background) cells were used for tumor challenge studies. TRAMP-C2 cells were grown
and expanded in vitro with IMDM medium supplemented with 5% Fetal bovine serum
(FBS; Gemini, Sacramento, CA), 5% Nu Serum IV (BD Biosciences, San Jose, CA), 0.01
nM dihydrotestosterone (Sigma Chemical Co.), and 5 g/ml insulin (Sigma Chemical
Co.). All in vivo studies were in compliance and approved by University of Southern
California Institutional Animal Care and Use Committee (USC IACUC).
Antibodies and Reagents.
The following antibodies were purchased from BD Bioscience (San Jose, California):
mu-CD4 FITC, mu-CD25 PE-Cy5, mu-FoxP3 PE-Cy7, mu-CD3 PE-Cy7, and
mu-CD8 PE. Goat mu-IgG FITC antibodies were purchased from Biolegend (San
Diego, CA). LTβR-Fc antibody was purchased from R&D Systems (Minneapolis, MN).
Appropriate isotype controls were purchased from either BD Bioscience or Biolegend.
Tumor Challenge, Treatments and Immunizations.
Groups of 6 to 8 week old C57BL/6 male mice were challenged subcutaneously with
5x10
5
TRAMP-C2 tumor cells in PBS. Tumor growth was measured three times per
29

week with manual calipers by measuring tumor length, height, and depth to generate a
tumor volume. Tumor volumes exceeding 1500 mm
3
or ulcerated tumors resulted in
euthanasia as per USC IACUC guidelines. For studies evaluating the effect of LIGHT in
vivo, recombinant adenovirus carrying DNA encoding the murine LIGHT gene (Ad-
LIGHT) were injected intratumorally using a 31 gauge insulin syringe (98). For every in
vivo experiment with LIGHT treatment, injections were performed when average tumor
volumes in randomized groups were approximately 30 mm
3
(25-30 days post challenge)
(Figure 2.1). Ad-LIGHT treatment was given twice, three days apart with 2x10
10
viral
particles (vp) per intratumoral injection. Control adenovirus particles (Ad-Control) were
used as a control.

Figure. 2.1 Vaccination scheme with LIGHT injections alone. C57BL/6 mice will be
challenged with 5x105 tumor cells and subsequently treated with intratumoral Ad-LIGHT
injections when tumors are approximately 30 mm
3
.

In studies evaluating the synergistic properties of both Ad-LIGHT and therapeutic
vaccination PSCA TriVax, mice were treated with two doses of Ad-LIGHT given three
day apart when average tumor volumes in randomized groups reached 30mm
3
, and were
subsequently vaccinated i.m. with PSCA TriVax 7 days and 14 days after the first LIGHT
injection. PSCA TriVax consist of a mixture of 50 µg of synthetic peptide PSCA
83-91
, 100
30

µg anti-CD40 mAb (BioXCell) and 50 µg of Poly-ICLC (Hiltonol, Oncovir, Inc.).
Control immunizations were conducted with a mixture of 100 µg of anti-CD40 mAb and
50 µg of Poly-ICLC alone. Tumor burden was recorded three times per week. Euthanasia
was conducted as per USC IACUC guidelines.
IFN-γ Enzyme Linked Immunospot (ELISpot) Assay.
96-well ELISpot plates (Millipore Multiscreen HTS IP) were coated with 10 µg/ml IFN 
capture Ab (IFN  R406A2, BD Pharmingen) in sterile PBS overnight at 4
o
C. Plates were
washed once with 0.5% PBS-T and then twice with sterile PBS. Complete RMPI medium
was then used to block plates for 2 hours at 37
o
C. Splenocytes isolated from treated mice
were plated in serial dilutions ranging from 5x10
5
to 1.25x10
5
cells per well in medium
containing either 50 µg/mL of PSCA
83-91
peptide, DMSO control or 10 µg/ml of PHA-L.
After 48 h of incubation at 37
o
C, plates were washed 6 times with 0.05% PBST and were
incubated with 1 µg/ml of biotinylated IFN-  antibody (BD Pharmingen) in 0.05%
PBST/1% BSA for 2 h at room temperature. Plates were washed 6 times with 0.05%
PBST and wells were subsequently incubated with 100 µl of 1:4000 diluted streptavidin-
horseradish peroxidase (Sigma Chemical Co.) for 1 h at room temperature. Spots were
developed with 3-amino-9-ethylcarbazole (Sigma Chemical Co.) for 5 minutes and
reactions were quenched with deionized water. A Zeiss KS ELISPOT microscope was
used to determine the number of spots per well. Numbers of spots were normalized to
background control (DMSO control) then each treatment group was further compared to
the untreated study arm.
31

Treg Suppression Assay.
Tumor draining lymph nodes from individual treatment groups were pooled together and
isolated for CD4+CD25
hi
(suppressive cells) populations via a CD4+CD25
hi
Regulatory
T cell magnetic activated cell separation (MACS) kit (Miltenyi Biotec). CD4+CD25-
(responder cells) population was isolated from splenocytes of naïve C57BL/6 mice. 5x10
4

Responder cells were co-cultured with a decreasing ratio of suppressor cells (Tresp:Treg
ratios: 1:1, 2:1, 4:1 and 8:1), 1 µg/ml anti-CD3, 2 µg/ml anti-CD28 and 5x10
5
irradiated
accessory cells (allogenic T cell cells) that were isolated from C3H mice. After 48 h, 1 µg
of 3H-thymidine was added into each well for an additional 24 h. Responder cell
proliferation was measured by thymidine incorporation using a TopCount NXT
microplate scintillation counter (PerkinElmer, Shelton, CT). The proliferation index was
calculated for each Tresp:Treg ratio and was normalized to maximum proliferation
(Tresp cultured in the absence of Tregs).
Treg Induction Assay.
Naïve CD4+ T cells were isolated from splenocytes of a naïve C57BL/6 mouse via the
Mouse CD4+CD62L+ T cell MACS kit (Miltenyi Biotec). Untreated TRAMP-C2 cells or
TRAMP-C2 cells infected with 2x10
3
vp/cell of Ad-LIGHT were irradiated at 30 Gray
prior to co-cultures. 5x10
5
naïve CD4+ T cells were plated out into each well of a 6 well
plate in complete T cell medium (RPMI medium supplemented with 10% FBS). Treg-
inducing factors and tumor cells were added to appropriate wells; 100 units/mL rhIL-2, 5
ng/ml rhTGF-b, 1:1 bead to cell ratio of CD3/CD28 dynabeads (Invitrogen, Oslo,
32

