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Oncolytic adenovirus based vaccine in cancer immunotherapy
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Oncolytic adenovirus based vaccine in cancer immunotherapy
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Content
Oncolytic Adenovirus Based Vaccine in
cancer immunotherapy.
By
Aazam Ghelani
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
In Partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
December 2015
2
ACKNOWLEDGEMENT
It gives me immense pleasure to convey my gratitude and regards to all those
who have been a part of my journey in successfully completing my Master’s Degree.
I would like to express my gratitude and gratefulness to my advisor, Dr. Xue
Huang, for her constant moral support, guidance, patience, motivation and immense
knowledge throughout my research work and writing my thesis. Besides my advisor,
I would like to thank the rest of my thesis committee Dr. Si Yi Chen and Dr. Andre
Ouellette for their time and consideration.
My special thanks to our lab manager Lindsey Woodham, who has been a
constant support and a great colleague.
I wish to specially acknowledge Dr. Martin Kast and his lab members, Dr.
Diane Da Silva, Dr. Andrew Woodham, Joseph Skeate and Brand Heike for helping
me through the entire project
I would like to thank members of Dr. Pinghui Feng’s lab, Dr. Chengyu
Liang’s lab and Brooke Nakamoora for their constant help with materials and
protocols.
Last but not the least, I would like to thank my family: my parents Mrs.
Maherbanu Ghelani, Mr. Parvez Ghelani, my brother Aadil, and all of my friends for
supporting me throughout my life.
3
TABLE OF CONTENTS
ACKNOWLEDGEMENT ...................................................................................................2
LIST OF FIGURES .............................................................................................................5
LIST OF TABLES ...............................................................................................................6
ABSTRACT .........................................................................................................................7
CHAPTER 1: INTRODUCTION .......................................................................................8
1.1 Oncolytic viruses ....................................................................................................8
1.2 Immuno-stimulation by oncolytic virus ...............................................................10
1.3 Adenovirus as an oncolytic virus .........................................................................12
1.4 Heat Shock Protein – Oncolytic adenovirus expressing Heat Shock protein 70
(AD-HE) .....................................................................................................................15
1.5 Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) – Oncolytic
virus expressing GM-CSF ..........................................................................................18
CHAPTER 2: GOALS .......................................................................................................20
CHAPTER 3: MATERIALS AND METHODS ...............................................................21
3.2 Methods for cloning .............................................................................................22
3.3. Cell culture ..........................................................................................................24
3.4 Transfection ..........................................................................................................24
3.5 Construction and cloning of pShuttle-mGMCSF-IRES-E1a (PGE) ....................25
3.6 Construction of adenoviral vector backbone expressing mGMCSF-E1a (AD-
PGE) from PGE ..........................................................................................................26
3.7 Generating recombinant oncolytic adenovirus expressing mGM-CSF (AD-
GMCSF) from AD-PGE .............................................................................................28
3.8 Western blot Analysis to determine E1a expression ............................................31
4
3.9 Intra-cellular cytokine staining to determine mGM-CSF expression ..................33
3.10 Construction and cloning pShuttle-CMV-E1a (PE1a) .......................................34
3.11 Transfection efficiency in B16 cells ...................................................................34
3.12 Tumor growth assay in mice by injecting plasmid transfected cells ..................35
CHAPTER 4: RESULTS ...................................................................................................37
4.1 Confirm Successful cloning of pShuttle-mGMCSF- IRES-E1a ..........................37
4.2 Confirm successful cloning of AD-GMCSF vector backbone AD-PGE .............41
4.3 GM-CSF and E1a expression by oncolytic adenovirus AD-GM-CSF .................42
4.4 Transfection efficiency in B16 melanoma cells ...................................................44
4.5 Effect of GM-CSF expression and HSP70 expression on in vivo tumor growth in
mice ...........................................................................................................................46
CHAPTER 5: DISCUSSION .............................................................................................49
CHAPTER 6: FUTURE DIRECTIONS ............................................................................50
REFERENCES ..................................................................................................................52
5
LIST OF FIGURES
Figure 1: Mechanistic representation of oncolysis by oncolytic virus................................9
Figure 2: Schematic representation of mechanism of immune-stimulation by oncolytic
virus ...................................................................................................................................12
Figure 3: Diagrammatic representation of Adenovirus replication cycle. ........................13
Figure 4: Mechanism of Oncolysis by oncolytic Adenovirus ..........................................14
Figure 5: Mechanism of action of AD-HE ......................................................................16
Figure 6: Anti-tumor and Anti-tumor immune response by AD-HE. ...............................17
Figure 7: Functions of GM-CSF (A) and role of GM-CSF at the tumor site (B). ............19
Figure 8: Summary of method for generating AD-PGE from PGE .................................28
Figure 9: Summary of protocol for generating AD-GMCSF from AD-PGE using AdEasy
protocol ..............................................................................................................................31
Figure 10: Construction and confirmation of cloning of PGE. .........................................40
Figure 11: Confirmation of successful cloning of AD-PGE. PCR bands for mGM-CSF
and IRES-E1a ....................................................................................................................41
Figure 12: Confirm successful generation of AD-GMCSF from AD-PGE. .....................44
Figure 13: Transfection efficiency by GFP expression confirms 10 µgm Plasmid
transfection efficiently transfects about 40-50% cells. ......................................................46
Figure 14: Mean tumor volume measurement for determining tumor growth of PGE,
PHE, PE1a, PCMV transfected and non-transfected tumor cells in mice. ........................48
Figure 15: Summary of Hypothesis of using AD-HE in combination with anti PD-1/PD-
L1 checkpoint therapy........................................................................................................51
6
LIST OF TABLES
Table 1: Primers used for PCR amplification ..................................................................22
7
ABSTRACT
Cancers have been a leading cause of mortality amongst humans for several years.
Even persistent innovations in the field of medicine, around the globe, have not yet been
able to overcome consistently high mortality rate due to cancers. One reason attributed to
being a major hurdle in treating cancers is difficulty in developing tumor specific and
selective therapy. Also, severe side effects have made several therapies undesirable. Recent
advances made in the field of gene therapy are still difficult to be translated to clinic, again
because of inability to achieve safe and tumor specific delivery. Oncolytic viruses have
been known since 19
th
century. Moreover, recent advances in genetic engineering have
made it possible to engineer an otherwise pathogenic virus to selectively replicate inside
and kill cancer cells. Such a therapy can also help to overcome undesirable side effects
associated with traditional therapy. Advances in gene therapy and molecular engineering
has made it possible to develop viruses which can not only selectively kill cancer cells, but
also express gene of interest inside cancer cells. The goal of this project was to compare
efficacy of oncolytic adenovirus expressing HSP70 and another similar virus expressing
GM-CSF in inducing anti-tumor and anti-tumor immune response. By western blot
analysis and intracellular cytokine staining it was confirmed that AD-GMCF was
successfully generated for this purpose. In in vivo tumor growth studies, tumor cells
transfected with plasmid expressing GM-CSF and HSP70 grew significantly slowly in
mice and their growth was more inhibited in mice compared to the control groups.
However, high titer virus needs to be generated in order to compare the efficacy of both
viruses in generating anti-tumor and anti-tumor immune response.
8
CHAPTER 1: INTRODUCTION
The progress made in different strategies such as chemotherapy, radiation therapy,
and surgery, to combat cancer has not been able to overcome high mortality rate amongst
cancer patients. During 2013, cancer was the second highest leading cause of deaths in the
USA with 584,881 deaths being reported due to malignant neoplasms
1
. These strategies
have particularly failed in late stage cancers and hard to cure cancers such as lung, brain,
and pancreatic cancers. Chemotherapy and radiation therapy have also been associated
with poor tumor selectivity and undesirable side effects. Oncolytic viruses have made it
possible to achieve tumor specific killing effect, without producing undesirable side effects
2
. Gene therapy and siRNA based therapy is gaining popularity amongst scientists for
tumor therapy. Oncolytic viruses serve as a novel method to deliver and express a gene or
shRNA inside tumor cells
2, 17, 19
.
1.1 Oncolytic viruses:
Oncolytic viruses specifically replicate inside tumor cells and lyse tumor cells
selectively. They might infect healthy host cells, however, because of their intrinsic
property (wild type oncolytic virus) or because of genetic recombination (genetically
engineered oncolytic virus), they specifically complete their replication cycle only inside
tumor cells and, subsequently, lyses tumor cells and not healthy host cells
3
. The released
progeny viruses, from the lysed tumor cells, infects and lyses neighboring tumor cells and,
thus, magnifies anti-tumor activity
2
. Figure 1 describes the mechanism of oncolysis by an
oncolytic virus.
9
Figure 1: Mechanistic representation of oncolysis by oncolytic virus
2
Oncolytic property of viruses has been known since the middle of 19
th
century
4
.
However, it came to light for the first time when in 1949 Moore showed the anti-tumor
effect of Russian Far East encephalitis virus in mice tumor model
5, 6
. Over the period of
1950s to 1980s several wild type viruses including Measles Virus, Newcastle Disease
Virus, Vesicular Stomatitis virus, etc., were used in clinical trials
7, 8
, and, with the advent
of molecular gene engineering techniques, over the last 2 decades, recombinant viruses
such as Adenovirus, Herpes Simplex virus, Vaccinia virus, etc., have been used as
oncolytic virus in several clinical and pre-clinical studies
9
. The first oncolytic virus to be
approved for clinical use was ONYX -015 in China, for oropharyngeal cancer treatment in
China
10
.
10
Oncolytic viruses can also potentiate an immune response against the tumor
indirectly by making tumor associated antigen available, released through tumor cell lysis,
to the local antigen presenting cells. The anti-tumor immune response, generated due to the
action of the oncolytic virus, is described in more detail later. Recombinant oncolytic
virus cloned to express a protein or shRNA, particularly cytokines, in cancer cells have
been shown to be a promising strategy for cancer immunotherapy
10
.
1.2 Immuno-stimulation by oncolytic virus:
Tumor cell lysis by oncolytic virus also potentiates an immune response against
tumor cells, by making tumor associated antigens accessible to the local antigen presenting
cells. Infection of tumor cells by oncolytic virus changes local innate and adaptive immune
response.
Cancer Immune environment:
Cancer microenvironment has shown to be highly immune-suppressive in nature.
Tumor cells upregulate secretion of immune inhibitory cytokines such as IL-10 and TGF-
β
11
. Cytokine such as VEGF, secreted by tumor cells, also promotes tumor growth and
proliferation. Tumor microenvironment consists of high numbers of immunosuppressive
cells such as T-regulatory cells, and Myeloid derived Suppressor Cells (MDSCs)
11
. Danger
signals such as Heat Shock Protein (HSPs), uric acid, bradykinin, etc., are known to
activate antigen presenting cells
12
. Tumor cells adapt to downregulate their expression
13
.
Hence, to summarize tumor microenvironment tends to be immune-suppressive by nature
and, thus, supports tumor proliferation.
11
Immuno-stimulation by oncolytic virus:
The oncolytic virus infects and selectively kills cancer cells and thereby releases
tumor associated antigens (TAAs) in tumor microenvironment
2
. TAAs are phagocytized
by antigen presenting cells, chiefly Dendritic cells (DCs), and these activated dendritic
cells primes T cells to produce an anti-tumor response. Indeed it has been shown in several
pre-clinical studies that treating tumor with oncolytic virus leads to high infiltration of
cytotoxic T cells, T helper cells (Th cells), Natural Killer cells (NK cells), macrophages
and neutrophils into the tumor
14, 15, 16, 17
. These cells produce an anti-tumor immune
response. Moreover, in a study involving Reovirus as an oncolytic virus, it was found that
reovirus treated melanoma cells produced higher amounts of IL-8 and RANTES, which
plays a role in recruiting DCs and neutrophils. Reovirus treated melanoma cells also
downregulated secretion of IL-10, which is an immune-suppressive cytokine
18
. A previous
study in our lab has shown that oncolytic adenovirus expressing RANTES when injected
into the tumor, dendritic cells infiltrate inside the tumor
19
. Several in vivo studies,
involving intra tumor injection of oncolytic virus, have shown that oncolytic virus treated
tumors produce tumor specific cytotoxic T cell response
16, 17, 18, 19
. Treatment of tumor
cells with oncolytic virus results in higher production of damage signals from tumor cells.
Higher production of damage signals can also act as an adjuvant on antigen presenting cells
that are exposed to TAAs. Thus, several studies have proved that treatment of tumor using
oncolytic virus not only leads to direct tumor cell lysis but also generates an anti-tumor
immune response. Figure 2 summarizes immune-stimulation by an oncolytic virus.
12
Figure 2: Schematic representation of mechanism of immune-stimulation by oncolytic
virus
2
.
1.3 Adenovirus as an oncolytic virus:
Adenoviruses have been identified as early as 1953
20
. It is a non-enveloped virus
containing icosahedral nucleocapsid and double stranded DNA genome. It infects host cells
by attachment of the knob domain of its capsid spike CAR/CD46 receptor on host cells. It
is also speculated that adenovirus can also attach to the host cells by binding to MHC
molecules, sialic acid or integrins. Adenoviral particle gets internalized by endocytosis,
and after the nuclear capsid proteins are processed, the adenoviral genome is released
inside the nuclease. The adenoviral genome consists of early and late transcribed genes.
Upon entry into the nucleus early transcribed genes are expressed and early transcription
products then inhibit host transcription and translation, inhibit host cell apoptosis, evade
immune cell attack and facilitate late gene expression and adenoviral DNA replication
21
.
Chief amongst these early products are E1a and E1b
21
. E1a binds to and inhibits
13
retinoblastoma tumor suppressor protein (RB), whereas E1b binds to p53 protein and
inhibits its function. Inhibition of RB and p53 immortalizes host cell and promotes
transcription and replication of adenoviral DNA
21
. The progeny viruses are assembled near
nuclear pore, and progeny viruses are released by lysing the cells
21
. Figure 3 summarizes
the replication cycle of wild type adenovirus.
Figure 3: Diagrammatic representation of Adenovirus replication cycle
33
.