Norway), and 1:1 ratio of naïve T cells to tumor cells. Cultures were incubated for 5
days at 37
o
C prior to Treg phenotyping via flow cytometry.
Isolating tumor Infiltrating Lymphocytes (TIL), Ad-LIGHT infected TRAMP-C2
cells and Flow Cytometry.
Tumors were extracted and weighed from TRAMP-C2 bearing C57BL6 mice that were
treated with Ad-LIGHT and/or immunized with PSCA TriVax. Tumor tissues were
minced into small pieces prior to using the Miltenyi Tumor Dissociation Kit and
GentleMACS Dissociator. Cell suspension was passed through a 70 µm nylon strainer to
generate a single cell population and separated in a Lympholyte-M gradient (Cedarlane)
for the isolation of TIL from debris. TIL were then washed 3 times with PBS, stained
with antibodies and analyzed by flow cytometry to determine the phenotype of
infiltrating lymphocytes. For Treg population, we first gated on CD4+ cells and then
gated on CD25+ and Foxp3+ cells. Effector T cells were gated on CD8+ and CD3+
double positive population and helper T cells were gated on CD4+ and CD3+ double
positive populations.
TRAMP-C2 cells infected with Ad-LIGHT (1x10
3
or 2x10
3
viral particles per cell) were
collected 24, 48, 72, 96 and 120 h post infection, washed twice with FACS Buffer, prior
to being stained with primary antibody, LT R-Fc recombinant protein, and then with
secondary, goat anti-mouse FITC. TRAMP-C2 cells were gated on FITC expressing cells
and the mean fluorescence intensity was recorded. 
Quantitative real-time polymerase chain reaction.
33

Tumors harvested from treated mice were weighed and stored in RNAlater solution.
Fixed tumors were homogenized with the PolyTron PT2100 homogenizer in RLT buffer
solution at 4
o
C. Ad-LIGHT treated TRAMP-C2 cells were isolated subsequent to Ad-
LIGHT infection with either 1x10
3
or 2x10
3
viral particles per cell. Total RNA was
isolated using the QIAGEN RNAeasy Plus kit following manufacturer’s instructions. The
iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) was used to reverse transcribe the
isolated RNA to cDNA. RNA and cDNA concentrations (ng/ml) were quantified using a
NanoDrop 2000 (Waltham, MA). Quantitative real time PCRs were performed on a CFX-
96 real time PCR machine (Bio-Rad) using the Sensi-fast SYBR NO-ROX kits (BioLine,
Taunton, MA), following manufacturers protocol. Genes including GAPDH, mLIGHT,
NOS, Arg2 and IDO were analyzed. The relative expression of each gene was normalized
to the expression of GAPDH (ΔΔCq) and results from each treatment groups were
compared to the untreated control study arm. All primers were synthesized by IDT
(Coralville, IA) or the USC DNA Core.
GAPDH qPCR primers: forward 5’-TCA ATG AAG GGG TCG TTG AT-3’; reverse 5’-
CGT CCC GTA GAC AAA ATG GT-3.’
mLIGHT qPCR primers: forward 5’-CAA CCC AGC AGC ACA TCT TA-3’; reverse 5’-
GCT CAG CTG CAC TTT GGA G-3.’
NOS qPCR primers: forward 5’-GTC GAT GTC ACA TGC AGC TT-3’; reverse 5’-
GAA GAA AAC CCC TTG TGC TG-3.’
34

Arg2 qPCR primers: forward 5’-AGG GAT CAT CTT GTG GGA CA-3’; reverse 5’-
AGA AGC TGG CTT GCT GAA GA-3.’
IDO qPCR primers: forward 5’-GTG GGC AGC TTT TCA ACT TC-3’; reverse 5’-GGG
CTT TGC TCT ACC ACA TC-3’
Measuring intratumoral cytokines.
Tumors harvested from treated mice were weighed and homogenized with the PolyTron
PT2100 homogenizer (Kinematica AG, Switzerland) in a 1x Halt Proteinase Inhibitor
Cocktail (Pierce, Rockford, IL)/PBS solution for 20 minutes at 4
o
C. Homogenate was
centrifuged and supernatants were collected to quantify cytokine levels with a custom 22-
plex Milliplex mouse cytokine immunoassay (Millipore, Billerica, MA) using the Bio-
Plex multiplex system (Bio-Rad).
Tumor resection and challenge

Figure 2.2 Tumor excision and rechallenge study. C57BL/6 mice will be challenged
with 5x105 tumor cells. Treated with Ad-LIGHT and/or PSCA TriVax. Primary tumors
will be excised and mice will subsequently be rechallenged.

35

Groups of C57BL/6 mice were challenged on their (right) side flank with 5x10
5
TRAMP-
C2 tumor cells in PBS. Once average tumor volumes reached 30 mm
3
, mice were
normalized into 6 treatment groups: Untreated, Ad-LIGHT, Ad-Control, PSCA TriVax,
Ad-LIGHT/PSCA TriVax and Ad-Control/PSCA TriVax. Ad-LIGHT treatment was
given twice, three days apart with 2x10
10
viral particles (vp) per intratumoral injection.
Control adenovirus particles (Ad-Control) were used as a control. Evaluating the
synergistic properties of both Ad-LIGHT and therapeutic vaccination PSCA TriVax,
mice were treated with two doses of Ad-LIGHT given three day apart when average
tumor volumes in randomized groups reached 30mm
3
, and were subsequently vaccinated
i.m. with PSCA TriVax 7 days and 14 days after the first LIGHT injection (Figure 2.2).
Two weeks after the last treatment, subcutaneously challenged mice were anesthetized
first with 4% isofurane and maintained at 1-2% isoflurane during tumor resection
surgery; the fur was clipped on the side flank, and a presurgical prep was performed
which includes 3 alternating scrubs of betadine/chlorhexidine and 70% ethanol. The
animal was draped aseptically and all instruments used were autoclaved or bead sterilized
prior to and in-between surgeries. An incision was made on the side flank to remove the
existing subcutaneous tumor. Interrupted sutures of 6-0 adsorbable synthetic material
were used to close the cutaneous layer. Mice were placed and observed in a clean cage
individually until full recovery. Mice received a dose of meloxicam (1-2mg/kg) PO for
the following 3 days. Sterile technique was used during surgery to minimize the risk of
infection. Animals were monitored daily and sutures were removed 2 weeks post
operation. Two weeks after tumor resection, the (left) side flank was then re-challenged
36

with 1x10
6
TRAMP-C2 tumor cells in PBS. Mice with tumors that were not completely
resected were censored from the study. The number of mice with tumors then determined
memory.
Survival surgery: Intraprostatic Ad-LIGHT injections
Groups of 10-12 week old TRAMP mice were distributed into the following treatment
groups for survival surgeries: Untreated (Mock), Ad-Control, Ad-LIGHT, PSCA TriVax,
PSCA TriVax with Ad-LIGHT and PSCA TriVax with Ad-Control. Mice were
anesthetized with 4% isoflurane and maintained at 1-2% isoflurane during surgery; the
fur was clipped on the abdomen, a presurgical prep was performed that includes 3
alternating scrubs of betadine/chlorhexidine and 70% ethanol. Mice were draped
aseptically and all instruments were either autoclaved or bead sterilized prior to and in-
between surgeries. An incision was made in the lower abdominal cavity above the
copulatory organ with iris scissors. The prostate was located and 20ul of treatment was
injected via a 31 gauge insulin syringe. Continuous sutures of 6-0 adsorbable synthetic
material was used to appose the peritoneal cavity, while using interrupted sutures to close
the cutaneous layer. Mice were placed and observed in a clean cage individually until full
recovery. Mice received a dose of meloxicam (1-2mg/kg) PO for the following 3 days.
Sterile technique was used during surgery to minimize the risk of infection. Animals were
monitored daily. Sutures were removed 2 weeks after surgery. Mice were euthanized at
20 weeks of age, and tumor weight was measured (Figure 2.3).