The fact that adenovirus requires both the proteins, p53, and RB, to be inhibited to
promote adenovirus production and amplification, it is an ideal candidate to be engineered
to inhibit only one of the 2 proteins and be used as an oncolytic virus. Cancer cells usually
lack a functional p53 protein and hence all an adenovirus needs to do is to inhibit only RB
protein to replicate and lyse cancer cells. However, such an adenovirus that can only inhibit
RB protein cannot replicate inside a healthy cell as a functional p53 will induce the host
cell to undergo apoptosis and hence, no viral progeny is produced. Adenovirus can be
14
designed to express only either of E1a or E1b, and it will selectively replicate inside cancer
cells
22
. Indeed, it has been shown experimentally that only E1a expressing virus selectively
replicates inside and lyses cancer cells
23
. However, it is necessary that adenovirus should
not express E3, as E3 interferes with MHC class I molecule translocation to cell surface.
MHC I translocation is needed by cytotoxic T cells to attack cancer cells. Moreover, the
cloning gene of interest is also comparatively easier in adenovirus. Adenovirus can also
bind and infect a variety of cell lines which also makes it very desirable. Even though,
adenovirus expressing either of E1a and E1b can be oncolytic in nature, our lab is interested
in developing E1a expressing oncolytic adenovirus as they have higher efficiency
24
.
Figure 4 explains the mechanism by which oncolytic adenovirus selectively kills cancer
cells.
Figure 4: Mechanism of Oncolysis by Oncolytic Adenovirus
24
15
1.4 Heat Shock Protein – Oncolytic adenovirus expressing Heat Shock Protein 70
(AD-HE):
Heat Shock Protein 70 (HSP70) is expressed in several cell types, and it has an inherent
property of binding to and chaperone several peptides on its surface
25, 26
. These peptides
can be antigens as well. HSPs can also enter dendritic cells via receptor mediated pathway
27
. HSPs also have an ability to gain access to MHC class I and class II antigen loading site
and hence, they can play a role as a carrier by carrying antigens bound on its surface to the
antigen loading site of MHC class I and II molecules and aid in generating an immune
response against those antigens
27
. Moreover, HSPs are also known damage associated
molecular patterns. They are recognized by TLR2 and TLR4 receptors
28
, and hence, they
provide a maturation signal to antigen presenting cells, stimulating them to upregulate
expression of co-stimulator and antigen presenting molecules such as CD80 and CD86 as
well as MHC II molecules
25, 26
. Hence, if expressed inside cancer cells and released from
cancer cells, heat shock protein can aid in generating an immune response against tumor
antigens and act as an adjuvant by giving stimulatory signals to tumor resident antigen
presenting cells. In a previous study, HSP, purified from the tumor, when injected back in
the tumor resulted in stimulation of immune response
25
. Our lab has previously developed
and tested an oncolytic adenovirus expressing HSP70 (AD-HE). Injection of AD-HE into
B16 melanoma tumor resulted in mice becoming tumor free
17
. Moreover, it also produced
an anti-tumor immune response that was confirmed by ELISpot assay and cell cytotoxicity
assay
17
. Figure 6 describes some of the results obtained by injecting AD-HE into B16
tumors. Figure 5 describes the mechanism by which AD-HE produces an anti-tumor
response.
16
Figure 5: Mechanism of action of AD-HE
17
.
17
Figure 6: Anti-tumor and Anti-tumor immune response by AD-HE represented by direct
recession in tumor growth in AD-HE treated mice (A), higher IFN spots in AD-HE
treated mice (B), higher percentage of B16 cell lysis by splenocytes from AD-HE treated
mice (C)
17
.
A
B
C
18
1.5 Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) – Oncolytic
virus expressing GM-CSF:
GM-CSF plays a role in activating and recruiting dendritic cells, macrophages and
monocytes at the site of injury
29
. GM-CSF plays a key role in providing a differentiation
and maturation signal to antigen presenting cells
29
. It stimulates stem cells to produce more
granulocytes and monocytes. GM-CSF, when secreted in the tumor site, induces
macrophages and dendritic cells to exit from circulation or tissue resident sites and enter
the tumor microenvironment
30
. This is very important in breaking tolerance and initiating
an anti-tumor T cell and immune response. In studies involving cancer cell vaccines
expressing GM-CSF, the anti-tumor immune response was stimulated, and it is also tested
in clinical trials. GM-CSF stimulates one specific type of dendritic cells which are more
potent in phagocytosis and adaptive immune cell stimulation
30
. Several oncolytic viruses
expressing GM-CSF have been developed, T-VEC (Herpes simplex virus based oncolytic
virus) and JX-594 (Vaccinia virus based vaccine)
31, 32
, and they are currently under clinical
trials. Figure 7 summarizes functions of GM-CSF and its role at the tumor site.
19
Figure 7: Functions of GM-CSF (A) and role of GM-CSF at the tumor site (B)
20
CHAPTER 2: GOALS
The lab’s overall goal is to develop novel immuno-therapeutic approaches for
treatment of cancer. One strategy is the AD-HE vaccine combined with anti PD-1/ PD-L1
checkpoint therapy
• As mentioned, our lab previously generated the AD-HE vaccine, which was
an oncolytic adenovirus to express HSP70 that possessed dual functions:
oncolytic activity against the primary tumor followed by the release of
tumor antigens, and HSP-associated adjuvant function on APC to prime T
cells against these tumor antigens.
• Given that a few of oncolytic viruses encoding human GM-CSF have
recently entered clinical trials and showed an antitumor effect through a
direct lytic activity and induction of antitumor immunity, we will construct
a recombinant oncolytic adenovirus expressing GM-CSF and compare this
with the AD-HE vaccine.
21
CHAPTER 3: MATERIALS AND METHODS
3.1 PCR, Primers, and Gel Electrophoresis:
All PCR reactions were carried out by following protocol using Applied
Biosystems GeneAmp PCR system 9700.
Reaction mix:
200 ngm Template DNA
+ 0.5 µl Forward primer
+ 0.5 µl Reverse Primer
+ 25 µl 2X PCR Master mix (Dream Taq Green, Frementas)
Nuclease free water (Growcells, inc.) to make 50 µl
PCR Temperature cycle:
95
o
C - 5 minutes
95
o
C - 30 seconds
55
o
C - 30 seconds
72
o
C - 30 seconds
72
o
C - 30 seconds
4
o
C - ∞
For gel electrophoresis, 1 % Agarose gel (Applied Biosystems) was prepared in 1X TAE
buffer containing 0.5 µg/ml ethidium bromide (Sigma). Electrophoresis was conducted at
constant 120V for 30 min. Samples, containing 1 X solution of loading dye (NEB) were
added into the well and appropriate size DNA ladder (NEB) was used. After
electrophoresis gels were analyzed using Bio-Rad gel analyzer machine.
30 Cycles
22
Table 1: Primers used for PCR amplification
Primer Name Company Sequence (5’ to 3’)
mGM-CSF
forward
Valuegene
ACT GCG GCC GCA TGT GGC TGC AGA ATT
TA
mGM-CSF
reverse
Valuegene CCG ACT AGT TCA TTT TTG GAC TGG TTT
IRES forward Valuegene GAT CCT CGA GGC CCC TCT CCC TCC CCC C
E1a forward Valuegene
AT CGC GGC CGC ATG AGA CAT ATT ATC
TGC CAC
E1a reverse Valuegene
GGA ATG AAT GTT GTA TAG GTG GCT TAA
GAT ATC CTC GAG ACT CCT TAC TTA CAA
CAT ATC CAC CGA ATT CTA TAG GAG CTC
TGA
3.2 Methods for cloning:
Digestion: All restriction enzyme digestion reactions were carried out by following
procedure in a 1.5 ml microcentrifuge tube. 4 µg template DNA, 1 µl R1 (restriction
enzyme 1), 1 µl R2 (restriction enzyme 2), 2 µl 10x cutsmart buffer (NEB), nuclease free
water to make 20 µl. The digestion mix was incubated at 37
o
C overnight in a water bath
(Fischer Scientific, Isotemp 205). All restriction enzyme digestion reactions were carried
out using 2 enzymes unless otherwise indicated. After overnight digestion 1 µl alklaline
phosphatase was added into the mix and samples were incubated for 30 minutes on water
23
bath at 37
o
C for dephosphorylation, unless otherwise indicated. Digested samples were
purified through gel electrophoresis or PCR purification kit.
Ligation: All ligation reaction were carried out using following protocol. 100 ngm digested
vector, insert (1:9 molar ratio of vector:insert), 1 µl T4 DNA ligase (Roche), 1 µl 10X T4
DNA ligase buffer, nuclease free water to make 10 µl. The ligation mix was incubated at
16
o
C overnight (PCR machine).
Bacterial transformation: Top 10 competent cells (obtained as a gift from Dr. Pinghui
Feng’s lab) were allowed to thaw on ice for 10 minutes. 10 µl ligation mix was added to
50 µl of cells. The mix was then incubated on ice for 30 minutes. The mix was then
subjected to heat shock by incubating it at 42
o
C for 1 minute in a water bath and
immediately transferring it back to ice. After allowing it to stand for 5 min in ice, 1 ml LB
medium was added to it, and the suspension was transferred to 15-ml Falcon 2059
polypropylene tube. The tube containing the cell suspension was transferred to orbital
shaker (Thermo scientific, Forma orbital shaker), and cells were subjected to shaking at
225 rpm and 37
o
C for one hour. Cell suspension was transferred to 1.5 ml micro-centrifuge
tubes and centrifuged at 140000 rpm for 10 seconds (5417R, Eppendorf). All but 100 µl of
supernatant media was aspirated. The cells were re-suspended in 100 µl media and plated
on LB-Agar-Kanamycin (50 µgm/l) plates and incubated overnight at 37
o
C. Next day,
positive colonies were picked up and were cultured overnight in 5 ml LB medium and by
incubating it in a shaker at 37
o
C. Positive plasmids were purified by miniprep by using
QIAprep spin mini prep kit (Qiagen by following manufacturer’s protocol. Plasmids were
analyzed by PCR and if correct maxi-prep was performed using QIAprep endo free plasmid
maxiprep kit (Qiagen) by following manufacturer’s protocol.
24
3.3. Cell culture:
All cells were cultured using following protocol. All cells were cultured using
DMEM (Corning, high glucose) containing 10 % fetal bovine serum (Hyclone) and 1 %
penicillin-streptamycin (Gibco). For 293A and 293 T (ATCC) cell culture, DMEM
(Corning, high glucose) containing 10 % fetal bovine serum (Hyclone) 1 % anti-anti
(Gibco) and 1 % L-glutamine (Gibco). All cells were incubated at 37
o
C and 5 % CO2 in a
sterile humidified incubator. Cell passaging was done either by trypsinization or by
vigorous pipetting. Detached cells were centrifuged at 500 g for 5 minutes, and the
supernatant was aspirated. Pelleted cells were re-suspended in 1ml fresh medium, and 0.5
ml or 0.7 ml of suspension was added into fresh medium containing cell culture dish
(Genesee Scientific). To obtain 50 % confluency in a 6-well dish 5 x 10
5
cells were added
to each well. For culturing in 6 well, 60 cm
2
, or 145 cm
2
dish 2, 10 or 20 ml complete
medium was used. Cell culture work was conducted in a sterile biosafety cabinet.
3.4 Transfection:
All transfection reactions were carried out using Polyethyleneimie (PEI) (Polysciences).
16-24 hours prior to transfection cells were passaged in order to achieve 50%-70%
confluency on the day of transfection. For transfection, vector DNA and PEI were added
in a ratio of 1:3 in serum free DMEM medium and properly mixed. The transfection mix
was allowed to stand for 15 minutes at room temperature and in the meantime old medium
was replaced with fresh medium in the cell culture dish to be transfected. After 15 minutes,
1 ml of complete medium was added and mixed in the transfection mix, and the mix was
then added to cell culture dish drop by drop. The cells were incubated overnight and the
25
next day medium was replaced with fresh medium. All transfection work was carried out
in the sterile biosafety hood.
3.5 Construction and cloning of pShuttle-mGMCSF-IRES-E1a (PGE):
AD-GMCSF will be generated using ADEASY protocol. As per ADEASY
protocol to generate recombinant AD-GMCSF, we need to first clone the gene of interest,
mGM-CSF cDNA and E1a cDNA, into pShuttle CMV vector (henceforth referred to as
PCMV) because PCMV has sequences which undergoes homologous recombination with
regions on pAD-EASY 1 to form adenoviral vector backbone containing gene of interest.
pShuttle-mGMCSF-IRES-E1a (henceforth referred to as PGE) was constructed from
pShuttle-HSP70-IRES-E1a (henceforth referred to as PHE), which was previously
prepared in our lab
17
. mGM-CSF cDNA, required for cloning into PGE, was obtained by
PCR amplification of mGM-CSF cDNA present in pcDNA-mGMCSF vector (provided as
a gift by Dr. Martin Kast’s lab, MMI Department, University of Southern California) using
mGM-CSF forward and reverse primers (sequence mentioned in Table 1) and by following
PCR protocol mentioned in section 3.1. PCR amplified mGM-CSF cDNA was purified
using QIAquick PCR purification kit (Qiagen) by following manufacturer’s protocol.
Purified cDNA was dissolved in 25 µl nuclease free water (Growcells, inc.), and its
concentration was determined using Nanodrop machine (Thermo Nanodrop 2000 c). PHE
and PCR amplified mGM-CSF cDNA were digested by restriction enzymes in separate 1.5
ml tubes by following protocol mentioned in section 3.2. In this case, the enzymes R1 and
R2 were NotI (NEB) and SpeI (NEB). The digested PHE vector was subjected to gel
electrophoresis to separate digested fragments based on their size by following protocol
mentioned in section 3.1 for gel electrophoresis. After electrophoretic separation digested
26
and linearized PHE vector was purified from 1% agarose using QIAquick gel extraction
kit II (Qiagen). Digested mGM-CSF cDNA was purified using QIAquick PCR purification
kit by following manufacturer’s protocol. Digested mGM-CSF was not dephosphorylated.