37

Figure 2.3. Surgical procedure of intraprostatic injections of Ad-LIGHT into
TRAMP mice. A) Outline of vaccination schedule and tumor harvest. B) Surgical area is
prepared under aseptic techniques. Trays and surgical tools are autoclaved and/or
sterilized via glass beads. C) Isoflurane machine is used to maintain stable release of
anesthetics. D) Mice are anesthetized and surgically draped.

38

Statistical Analysis.
All statistical analyses were performed on Prism, Graphpad 5.0 (GraphPad Software Inc.,
San Diego, CA). Tumor growth, ELISpots, Cytokine levels and flow cytometry results
were assessed with either a student t test, one-way ANOVA or a two-way ANOVA
comparing data to untreated controls. Significance was defined at p≤0.05 for all
experiments.
39

2.4 Results
2.4.1 Combined Ad-LIGHT and PSCA TriVax therapies synergize to reduce tumor
burden
The main goal of this study was to determine the effects on tumor burden using Ad-
LIGHT in conjunction with a therapeutic prostate cancer vaccine, PSCA TriVax. Prior to
in vivo studies with intratumoral LIGHT injections, we determined whether TRAMP-C2
prostate cancer cells were capable of taking up an adenovirus vector coding for LIGHT
DNA and then expressing membrane bound LIGHT on the tumor cell surface. To
examine this, TRAMP-C2 tumors were incubated with either 1x10
3
viral particles/cell
(vp/cell) or 2x10
3
vp/cell for various time points and analyzed for mRNA and surface
expression of LIGHT. The mRNA and surface protein expression of LIGHT peaked
within 24-48 hours post-infection as shown by qRT-PCR and flow cytometry (Figure
2.4). These results demonstrate the ability of TRAMP-C2 cells to take up Ad-LIGHT and
express membrane bound LIGHT on the tumor cell surface.

40

Figure 2.4 Membrane bound LIGHT expression in TRAMP-C2 cells peaks within the
first 48 hours of infection. (A) 5x10
5
TRAMP-C2 cells were infected with either 1x10
3
or
2x10
3
Ad-LIGHT viral particles per cell. mRNA was isolated and showed a higher
mRNA level of LIGHT with 2x10
3
viral particles as compared to 1x10
3
viral particles.
Expression peaked at 24 and 48 hours. Shown is the relative expression of LIGHT
mRNA normalized to GAPDH (± SD) in Ad-LIGHT infected cells measured by RT-
qPCR. (B) Membrane bound LIGHT was detected via flow cytometry with the LTβR-Fc
ligand. Expression of LIGHT protein correlates with the mRNA expression level, where
24 hours shows the highest levels of LIGHT expression. All experiments were repeated
once and representative data are shown.

We then determined whether intratumoral forced LIGHT expression combined with
PSCA TriVax would inhibit prostate tumor growth. C57BL/6 mice were challenged with
TRAMP-C2 prostate cancer cells and divided into groups with mean tumor volume of 30
mm
3
. Groups were then treated as follows: Ad-LIGHT with PSCA TriVax, PSCA
TriVax, or untreated. When compared to mice in the untreated and PSCA TriVax groups,
mice treated with Ad-LIGHT and PSCA TriVax combined showed a sustained reduction
in tumor burden until day 50 (Figure 2.5). Tumor burden in Ad-LIGHT treated mice was
not significantly different from Ad-Control treated or untreated mice (Figure 2.6). Taken
41

together, these results indicate that LIGHT treatment and PSCA TriVax vaccination acted
synergistically to reduce prostate tumor burden in vivo.

Figure 2.5 Ad-LIGHT and PSCA TriVax therapeutic vaccination delays TRAMP-C2
tumor growth. Mice were first treated with two doses of Ad-LIGHT (or Ad-control) prior
to receiving PSCA TriVax. 2 weeks post treatment, animals whom received Ad-LIGHT
followed by PSCA TriVax showed a delay in tumor growth. (Two-way ANOVA on
single time-point, ***p<0.001). All experiments were repeated once and representative
data are shown.

42

Figure 2.6 Tumor growth curve of untreated, Ad-Control and Ad-LIGHT treated tumor
bearing mice. Tumor volumes were not significantly different from each treatment group,
suggesting that Ad-LIGHT as a standalone treatment is not powerful enough in a non-
immunogenic prostate cancer setting to induce tumor regression. (No statistical
significance, Two-way ANOVA on single time-point).

2.4.2 Ad-LIGHT synergizes with PSCA TriVax by increasing TIL numbers.
Positive therapeutic outcome for cancer vaccines are associated with higher levels of
tumor infiltration by lymphocytes. Given the role of LIGHT in recruiting lymphocytes to
its area of expression via CXCR4/CCL21 signaling (99), we hypothesized that forced
tumor LIGHT expression may reduce tumor burden by increasing the numbers of TIL per
unit mass of tumor. To investigate this, C57BL/6 mice were challenged, normalized for
tumor volume and then treated with either PSCA TriVax, Ad-LIGHT with PSCA TriVax,
or left untreated. The number of TIL per gram of tumor was determined by flow
cytometric analysis. The combination of Ad-LIGHT with PSCA TriVax resulted in an
influx of TIL as compared to PSCA TriVax alone (Figure 2.7). The number of CD8+ T
cells per gram of tumor was significantly higher in the Ad-LIGHT with PSCA TriVax
43

group compared to the PSCA TriVax alone and untreated groups. Additionally, there was
a trend towards increased numbers of infiltrating CD4+CD3+ T cells. These data suggest
that forced LIGHT expression on tumor cells increases the frequency of TIL in the tumor
microenvironment.

Figure 2.7 Intratumoral LIGHT treatment in combination with PSCA TriVax shows an
increased number of CD4+ and CD8+ in a prostate cancer tumor model. Expression of
LIGHT in the tumor microenvironment recruited CD4+/CD3+ and CD8+/CD3+ T cells
into the tumor microenvironment of PSCA TriVax vaccinated mice. Representative data
are shown from two experiments. (*p<0.05, one-way ANOVA).

We then examined the effect of LIGHT treatment on the induction of peripheral TAA
specific T cells. Three groups of C57BL/6 mice were challenged subcutaneously with
TRAMP-C2 cells, normalized into groups and were treated with either PSCA TriVax,
Ad-LIGHT with PSCA TriVax, or were left untreated. Splenocytes were isolated one
week after the last treatment, then PSCA-specific T cells were enumerated via IFN-γ
ELISpot assay with PSCA
83-91
peptide. Combination treatment with Ad-LIGHT and
PSCA TriVax did not lead to an increase in number of PSCA-specific IFN-γ secreting T
44

cells induced compared to PSCA TriVax alone (Figure 2.8). This suggests that
expression of LIGHT in the tumor enhances vaccine-induced TAA-specific T cells
through a mechanism distinct from increasing their total frequency. Supporting this
theory is the observation that Ad-LIGHT treatment alone did not induce PSCA specific T
cells (Figure 2.9).