Purified digested vector and cDNA were dissolved in 25 µl nuclease free water and the
final concentration was determined using the same nanodrop machine. Linearized vector
and cDNA insert were then ligated by following ligation protocol mentioned in section 3.2.
Ligated mix was then used to transform TOP 10 chemical competent bacterial cells using
LB/Agar plates containing 50 µgm/ml kanamycin as pShuttle vector has kanamycin
resistance gene. The protocol for the same is mentioned in section 3.2. Transformed
colonies were cultured to obtain successfully constructed and cloned PGE; plasmid
purification was done using mini-preparation. To confirm successful cloning of PGE, PCR
was conducted using mGM-CSF forward and reverse primers (Table 1) and IRES forward
and E1a reverse primers (Table 1). After confirming positive clones, endo-free maxi-
preparation was performed for one of the positive clone using QIAprep endo free maxi
preparation kit (Qiagen) using manufacturer’s protocol.
3.6 Construction of adenoviral vector backbone expressing mGMCSF-E1a (AD-
PGE) from PGE:
According to AdEasy protocol, to generate adenoviral vector backbone containing the
gene of interest, PCMV containing the gene of interest and pAdEasy1 are facilitated to
undergo homologous recombination in BJ5183 recombination competent cells. Figure 8
summarizes the protocol for the construction of AD-PGE. PGE was digested and linearized
using protocol mentioned in section 3.1. However, 2 modifications were made to that
protocol. Instead of 2 only one restriction enzyme was utilized PmeI (NEB) and digested
27
vector was not dephosphorylated. Linearized PGE was purified using QIAquick PCR
purification kit (Qiagen) and dissolved into nuclease free water (Growcells inc.). BJ5183
AD 1 cells (Agilent Technologies), which are already transformed with pAdEasy 1
plasmid, were used to construct AD-PGE. After removing BJ5183 AD 1 cells from –80
o
C, they were allowed to thaw on ice for about 8 – 10 minutes. 40 µl of thawed BJ5183 AD
1 cells were transferred to sterile and pre-chilled 2 mm gap electroporation cuvette (Bio-
Rad). 0.05 µgm linearized PGE was added to cell suspension in an electroporation cuvette.
The suspension was tapped to make sure there were no bubbles in the cuvette and cuvette
was wiped to dryness from outside by using a Kim wipe. The cuvette was then subjected
to electric pulse (200 Ω, 2.5 kV, 25 µF) using Bio – Rad Gene Pulser Xcell. Immediately
1 ml LB medium was added to it, and the suspension was transferred to 15-ml Falcon 2059
polypropylene tube. The tube containing the pulsed cell suspension was transferred to
orbital shaker (Thermo scientific, Forma orbital shaker), and cells were subjected to
shaking at 225 rpm and 37
o
C for one hour. The cell suspension was then transferred to 1.5
ml micro-centrifuge tubes and centrifuged at 140000 rpm for 10 seconds (5417R,
Eppendorf). All but 100 µl of supernatant media was aspirated. The cells were resuspended
in 100 µl media and plated on LB-Agar-Kanamycin (50 µgm/l) plates and incubated
overnight at 37
o
C. 3 types of bacterial colonies are produced big, intermediate and small.
Small colonies are considered to be the positive colonies, and others are background. Small
colonies were picked up and cultured to purify positively cloned AD-PGE by following
protocol mentioned in section 3.2. Purified plasmids were verified for successful cloning
by PCR and gel electrophoresis using mGM-CSF forward and reverse primers and IRES
forward, and E1a reverse primers (Table 1). Protocol for the same is mentioned in section
28
3.1. Endo-free maxi-preparation was performed for positive clones using QIAprep endo
free maxi preparation kit (Qiagen) using manufacturer’s protocol.
Figure 8: Summary of method for generating AD-PGE from PGE
3.7 Generating recombinant oncolytic adenovirus expressing mGM-CSF (AD-
GMCSF) from AD-PGE:
AD-GMCSF was generated from AD-PGE using AdEasy protocol. AD-PGE was
digested by restriction enzyme PacI (NEB). To perform the enzyme mediated digestion 15
µgm AD-PGE plasmid was added in a 1.5 ml micro-centrifuge tube. 4 µl of enzyme, 5 µl
of 10X cutsmart (NEB) buffer and nuclease free water was added to make the volume up
to 50 µl. The digestion mix was incubated overnight in a 37
o
C water bath (Fischer
scientific, Isotemp 205). Linearized AD-PGE was purified using QIAquick PCR
29
purification kit (Qiagen) by following manufacturer’s protocol. Purified AD-PGE was
dissolved in nuclease free water. 10 µgm of linearized AD-PGE was transfected into 2-3
times passaged 70% confluent 293A (ATCC) cells in a 60 cm
2
culture dish. 293A cells
were passaged 16-24 hour prior to transfection. Culturing and passaging 293A cells was
carried out by following protocol mentioned in section 3.3. Transfection was carried out
by following protocol mentioned in section 3.4. Transfected 293A cells were incubated at
37
o
C and 5% CO2 in an incubator until they developed cytopathic effect (henceforth
referred to as CPE) which occurs 8-10 days after transfection. After 8-10 days, once almost
all the cells developed CPE but were not yet detached from the bottom of the dish, the cells
were re-suspended in culture medium by vigorous pipetting. The medium containing cells
was transferred to 15 ml centrifuge tube (BD Falcon). The cells were centrifuged at 1000
g for 5 min (Thermo Scientific Sorvall Stratos series) at 4
o
C. After pelleting the cells, all
but 5 ml of media was aspirated. Cells were re-suspended in 5 ml media. The cell
suspension was then subjected to 3 cycles of freeze and thaw, to lyse the cells and release
enclosed progeny virus into the media, by using dry ice-ethanol bath to freeze and 37
o
C
water bath (Fischer Scientific Isotemp 205) to thaw. After the 3
rd
cycle, thawed cell
suspension was centrifuged at 3000 g for 10 min at 4
o
C (Thermo Scientific Sorvall Stratos
series). After centrifugation supernatant virus containing media (approximately 5 ml) was
transferred to a clean and sterile 15 ml centrifuge tube in a biosafety cabinet. AD-GMCSF
virus containing supernatant was then added to a single 70% confluent 293A cell culture
in a 60 cm
2
cell culture dish. 293A cells were passaged 16-24 hours prior to addition of
virus containing media and were cultured by following protocol mentioned in section 3.3.