Figure 2.8 Combined Ad-LIGHT and PSCA TriVax vaccination shows an increased
trend in TAA specific T cells as compared to PSCA TriVax alone but was not
significantly different. Splenocytes were plated against 50 μg/ml of PSCA
83-91
.
Representative data are shown from two experiments. (**p<0.01, one-way ANOVA).

45

Figure 2.9 TAA specific T cells after untreated, Ad-Control, or Ad-LIGHT treatment.
IFN-γ ELISpot assay show that untreated, Ad-Control, and Ad-LIGHT do not induce
TAA specific T cells against PSCA
83-91
peptide. (No statistical significance, one-way
ANOVA).

2.4.3 LIGHT treatment alters the tumor microenvironment from an
immunosuppressive to an immunostimulatory milieu.
To further explore the potential reasons for the synergistic results seen in Figure 1, we
investigated the effect of Ad-LIGHT treatment alone on T cell tumor infiltration, tumor
cytokine expression and expression of immunoregulatory genes in the tumor. We first
investigated whether forcing LIGHT expression in implanted TRAMP-C2 prostate
tumors alone (i.e., in the absence of a vaccine) was able to recruit T cells to the tumor
microenvironment. To this end, mice were challenged subcutaneously with 5x10
5

TRAMP-C2 cells. When tumors were palpable and large enough to inject, mice were
randomized into treatment groups with an average tumor volume of 30 mm
3
. Mice were
then given two intratumoral injections of either Ad-LIGHT or Ad-Control given three
days apart. Tumors were harvested one week after the last treatment. TIL were released
from tumors using the Miltenyi tumor dissociation kit and phenotyped by flow
46

cytometry. An increase in the mean number of infiltrating CD8+/CD3+ and CD4+/CD3+
T cells per gram of tumor was observed in Ad-LIGHT treated tumors compared to
untreated controls (Figure 2.10A). In contrast, the mean number of suppressive Treg per
gram of tumor was not statistically significantly different between any of the three
treatment groups (Figure 2.10B). These data show that expression of intratumoral
LIGHT increases the number of infiltrating effector T cells but does not change the total
number of Tregs in the tumor microenvironment, creating a more favorable balance of
the intratumoral Teffector:Treg ratio.

Figure 2.10. Phenotype of tumor infiltrating lymphocytes. Increase in intratumoral CD4+
and CD8+ T cells following forced expression of membrane bound LIGHT in a prostate
cancer tumor model. (A) Tumor infiltrating lymphocytes were released from untreated or
treated tumors 7 days after Ad-Control or Ad-LIGHT injection. Cells were stained with
CD4, CD8 and CD3 Ab and analyzed via flow cytometry. The number of TIL/gram of
tumor from CD8+/CD3+ and CD4+/CD3+ T cells were significantly higher in Ad-
LIGHT treated mice compared to untreated. (p<0.05, one-way ANOVA). (B) The
number of CD4+CD25+Foxp3+ Tregs per gram of tumor were not significantly
differently, despite the increase in total number of infiltrating lymphocytes in the Ad-
LIGHT samples. Shown is the average number of FoxP3+ TIL (±SD) from 5 treated
mice/group. Data are representative of two individual experiments.
47

We next evaluated the chemokine and cytokine levels in the microenvironments of
untreated, Ad-Control and Ad-LIGHT treated tumors. Tumors lysates from treated mice
were prepared and analyzed using a 22-plex chemokine and cytokine ELISA.
Macrophage inflammatory protein (MIP)-1 , MIP-1 , and vascular endothelial growth
factor (VEGF) showed an increased trend in Ad-LIGHT treated tumors (Figure 2.11A).
All other analytes tested (GM-CSF, IFN- , IL-1 , IL1 , IL-2, IL-4, IL-5, IL-6, IL-9, IL-
10, IL-12(p70), IL-13, IL-15, IL-17, KC, MCP-1, M-CSF, MIP-2, TNF- ) were
equivalent between treatment groups (data not shown).
Next, we measured the mRNA expression of nitric oxide synthase (NOS), indolamine
2,3-dioxygenase (IDO) and arginase-2 (Arg-2), which are immunoregulatory proteins
that can affect T cell and tumor metabolism, in treated tumors to determine whether these
were modulated in LIGHT-expressing tumors. Tumors from Ad-LIGHT or Ad-Control
treated mice were isolated one week after treatment and RNA isolated to analyze gene
expression in the tumors. First we wanted to confirm that the presence of LIGHT
expression was only evident within LIGHT treated tumors with the relative expression of
LIGHT mRNA normalized to GAPDH. As expected, LIGHT expression was only
present within LIGHT treated tumors, whereas untreated and vector-control treated
animals showed no expression of LIGHT (Figure 2.11B). Relative mRNA expression of
NOS, normalized to GAPDH, was increased in LIGHT treated mice compared to
untreated and vector control. Additionally, the relative mRNA levels of both IDO and
Arg-2 were decreased in both LIGHT and adenovirus-vector control groups, suggesting
an adenovirus-vector effect rather than a LIGHT-induced effect.
48

Figure 2.11 Ad-LIGHT treatment modulates the tumor microenvironment to an
immunostimulatory environment. (A) There is an increase trend in concentration of MIP-
1α, MIP-1 𝛽 and VEGF. MIP-1α is statistically higher in Ad-LIGHT treated groups when
compared to combined controls of untreated and Ad-Control (student t test, p=0.046). (B)
Expression of intratumoral LIGHT alters gene expression in the tumor
microenvironment. Tumors were isolated from TRAMP-C2 challenged mice that were
given Ad-Control, Ad-LIGHT or left untreated. Various gene were examined by RT-
qPCR including LIGHT, NOS, IDO and Arg2. Shown is the relative gene expression ±
S.D. (Two-way ANOVA, *p<0.05) All experiments were repeated once and
representative data are shown.

49

2.4.4 LIGHT prevents the maturation of Tregs from naïve CD4+ T cells and
compromises the suppressive function of existing Tregs.
Induced Tregs (iTregs) are derived from naïve CD4+ T cells that receive stimulatory
signals of IL-2, TGF-  and weak co-stimulation, which commonly occurs in the tumor
microenvironment. We hypothesized that because LIGHT provides a strong co-
stimulatory signal, intratumoral LIGHT expression would prevent the induction of
infiltrating naïve T cells into Treg, potentially explaining the data presented in Figure 4.
To investigate this possibility, we isolated naïve CD4+CD62L+ T cells from the spleens
of naïve mice and performed an iTreg induction assay. The maturation of these naïve
CD4+ T cells to Tregs was induced by providing IL-2, TGF-  and CD3/CD28
stimulation. This was also done with the addition of either irradiated TRAMP-C2 cells or
irradiated TRAMP-C2-LIGHT expressing cells to the Treg induction cultures. After 5
days in Treg-inducing co-cultures, the percentage of naïve CD4+ T cells that had been
induced to become CD4+CD25+FoxP3+ Tregs was quantitated by flow cytometry. In the
positive control group (no tumor cells added), 16.7% of naïve T cells had been converted
to iTregs (Figure 2.12A). Using the same conditions but with the addition of TRAMP-
C2-LIGHT cells; the frequency of iTregs was limited to 2.7%, compared to 7.3 % with
the addition of untreated TRAMP-C2 cells (Figure 2.12A). These data suggest that
LIGHT expression on tumor cells prevented the maturation of naïve T cells to iTregs.
50

Figure 2.12. Intratumoral expression of LIGHT reduces the frequency of induced Tregs
and causes existing Tregs to lose their suppressive capacity. (A) Naïve CD4+CD62L+ T
cells were cultured with Treg inducing factors. Flow cytometry data represented in table.
Results in each column show the frequency of Tregs induced in different treatment arms
and growth factors and cytokines that were added. Results demonstrate that the presence
of LIGHT reduces the frequency of iTregs. Experiment was repeated once and
representative frequency is shown. (B) Tresp cells alone (1:0 Tresp:Treg ratio) was taken
as 100% proliferation (Set to 1). Tregs isolated from Ad-Control treated mice suppressed
Tresp proliferation at all co-culture ratios. Tregs isolated from Ad-LIGHT treated mice
lose the ability to suppress Tresp proliferation. Tregs isolated from untreated tumor-
bearing mice or naïve mice showed statistically similar suppressive capacity to Ad-
Control treated mice (Supplemental Fig. 5). N=10 per experiment, one-way ANOVA,
*p<0.05, **p<0.01, ***p<0.001). Experiment was repeated twice.

Finally, we evaluated the suppressive capacity of Tregs isolated from mice bearing Ad-
LIGHT-treated TRAMP-C2 tumors via a Treg suppression assay. Tregs were isolated
from tumor draining lymph nodes of tumor-bearing mice that had been treated with either
Ad-Control, Ad-LIGHT or left untreated, and from the lymph nodes of naïve mice
(untreated and tumor-free). We performed co-culture experiments with various ratios
using Tregs isolated from tumor-bearing animals and naïve T responder cells (Tresp)
isolated from naïve mice. The proliferation of Tresp in these co-cultures was expected to
51

inversely correlate to the suppressive function of Tregs. The proliferation index of cells at
all Tresp:Treg ratios were compared to the maximum proliferation of Tresp (i.e., Tresp
cells cultured alone, without the influence of Treg). As expected, the proliferation of co-
cultures containing Tregs isolated from naïve, untreated and Ad-Control mice were
inhibited proportional to the number of Tregs in the co-cultures. (Figure 2.12B and
Figure 2.13). In contrast, Tregs isolated from Ad-LIGHT treated mice were incapable of
suppressing the proliferation of Tresp; maximum proliferation was observed at all
Tresp:Treg ratios from 1:1 to 8:1. These data indicate that Tregs isolated from Ad-
LIGHT treated mice had lost their suppressive capacity.

Figure 2.13 Untreated and Naïve B6 mice display functional Tregs. Proliferation of
Tresp co-cultured with Tregs isolated from untreated and naïve C57BL/6 mice show an
increased proliferation with increased ratio of Tresp:Treg. (one-way ANOVA, *p<0.05,
**p<0.01, ***p<0.001. Experiment was repeated twice).
52

2.4.5 PSCA TriVAX induces memory T cells in TRAMP-C2 re-challenge study
To investigate whether PSCA TriVax or LIGHT, or the combination of both may induce
memory T cells against TRAMP-C2 cells, we performed a tumor challenge and re-
challenge study after treatment. During the first challenge, 100% of the mice took
(Column TRAMP-C2 Challenge 5x10
5
cells), when tumors were approximately 30 mm
3
,
tumors were normalized amongst groups and vaccinated with the appropriate treatment.

Table 2.1. Number of Mice undergoing tumor challenge, tumor resection and tumor
re-challenge. The TRAMP-C2 challenge column represents the number of mice that
were inoculated with 5x10
5
TRAMP-C2 tumor cells and took the tumor. The tumor
resection presents the number of mice that had their tumor resected from the previous
column. The number of mice that took the tumor re-challenged are noted in the TRAMP-
C2 re-challenge column. (*) represents groups where mice were censored from the study
due to the regrowth of the original tumor after tumor resection.

A week after the last treatment, tumors were harvested and mice were subsequently re-
challenged two weeks later. The naïve group acted as a control for the re-challenge to
ensure the effective TRAMP-C2 tumor inoculation. The % of mice with tumor growth
53

was noted and converted to the % tumor regrowth for the treatment group. A number of
mice were censored (*) after tumor resection due to the regrowth of the original tumor.
Amongst the treatment groups, mice treated with PSCA TriVax had a reduction in tumor
growth (Table 2.1). In contrast, Ad-LIGHT did not appear to induce memory T cells due
to the high percentage of tumor regrowth in the re-challenge.
2.4.6 Induction of pro-inflammatory responses improves the efficacy of PSCA
TriVax in the TRAMP model

Figure 2.14. Prostate tumor weights of TRAMP mice at 20 weeks of age. Tumor weights
(seminal vesicles and prostate tumor) were recorded at 20 weeks of age in TRAMP mice
treated with various vaccination regimens.

To investigate the role of forced LIGHT expression in a prostate cancer model that
mirrors the pathogenesis of a subset of prostate cancer patient disease, TRAMP mice
were used. In this study, 10-12 week old TRAMP mice were anesthetized and surgically
prepped for an incision on the abdomen where Ad-LIGHT was delivered
intraprostatically. Mice were followed through to 20 weeks of age, where they were
54

euthanized and the tumor weights were recorded. The tumor weights were not
statistically different from each treatment group, however tumor weights of mice that
received PSCA TriVax and an adenovirus injection trended down (Figure 2.14). This
suggests that the combination of PSCA TriVax and adenovirus mediated an
immunogenic response that reduced tumor burden.
55

2.5 Discussion

Although immunotherapeutic vaccines against cancers are promising, these treatments
often fail to elicit an effective response that results in tumor regression due to active
immunosuppression within the prostate tumor microenvironment, stressing the need for
concomitant treatment with agents that can overcome immunosuppression. The
identification of such agents for use in therapeutic modalities is crucial for increasing
TAA specific T cell responses, while simultaneously reducing tumor immunosuppression
so as to reduce tumor burden. Ultimately this may improve prostate cancer patient
survival. With that goal in mind, we evaluated the role of concomitant LIGHT treatment
and PSCA TriVax immunization. This was done by performing intratumoral injections of
Ad-LIGHT into the tumors of TRAMP-C2 challenged mice in an effort to stimulate
LIGHT-mediated lymphogenesis in the tumor microenvironment, followed by PSCA
TriVax administration. TriVax has been used therapeutically in other tumor models and
has proven to be an effective cancer vaccine (100,101). PSCA TriVax treatment is a
three-component cocktail that elicits cytotoxic CD8+ T cell response against PSCA, a
known prostate TAA. This cocktail contains the TAA peptide PSCA
83-91
and two antigen
presenting cell stimulators, anti-CD40 mAb and Poly-ICLC (101), a combination that has
been demonstrated by our lab to result in extended tumor-free survival when used as a
vaccine 20 days post TRAMP-C2 tumor challenge (Kast lab, unpublished data). Prostate
cancer TAAs (including PSCA) are all self-antigens, making prostate cancer a difficult
disease to induce an immune response against. However, the combination of LIGHT and
PSCA TriVax reduced tumor burden significantly by day 50 when compared to untreated
56