Transfected cell culture dish was incubated at 37
o
C and 5% CO2 in an incubator until they
30
developed CPE. CPE becomes evident after 3-4 days. After 3-4 days, once almost all the
cells developed CPE but were not yet detached from the bottom of the dish, cells were re-
suspended in media and were centrifuged, subjected to 3 freeze-thaw cycles, again
centrifuged to pellet lysed cell debris as mentioned previously in this section. As mentioned
previously, 5 ml supernatant containing virus was obtained. In the next step of viral
amplification, 5 ml supernatant was split into 2 halves, each half was used to transfect one
70% confluent 293A cell culture in a 60 cm
2
as mentioned previously. Hence, 2 halves
were used to transfect 2 60 cm
2
dishes of 293A cells. By following above mentioned
protocol, obtaining cells after CPE and freeze-thawing 3 times and centrifuging, 10 ml (5
ml from one dish) of AD-GMCSF containing medium is obtained for further amplification
of AD-GMCSF. For further amplification, 10 ml virus containing medium, obtained from
the previous step, was split into 2, and it was used to transfect 2 70% confluent 293A cell
culture in a 145 cm
2
dish. By following previously mentioned protocol, 10 ml virus
containing medium is obtained for large scale amplification. For large scale amplification,
10 ml virus containing medium, obtained from the previous step, was split into four equal
parts. Each part was used to transfect one 145 cm
2
culture dish containing 70% confluent
293A cell culture. After following above mentioned steps to release virus into the medium
from cells 20 ml (5 ml from each dish) AD-GMCSF containing medium was obtained. 20
ml virus containing medium was split into 4 and was used to generate more AD-GMCSF
by transfecting 4 145 cm
2
70% confluent 293A cell culture dishes. This cycle was repeated
10 times to obtain high titer virus. At the end of 10 cycles of transfecting 4 145 cm
2
cell
culture dishes and subsequently isolating virus from cells by freeze-thaw cycles, 20 ml of
medium containing high titer AD-GMCSF was obtained. This stock was used for further
31
experiments and virus purification. The stock can be stored in a -80
o
C freezer for upto 6
months by avoiding repeated thawing and freezing. Figure 9 summarizes the protocol for
generating recombinant AD-GMCSF.
Figure 9: Summary of protocol for generating AD-GMCSF from AD-PGE using AdEasy
protocol
3.8 Western blot Analysis to determine E1a expression:
Western blot analysis was performed to determine E1a expression by PGE and AD-
GMCSF virus. To determine E1a expression by PGE, 3 wells of 50% confluent 293T cells
32
(ATCC) were transfected with either of PGE, PCMV or PEI alone by using PEI as a
transfection vehicle, in a 6 well culture dish. To determine E1a expression by AD-GMCSF,
3 wells of 50% confluent B16 melanoma cells (ATCC) were transfected with either of 1ml
medium containing AD-GMCSF, AD-E1a (10
5
pfu) or empty medium, in a 6 well culture
dish. Cell culture and transfection were carried out by following protocol mentioned in
section 3.3 and 3.3 respectively. Transfected cells were incubated for 48 hours at 37
o
C and
5% CO2 in an incubator. After 48 hours, supernatant medium was removed by aspiration.
Adherent Cells were detached from the bottom of the well by adding ice cold PBS and
vigorous pipetting. The cell suspension was centrifuged at 500g for 5 min (Eppendorf,
5417R). Supernatant was aspirated, and the cells were suspended in 100 µl of cell lysis
buffer (RIPA, Millipore) containing protease and phosphatase inhibitor (Halt inhibitor,
Thermo Scientific). The cell suspension was incubated on ice for 10-15 minutes. After 10-
15 minutes of lysing, the cell suspension was centrifuged at 14000 rpm for 5 minutes
(Eppendorf, 5417R). Supernatant containing cellular proteins was collected into a different
1.5 ml tube. Concentration of proteins in the lysate was determined by using Bio-Rad
protein Dye assay (500-0006, Bio-Rad) by following manufacturer’s protocol. After
determining the concentration, cell lysate were boiled for 7-10 minutes after adding SDS
boiling buffer, reducing (gbiosciences), to make its 1X solution in cell lysate. Equal
amounts of boiled cell lysate from different samples were subjected to electrophoresis on
Nupage 4-12% bis-tris gel (Novex, Life technologies) and 1X solution of Nupage MES
SDS running buffer (life technologies) in distilled water at constant 120V. Nupage MES
protein standard (life technologies) was used as a ladder and marker. After electrophoresis
protein, samples were transferred to nitrocellulose membrane, using iblot electro-transfer
33
machine (Invitrogen) by following manufacturer’s protocol. The membrane containing
protein samples was blocked for 1 hour by rocking at room temperature by using Odyssey
blocking buffer. The membrane was then cut into 2 parts at 37 KDa band of the marker.
The membrane containing protein samples of molecular weight higher than 37 KDa was
stained overnight at 4
o
C by rocking by using rabbit anti-mouse actin antibody (4967S, Cell
signaling technologies). The part of the membrane containing protein samples of molecular
weight lower than 37 KDa was probed using mouse anti Ad5E1a (M58, sc-58658, Santa
Cruz Biotechnologies) antibody under the same conditions and procedure that was used for
actin staining. The antibodies were diluted by 1:500 dilution factor using TBST containing
10% v/v Odyssey blocking buffer. Stained membranes were washed 3 times by TBST
buffer under constant rocking. Each wash was conducted for 15 min under constant rocking
at room temperature. After washing, membranes were probed with secondary antibody anti
rabbit/anti mouse (Odyssey) (1:5000 dilution in TBST- 10% blocking buffer) at room
temperature for one hour by covering the container with aluminum foil. The bands on the
membrane were analyzed using Odyssey Licor scanner.
3.9 Intra-cellular cytokine staining to determine mGM-CSF expression:
mGM-CSF expression by PGE and AD-GMCSF was confirmed by intracellular
cytokine staining assay. For confirming expression by PGE, 293 T cells in 3 of 6 well
culture dish were transfected using either of PGE, PHE or PEI alone by following protocol
mentioned in section 3.4. For confirming expression by AD-GMCSF, B16 cells were
transfected with either of AD-GMCSF 500 µl, AD-GMCSF 1ml or AD-E1a (10
5
pfu). To
block cytokine release from the cells, Golgi Plug (BD bisosciences, 1:1000) was added in
the medium of transfected cells 4 hours prior to collecting the cells. After 4 hours, cells
34
were collected using PBS and stained with Fc block (ebiosciences, 1:100 dilution) in
staining buffer (0.5% v/v FBS in PBS). Cells were then permeabilized and washed using
BD cytofix/cytoperm & 1X BD perm wash (BD Biosciences). Cells were then stained with
fluorochrome PE conjugated anti-mouse GM-CSF (BD Biosciences, 1:100 dilution in 1X
BD Perm Wash). After staining the cells were washed using 1X BD Perm wash and fixed
using BD cytofix/cytoperm solution. Stained cells were analyzed using BD FACS Canto
II flow cytometer and results were analyzed using Flow Jo software version 10.0.8.
3.10 Construction and cloning pShuttle-CMV-E1a (PE1a):
As we could not generate high titer virus to conduct in vitro studies using a virus,
we decided to in vivo tumor growth studies by using plasmid transfected B16 cells. For the
same PE1a needed to be constructed as one of the control plasmid. E1a cDNA, obtained
by PCR from PHE, was digested and cloned by following the protocol mentioned in section
3.2. NotI (NEB) and EcoRV (NEB) restriction enzyme were used for digestion. After
cloning positive colonies were analyzed PCR to confirm successful cloning E1a into
pShuttle CMV, primers used for the same were A-91 and A-91 and A-95. One the
positively cloned plasmid was amplified and purified by maxi-preparation using QIAprep
endo free maxi-prep kit (Qiagen).