and PSCA TriVax vaccinated mice alone. Such effects were not seen in untreated, Ad-
LIGHT and Ad-Control alone treated groups, as tumor volumes were not significantly
different between the groups (p>0.05) (supplemental Figure 2). These data are consistent
with a previous study in which we demonstrated in an HPV induced cervical cancer
model that LIGHT in combination with an HPV therapeutic vaccine delayed tumor
growth and extended survival (67). Unfortunately in our study, accurate survival statistics
(e.g. log-rank survival curves) were hindered by the consistent occurrence of ulcerations,
necessitating euthanasia prior to the endpoint of maximum tumor volume. Nevertheless,
the tumor growth curves by themselves demonstrated reduced tumor burden as compared
to the untreated control, and suggests that the addition of LIGHT as an adjuvant enhances
the efficacy of PSCA TriVax. Other prime-boost immunization schedules were evaluated
(i.e. vaccination first followed by Ad-LIGHT intratumoral injection, increasing number
of days between injections), however none of these improved the anti-tumor immune
responses as judged by ELISpot, flow cytometry and tumor burden studies (data not
shown). The optimal immunization schedule in this particular model is therefore priming
with Ad-LIGHT first to induce changes in the tumor microenvironment, followed by
vaccination with a therapeutic vaccine.
The numbers and phenotypes of intratumoral lymphocytes in LIGHT and PSCA TriVax
treated mice were analyzed to determine the mechanism of action in reducing tumor
burden. A high frequency of infiltrating CD4+CD3+ and CD8+CD3+ T cells were found
in the tumors of LIGHT and PSCA TriVax treated mice. As this effect was not observed
in untreated or PSCA TriVax alone, these results suggest a central role for LIGHT in
57

recruiting T cells into the prostate tumor microenvironment. LIGHT in combination with
PSCA TriVax did not increase the peripheral frequency of TAA specific T cells as
compared to PSCA TriVax alone. This contrasted with our previous findings in the HPV
16 tumor model, where tumor antigen specific T lymphocytes were induced by
intratumoral Ad-LIGHT injections in the absence of vaccination. HPV 16 tumors have
foreign tumor specific antigens for which there is no tolerance; this provides an
advantage in immunotherapy where higher frequencies of T cell responses can be
generated due to lack of central and peripheral tolerance. This phenomenon suggests that
Ad-LIGHT may not break peripheral tolerance to self-antigens but may act to remodel
the tumor microenvironment through alterations in cytokines and chemokines, and in
changes in patterns of infiltrating lymphocytes. To investigate this, we evaluated the
mechanism of action of LIGHT treatment alone to elucidate its role in contributing to the
synergistic efficacy of combination treatment.
In a previous examination of the effects and mechanism of action of LIGHT in
melanoma, it was shown that LIGHT recruited and activated anti-neoplastic CD8+ T
cells in the tumor microenvironment and microvesicles (96). LIGHT protein has been
shown to recruit lymphocytes through chemokine signaling via CXCR4/CCL21 in
stromal cells and acts as a co-stimulatory molecule when engaging the HVEM receptor
on T cells (62,102,103). In agreement with these results, we discovered that significantly
higher numbers of TILs were recruited into the microenvironments of LIGHT-treated
prostate tumors compared to untreated tumors. In addition, the number of infiltrating
suppressive Tregs did not increase in LIGHT treated tumors. Therefore, forced LIGHT
58

expression increased the Teff:Treg ratio as compared to other treatment groups. Lower
Teff:Treg ratios were shown to be predictive of a poor prognosis in a colorectal cancer
model (104). A recent case-control study that examined the Teff:Treg ratio in men with
prostate cancer showed an increased risk of dying of prostate cancer when a high
frequency of Tregs were found within the tumor microenvironment, but no correlations
were generated between prognosis and frequency of infiltrating T effector cells (105).
Although the importance of TIL in prostate cancer patients are controversial in disease
prognosis (106), our data suggest an important role of infiltrating CD4+CD3+ and
CD8+CD3+ T cells in mounting an effective immune response, and thus in reducing
tumor burden in vivo.
The clinical outcome of immunotherapeutic treatments is tightly controlled by the
balance between co-stimulatory and inhibitory signals (107). Traditional therapeutic
vaccines induce T cells with antigen specificity but fail to target the inhibitory pathways
in cancers, leading to an immune imbalance that favors tumor growth. This is the first
study we are aware of that examines LIGHT’s effect upon Treg mediated
immunosuppression in a tumor microenvironment, and we show that LIGHT favorably
tips the balance away from an immunosuppressive and towards an immunostimulatory
milieu. LIGHT has the ability to prevent the maturation of naïve T cells to Tregs and
infiltration of Tregs into the tumor microenvironment, thereby inhibiting the suppressive
effects of Tregs that promote tumor growth. Moreover, in iTreg induction assays, the
addition of LIGHT-expressing TRAMP-C2 cells resulted in a dramatic inhibition of
naïve T cells being induced to become iTregs. This indicates that the effects of LIGHT
59

may directly counteract Treg inducing factors with positive co-stimulation. TRAMP-C2
cells alone in the Treg induction assay also reduced the frequency of induced Tregs. This
may be explained by the fact that TRAMP-C2 tumor cells express TGF-β receptors (108),
which may have sequestered TGF-β in the cocultures. This likely reduced the free TGF-β
in culture that is used to drive Treg maturation, thus fewer Tregs were induced in
TRAMP-C2 cocultures than would otherwise be expected. LIGHT has been shown to
play a dominant role in preventing the immunosuppressive interaction between HVEM
and BTLA, which has been shown to inhibit T cell activation and enhance Treg mediated
immunosuppression (109). Studies have indicated the interaction between LIGHT-
HVEM and HVEM-BTLA play opposing roles in the tumor microenvironment. This
phenomenon has been coined as the “molecular switch” of T cells, where LIGHT
functions to activate T cells whereas BTLA inhibits this activation mechanism (63). The
HVEM receptor has 3 cysteine rich domains (CRD); LIGHT has been shown to occupy
CRD 2 and 3 while BTLA occupies CRD 1 (110,111). Due to a higher binding avidity
and affinity, LIGHT is capable of dislodging the inhibitory interaction between HVEM-
BLTA (112,113), indicating LIGHT’s potential in providing a positive effect of
costimulation and T cell recruitment when bound to HVEM. These interactions may
explain why we observed increased TILs and reduced Treg mediated immunosuppression
in LIGHT-treated prostate tumors.
LIGHT further potentiates an immunostimulatory environment through the regulation of
immune-modulating genes and cytokines. Tumors treated with Ad-LIGHT also displayed
an increase in NOS expression as compared to vector control or untreated, and NOS has
60