3.11 Transfection efficiency in B16 cells:
As we could not generate higher titer control and experimental virus AD-GMCSF,
AD-HE and AD-E1a in time to do in vivo tumor growth experiments, we decided to
perform in vivo tumor growth studies by injecting B16 melanoma cells transfected with
either of PGE, PHE, PE1a, PCMV and non-transfected cells. However, we needed to
35
determine how much amount of plasmid was needed to be transfected to achieve a higher
number of cells positive for transfected plasmid and also produce a tumor in mice. To
determine the same, 5 µgm and 10 µgm of plasmid pmax-GFP (Lonza) were transfected in
2 separate 50% confluent B16 melanoma cell culture dish of 60 cm
2
area by using protocol
mentioned in section 3.4. Transfected cells were analyzed for GFP expression 2 days post
transfection by using Zeiss Axio Observer A1 inverted microscope.
3.12 Tumor growth assay in mice by injecting plasmid transfected cells:
As we were not able to generate high titer virus in time to conduct in vivo studies
using virus, we decided to conduct tumor growth studies in mice by injecting plasmid
transfected cells in mice in order to study the effect of expression of 2 proteins, mGM-CSF
and HSP70, in tumor cells. 5 50% confluent B16 melanoma cell culture in a 60 cm
2
dish
were transfected with 10 µgm of plasmids PGE, PHE, PE1a, PCMV and empty medium,
one dish for each one of the plasmid. Transfection and cell culture protocols are mentioned
in section 3.3 and 3.4. After 48 hours, transfected cells were collected and 2 X 10
6
plasmid
transfected cells were injected subcutaneously into the right lateral flank of mice. 25 (N=
5/group) age matched, 6 to 7 weeks old female C57BL6 mice (Jackson Laboratory) were
injected with tumor cells using tuberculin 1 ml syringe and 28 gauge ½ inch attached
needle. 5 mice per group for each of the plasmid transfected cells. Mice were monitored
for tumor growth, and size was measured using digital vernier caliper, first at 7 days after
the injection and then twice a week till the end of the study. Vertical and horizontal
diameter of the tumor was measured using caliper and volume of the tumor was determined
by using following formula V = w
2
x l where V = volume (mm
3
), w = smaller of the 2
diameter and l = larger of the 2 diameter. Statistical comparison of tumor size of differently
36
transfected B16 cells amongst 5 groups was conducted by one way ANOVA test (Alpha =
0.05) using IBM SPSS version 19. All animal work was conduct as per the guidelines of
USC, Keck School of Medicine, department of animal resources and protocol was
approved by IACUC committee.
37
CHAPTER 4: RESULTS
4.1 Confirm Successful cloning of pShuttle-mGMCSF- IRES-E1a:
We have used the Ad-Easy protocol to construct oncolytic adenovirus expressing
mGM-CSF and E1a. To generate an oncolytic adenovirus expressing a protein, it requires
cloning the gene of interest for that particular protein into pShuttle CMV vector. Protein of
interest is expressed under the effect of CMV promoter. After conducting cloning of
mGM—CSF cDNA into pShuttle-IRES-E1a vector as mentioned in the methods section,
PCR was conducted to confirm successful cloning of cDNA into pShuttle-IRES-E1a. PCR
using primers for mGM-CSF cDNA and IRES-E1a on samples PGE and PHE confirmed
that mGM-CSF cDNA was successful cloned into PGE, as PGE sample showed
approximately 400 base pairs (bp) band on 1% agarose gel (Figure 10 a), when PCR was
conducted using mGM-CSF primers for PGE sample and subsequently separated on 1%
agarose gel using gel electrophoresis. PHE sample subjected to PCR using mGM-CSF
primers and subsequent separation on 1% agarose gel, did not show a 400 bp band (Figure
10 a). Both PGE and PHE showed approximately 3.2 kilo bases (kb) band on 1% agarose
gel (Figure 10 a), when separated by electrophoresis after conducting PCR using IRES
forward and E1a reverse primer. Hence, it concludes that mGM-CSF cDNA was
successfully cloned into pShuttle CMV containing IRES-E1a sequence.
The next step was to confirm expression of cloned cDNA for mGM-CSF along with the
expression of E1a protein. 293 T cells were split into 3 wells and each of the 3 well were
transfected separately with either one of PGE, PHE and transfection agent PEI. Each of the
38
3 samples were permeabilized and intra cellular cytokine staining was performed using PE
conjugated antibody against mouse GM-CSF and all the 3 samples were analyzed for PE
fluorescence using BD FACS Canto II flow-cytometer. The results were analyzed using
Flow Jo Software version 10.0.8. The results indicate that PGE transfected cells had a clear
GM-CSF positive population (5.97%), PHE (0.82%) and PEI (0.6%) transfected cells did
not have GM-CSF positive cells (Figure 10 b). This confirms that PGE led to successful
expression of mGM-CSF. Similarly 3 wells of 293T cells, each of the 3 wells, separately
transfected with either one of PGE, PCMV, and PEI, when analyzed for E1a expression by
western blot analysis, stained using anti Adenovirus 2/5 E1a antibody, out of the 3 only
PGE transfected cell lysate showed a band for E1a ( ~32-35 KDa.) (Figure 10 c). Hence,
figure 10 confirms that PGE was successfully cloned and upon transfection with PGE,
mGM-CSF and E1a were expressed in transfected cells. Figure 10 d is a schematic
representation of cloned PGE cloned to express mGM-CSF and E1a.
39
A
B
40
Figure 10: Construction and confirmation of cloning of PGE. A) PCR results of PGE
when amplified by mGM-CSF and IRES-E1a primers confirms successful cloning. B)
Intracellular cytokine expression assay confirms mGM-CSF expression by PGE when
transfected into 293 T cells. C) Western blot analysis confirms E1a expression by PGE
when transfected into 293T cells. D) Schematic representation of PGE
C
D
41
4.2 Confirm successful cloning of AD-GMCSF vector backbone AD-PGE:
To produce oncolytic adenovirus expressing mGM-CSF from PGE, PGE is cloned
into pAD-EASY 1 adenoviral backbone to form AD-PGE vector. After doing the same by
using the procedure as described in the methods section, PCR analysis was conducted to
confirm successful cloning of PGE into pAD-EASY 1. PCR analysis of AD-PGE using
mGM-CSF forward and reverse primers and IRES forward and E1a reverse primers and
subsequent separation by gel electrophoresis using 1% agarose gel confirms that PGE was
successfully cloned into pAD-EASY 1 (Figure 11). PCR analysis of AD-PGE resulted into
bands for both mGM-CSF (400 bp) and IRES-E1a (3.2 kb) (Figure 11). Also, AD-PGE,
when subjected to gel electrophoresis on 1% agarose gel, did not travel along the gel
because of its large size (~33 kb) (Figure 11). These results confirm successful cloning of
PGE into pAD-EASY 1 to form AD-PGE vector.
Figure 11: Confirmation of successful cloning of AD-PGE. PCR bands for mGM-CSF
and IRES-E1a confirms cloning
42
4.3 GM-CSF and E1a expression by oncolytic adenovirus AD-GM-CSF
Successfully cloned AD-PGE adenoviral vector was then used to produce oncolytic
adenovirus expressing mGM-CSF and E1a (AD-GMCSF) using 293A cells by AD-EASY
protocol as mentioned in the methods section. After the final round of amplification, AD-
GMCSF virus was released into 20ml media by subjecting infected cells to 3 cycles of
freeze-thaw in 2 0 ml media and subsequently pelleting cell debris by centrifuging. AD-
GMCSF containing media was then used to confirm mGM-CSF expression and E1a
expression by AD-GMCSF. To do the same, 3 wells of B16 melanoma cells were
transfected with AD-GMCSF 500 µl, AD-GMCSF 1 ml, AD-E1a 10
5
ifu (infection
forming units) respectively. These samples were used to detect mGM-CSF expression by
intracellular cytokine staining. Intracellular cytokine staining of cells transfected wither
with AD-GMCSF or AD-E1a revealed that B16 melanoma cells transfected with AD-
GMCSF 1ml (47.7%) or AD-GMCSF 500 µl (26.7%) had a clear mGM-CSF expressing
population of B16 cells, whereas AD-E1a (1.23%) transfected cells did not have mGM-
CSF expressing population of B16 cells (Figure 12 a and b). In another experiment 3 wells
of B16 cells transfected with AD-GMCSF 1 ml, AD-E1a 10
5
pfu (plaque forming units)
and non-transfected cells were used for western blot analysis to determine E1a expression.
AD-GMCSF & AD-E1a transfected cell lysate were positive for E1a band (~32-35 KDa),
and non-transfected cells did not show band for E1a (Figure 12 c). Hence, figure 12
confirms that oncolytic adenovirus AD-GMCSF successfully expresses mGM-CSF as well
as E1a in B16 melanoma cells
43
A
B
44
Figure 12: Confirm successful generation of AD-GMCSF from AD-PGE. A) Half offset
overlaid histogram of intracellular cytokine stained B16 cells stained either with AD-
GMCSF or AD_E1a or unstained confirms mGM-CSF expression by AD-GMCSF. B)
Same intracellular cytokine staining results with histograms separated. C) Western blot
analysis for E1a confirms expression by AD-GMCSF.
4.4 Transfection efficiency in B16 melanoma cells
As we were not able to generate high titer experimental as well as control virus to carry out
in vivo experiment using viruses, we decided to conduct in vivo tumor growth studies using
B16 melanoma cells transfected with plasmid expressing mGM-CSF or HSP70, as a
preliminary experiment to determine the effect of 2 proteins, when overexpressed, in tumor
microenvironment. However, to conduct the same experiment, it was necessary first to
determine transfection efficiency of PEI in B16 cells and subsequently determining the
number of cells needed to produce a tumor in mice by subcutaneous injection. It is evident
from Figure 13 that 10 µgm plasmid transfection using PEI leads to transfection of up to
C
45
40-50% B16 cells, which is desirable. Transfection with 5µgm plasmid and PEI resulted
up to 20-30% cells being positively transfected (Figure 13). Moreover, in a separate
experiment number of transfected cells needed to produce a tumor in mice was determined
by injecting 10 µgm PCMV transfected cells in mice sub-cutaneously. The tumor was
produced by injecting a minimum of 2 x 10
6
transfected B16 melanoma cells (data not
shown). Moreover transfection efficiency with higher amount of plasmid, that is 20 µgm,
did increase the transfection efficiency, however, since it also involved increasing the
amount of PEI needed to carry out transfection, cell viability decreased and subsequently
the capacity to produce a tumor by transfected cells in mice was also lost (data not shown).
Hence, transfecting with 10 µgm plasmids using PEI was concluded to be ideal for in vivo
experiment.
46
Figure 13: Transfection efficiency by GFP expression confirms 10 µgm Plasmid
transfection efficiently transfects about 40-50% cells.
4.5 Effect of GM-CSF expression and HSP70 expression on in vivo tumor growth in
mice:
As determined previously, 10 µgm plasmid (either of PGE, PHE, PE1a, PCMV in
each group) transfected cells and non-transfected cells (B16 group) were injected
subcutaneously in right lateral flank of 6-7 weeks old age matched female C57Bl/6 mice
(n = 5/ group). As shown in Figure 14, tumor growth of B16 cells transfected with plasmid
expressing mGM-CSF (PGE) and plasmid expressing HSP70 (PHE) was significantly
slower and impaired (P < 0.005) compared to the growth kinetics of B16 cells transfected
with PE1a, PCM or non-transfected cells. However, the tumor growth was not completely
B16 Cells Non Transfected
B16 cells + 5
µgm pmax GFP
B16 cells + 10
µgm pmax GFP
47
inhibited which explains the need for oncolysis by an oncolytic virus. Tumors in groups
with cells transfected using PE1a, PCMV, or non-transfected (B16 group) crossed the
maximum acceptable limit of 1.5 cm
3
(as per USC DAR guidelines) by 17
th
day post tumor
injection and all the mice in these groups died by 20
th
day. However, tumors in PGE and
PHE transfected groups did not cross maximum acceptable limit until the 20
th
day, and
they did not die until euthanized. Moreover, on 15
th
day post tumor injection, tumors from
2 mice in each group were collected and analyzed using flow cytometer and appropriate
antibodies to determine presence of CD8 T cells, CD4 T cells, dendritic cells, macrophages
and myeloid suppressor cells at tumor site. However, no difference was seen in the number
of above mentioned immune cells, at the tumor site, between the 5 groups (data not shown).
This could be because of a technical error and hence, it will be confirmed by performing
H&E staining and immune-staining of tumor tissue sections.
48
Figure 14: Mean tumor volume measurement for determining tumor growth of PGE,
PHE, PE1a, PCMV transfected and non-transfected tumor cells in mice. 2 million cells of
each plasmid transfected B16 cells were injected into the right lateral flank of mice (n=
5/group). Tumor growth was monitored with a digital caliper. PGE and PHE transfected
tumors grew significantly slowly (* = P <0.005). Data is represented as Mean ± Standard
Error.
*
*
49
CHAPTER 5: DISCUSSION
The goal of this project was to develop an oncolytic adenovirus expressing GMCSF
and compare its anti-tumor efficiency and its efficiency in stimulating an anti-tumor
immune response with anti-tumor and anti-tumor immune response generated by AD-HE
treatment. After thoroughly following Ad-Easy protocol AD-GMCSF, oncolytic
adenovirus expressing GM-CSF was generated.
Transfection of AD-GMCSF in B16 melanoma cells resulted in GM-CSF and E1a
expression, which confirms that AD-GMCSF duly transfects B16 cells and expresses the
gene of interest. However, high titer experimental virus and control virus could not be
generated. Hence, we conducted in vivo experiments by injecting tumor cells transfected
with plasmids expressing the gene of interest. By monitoring tumor growth of transfected