been shown to be cytotoxic to tumor cells (114-117). It was also observed that Arg-2 and
IDO, immunosuppressive and pro-tumor factors associated with poor prognosis
(118,119), were reduced in both Ad-LIGHT and Ad-Control treated tumors. IDO has
become a gene of interest because expansion of Tregs has previously been shown to be
induced in the presence of IDO, leading to immune tolerance against TAA (119). Arg-2
is known to be highly elevated in cancer patients with aggressive tumors (118,120,121);
therefore, reduction in both IDO and Arg-2 expression can potentiate a better prognosis.
Although statistical significance in the cytokine levels between the three treatment groups
was not reached due to high mouse to mouse variation, the trend towards increased NOS
and decrease Arg-2 and IDO expression argues that LIGHT expression indeed changes
the tumor microenvironment towards a more immunostimulatory one, which can be
confirmed in future experiments by using larger groups of mice. Additionally, we show
that LIGHT expression in prostate tumors resulted in an increased trend in
immunostimulatory chemokines, MIP-1  and MIP-1. MIP’s are directly involved with
migration and activation of lymphocytes (122,123), potentially explaining the increased
trafficking of T lymphocytes to the LIGHT expressing prostate tumors. VEGF, normally
a pro-tumor factor, shows a trend toward increased expression in LIGHT-expressing
prostate tumors. This is in agreement with the HPV 16 induced-cervical cancer model
(67) where VEGF was also increased after LIGHT treatment. Although VEGF is known
to be associated with a poor prognosis in patients due to promotion of tumor angiogenesis
(124), we have yet to determine the role of VEGF in LIGHT-expressing tumors. In a
wound healing study, LIGHT has been demonstrated to promote macrophage apoptosis
61

through VEGF expression, a process that is crucial for the resolution of inflammation
(125). VEGF may play a similar role in LIGHT-treated tumors, which would reduce
inflammation and thus control tumor homeostasis.
The induction of memory T cells plays an essential role in cancer-fighting properties. The
idea of a successful vaccine provides an advantage to the immune system in mounting an
immune response against invasive diseases and pathogens. Memory T cells plays a
crucial role in orchestrating these immune responses, they have distinct activation and
intracellular markers with a lower threshold and a diverse cytokine, profile against
specific antigens (126-128). Some studies show that in-situ tumor destruction of
melanoma or fibrosarcomas (excision of the primary tumor), aid the immune system in
mounting an anti-tumoral response against the tumor re-challenge (129,130). In this
study, we show that the excision of the primary tumor did not protect against the tumor
re-challenge (untreated group). However, we demonstrate that the vaccination with PSCA
TriVax and subsequently the removal of the primary tumor, protected against the tumor
re-challenge. This data suggest that PSCA TriVax induced tumor antigen specific
immune response that were capable of mounting an immune response against TRAMP-
C2 cells. In a translational sense, patients diagnosed with prostate cancer may opt for a
prostatectomy in combination with a therapeutic vaccine that will further control the
future progression of the malignant disease (131). LIGHT was not capable of inducing
memory against TRAMP tumors, although this was not surprising due to the lack of TAA
specific T cells in LIGHT vaccinated mice.
62

The TRAMP model is widely used in the field of prostate cancer research, since the
progression of disease mirrors a subset of patient cases. In this study, the vaccination of
TRAMP mice with PSCA TriVax, Ad-Control and Ad-LIGHT alone did not improve the
disease status. However the combination of PSCA TriVax and an adenovirus vector
(either Ad-Control or Ad-LIGHT) induced an immunogenic response and resulted in a
lower tumor burden as compared to single treatments. Although the tumor weights were
not statistically different, the pitfall lies in the TRAMP model since the SV40 Large T
antigen is constitutively expressed after puberty is reached, therefore, despite a powerful
treatment option the continuous growth of the tumor will continue. Our results propose
that the use of an immunogenic vaccine with an inflammatory response via intraprostatic
injections may reduce tumor burden.

63

2.6 Conclusion
In summary, our results provide evidence that the treatment of prostate tumors with
LIGHT can synergize with a therapeutic cancer vaccine in enhancing the anti-tumor
response that it elicits. Our data indicate that LIGHT achieves this by remodeling the
tumor microenvironment, by increasing cytokine signaling and effector T cell infiltration
within the tumor, by reducing Treg-mediated immune suppression, and by increasing
NOS expression. These factors skew the tumor microenvironment to a more
immunostimulatory state. The use of LIGHT may therefore be advantageous for the
successful application of therapeutic vaccines in prostate and other cancers.

64

2.8 Acknowledgement
This research was supported by Department of Defense grant PC100519 (to W.M.K).
Contributions from the Karl H. and Ruth M. Balz Trust are also gratefully acknowledged.
Poly-ICLC was a generous gift from Dr. Andres Salazar (Oncovir). W. Martin Kast holds
the Walter A. Richter cancer researcher chair. Lisa Yan is a TL1 scholar and supported
by SC CTSI (NIH/NCCR/CATS) grant #TL1TR000132. Bhavna Verma is supported by
a Roche post-doctoral fellowship. We thank ASM Scientific Writing and Publishing
Institute for critical reading of the manuscript. Elispot and cytokine assays were run with
the assistance of the USC Norris Comprehensive Cancer Center Beckman Center for
Immune Monitoring, supported in part by award number P30CA014089 from the
National Cancer Institute. The content is solely the responsibility of the authors and does
not necessarily represent the official views of the National Cancer Institute or the
National Institutes of Health

65

Chapter 3

Working Model, Pitfalls and Future Directions

3.1 Working model: Current view of LIGHT’s role in remodeling the
tumor microenvironment

In the context of the current research, our originally proposed model founded on our
hypothesis has been revised based on the data generated in this study (Figure 3.1). The
revised model is supported by the results and incorporates a cohesive concept. In this
model, LIGHT is expressed on the tumor cell surface via the delivery of an adenovirus
encoding membrane bound LIGHT. LIGHT subsequently interacts with resident LT βR
on stroma cells that release chemokines that attract naïve and effector T cells into the
microenvironment. LIGHT promotes the release of inflammatory cytokines, MIP-1α and
MIP-1β. The addition of PSCA TriVax mounts an immune response on DC, which then
migrate to the tumor draining lymph nodes where DC prime naïve T cells against
PSCA
83-91
. These primed T cells then migrate to the tumor microenvironment and lyse
the tumor. The expression of LIGHT nullifies the suppressive ability of Tregs and
prevents the differentiation of naïve T cells to Tregs. What this model doesn’t answer is
the role of LIGHT in the differentiation of naïve T cells (6). It is still unclear whether
LIGHT aids the differentiation of naïve T cells to effector cell types in the tumor
microenvironment (Figure 3.1).

66

Figure 3.1 Revised working model. (1) LIGHT is delivered to the prostate cancer cell
via an adenovirus vector coding for membrane bound LIGHT. (2) Once membrane
LIGHT is expressed, LIGHT engages with LT βR on stroma cells resulting in the release
of chemokine CCL21 that attracts T cells. (3) LIGHT alters the tumor microenvironment,
increasing cytokines MIP-1α, MIP-1β and VEGF and shifting immune modulating genes
towards a proinflammatory setting. (4) PSCA TriVax activates DC and subsequently
migrates to the tumor draining lymph nodes, (5) where they prime naïve T cells against
PSCA. (6) Engagement of LIGHT with naïve T cells prevents the induction of Tregs. The
type of differentiated T cells are still to be determined. (7) Tregs that engage with LIGHT
become inactivated and lose their suppressive capacity.
67

Figure 3.2. Differentiation of Naïve CD4+ T cells to effector cell types. The
differentiation of naïve T cells to effector cell types are persuaded by the cytokines and
signals provided. Figure adopted from the National Institute of Arthritis and
Musculosketal and Skin Disease (132).

68

3.2 Pitfall: Delivery and intratumoral expression of Ad-LIGHT
The effects of LIGHT demonstrated an immunogenic response that synergized with
PSCA TriVax in ways of recruiting T cells while overcoming Treg mediated
immunosuppression in TRAMP-C2 challenged mice. Although the vaccination regimen
reduced tumor burden to a certain degree, an improved method of LIGHT delivery may
advance the overall outcome of our results. Adenovirus LIGHT serves as a great delivery
method in vitro, where plated cells have the opportunity to take up the virus. However,
the expression of LIGHT in the whole tumor is questionable (Figure 3.3). Transplantable
TRAMP-C2 tumors are solid in nature and have an extraordinarily high interstitial
pressure (133), posing a barrier to drug delivery.

Figure 3.3. Delivery of Ad-LIGHT via intratumoral injections. 2x10
12
vp are
delivered intratumorally via a 31 gauge insulin syringe. The needle is inserted into the
center of the tumor and 20 µl of Ad-LIGHT is injected. Figure adopted from glowm.com
69

The delivery of Ad-LIGHT into the tumor only exposes the virus to the needle line,
suggesting that the virus does not become distributed throughout the solid tumor due to
interstitial pressure, therefore, minimizing the potential effects of Ad-LIGHT. We have
explored immunohistochemistry of tumor sections treated with Ad-LIGHT, Ad-Control
and Untreated to explore the expression of LIGHT solid tumors. Due to the complex
staining and transient expression of Ad-LIGHT, an optimized protocol is needed.
3.3 Future direction: Targeted therapies: Development of Bi-specific
Fusion Proteins with LIGHT

Although Ad-LIGHT proved to be a synergistic option with a prostate cancer therapeutic
vaccine our delivery option is limited to the needle-line injection, therefore, to achieve
whole tumor expression of LIGHT an improved delivery method is needed. As seen in
literature, a critical barrier to achieving complete tumor eradication is due to the lack of
targeted therapies in a highly vascularized tumor microenvironment (134). Delta-like
Ligand 4 (DLL4) and EphB4 are two proteins involved in angiogenesis that are
upregulated in tumor vasculature and tumors. Their expression in the tumor
microenvironment allows specific targeting by antibodies or antibody fragments. Anti-
DLL4 antibodies target the tumor vasculature and have been demonstrated to induce non-
functional angiogenesis and in turn reduce tumor burden (135). Anti-EphB4 antibodies
target EphB4 expressed on tumor cells and likewise mediate tumor cell death (136).
Therefore the development of bi-functional fusion proteins with LIGHT are promising
candidates for targeted cancer therapy because they are able to engage two molecules that
may enhance anti-tumor T cell immunity. Expression of LIGHT in the tumor
70

microenvironment through DLL4 or EphB4 antibody targeting is a novel approach to
alter the primary tumor microenvironment and any distant metastases. The development
of the first bi-functional protein has been shown to be functional via an ELISA performed
by Dr. Bhavna Verma.
Specific aims of this future project:
Specific Aim 1: To produce and validate scFv-DLL4-LIGHT protein and scFv-EphB4-
LIGHT protein for in vivo studies.
Specific Aim 2: To determine the efficacy of scFv-DLL4-LIGHT and scFv-EphB4-
LIGHT, in TRAMP-C2 tumor bearing mice.
Specific Aim 3: Determine whether scFv-DLL4-LIGHT or scFv-EphB4-LIGHT
treatment in combination with a therapeutic prostate cancer vaccine can induce complete
regression of primary and advanced prostate tumors in the TRAMP model.

71

3.5 Research Summary

While the developments of novel treatments are ongoing, metastatic prostate cancer is an
advanced disease with unmet clinical needs. This doctoral thesis focused on improving
current vaccines with a strategy that can further boost immunogenic responses. Although
immunogenicity may be difficult to trigger, we witnessed inflammatory responses in the
tumor microenvironment with forced LIGHT expression. It was shown in our results that
LIGHT induced a limited immune response, most probably due to the expression of
LIGHT in a subsection of the tumor due to the limitations of the delivery method. Further
work needs to be carried out to enhance targeted therapies and to determine whether bi-
functional fusion proteins are better targeting therapies to deliver the benefits of LIGHT.
Taken together, this study achieved a few important objectives. It provided an excellent
gateway in establishing the mechanistic effects of forced LIGHT expression in tumor
microenvironments, providing information that can potentially lead to the development of
targeted treatments against Treg suppression. Secondly, LIGHT treatment may
potentially be a new therapy in cancer treatment without side effects of current standard
of care; chemotherapy, prostatectomy, etc. Overall, this study is pushing immunotherapy
one step forward, mitigate many unknown facts in tumor immunosuppression and
establish a useful augmenter for current and future cancer vaccines. We hope that the
findings here will aid future development of therapeutic vaccines and will soon be
translated to the clinic.
72

Chapter 4

Additional Projects

4.1 Publications by the author

1. Gray A, Yan L, Kast WM, Prevention is better than cure: the case for clinical
trials of therapeutic cancer vaccines in the prophylactic setting. Mol Interv. 2010
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Abstract (if available)

Abstract Prostate cancer affects approximately 200,000 men and account for 30,000 deaths annually in the United States alone. This major health problem cost Americans $255.6 million dollars in health care and even then, current treatments for prostate cancer including surgery, chemotherapy and radiotherapy, may lead to devastating side effects such as impotence and incontinence. Prostate cancer immunotherapy offers an advantage to patients, avoiding these potential side effects, where a patient’s own immune system is stimulated and taught to eradicate the existing tumor. The goal of this research is to improve current therapeutic vaccines by boosting the immune system with a new treatment, LIGHT, in combination with a prostate cancer therapeutic vaccine. This study will assess the efficacy of combination immunotherapy treatment and measure survival in a prostate cancer mouse model that mimics prostate cancer progression in a subset of patients. With this translational project, we will generate a synergistic combination therapy that will push the field of prostate cancer immunotherapy forward, providing a curative option for patients of this disease.

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Asset Metadata

Creator Yan, Lisa (author)

Core Title LIGHT-ing up prostate cancer: remodeling the tumor microenvironment and enhancing therapeutic vaccine efficacy through forced LIGHT expression in prostate cancer

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 01/24/2015

Defense Date 12/08/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag immunotherapy,Light,OAI-PMH Harvest,prostate cancer,regulatory T cells

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Kast, W. Martin (committee chair), Akbari, Omid (committee member), Wong, Michael K. (committee member)

Creator Email lisayan@usc.edu,lisayanphd@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-523698

Unique identifier UC11297385

Identifier etd-YanLisa-3126.pdf (filename),usctheses-c3-523698 (legacy record id)

Legacy Identifier etd-YanLisa-3126.pdf

Dmrecord 523698

Document Type Dissertation

Format application/pdf (imt)

Rights Yan, Lisa

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA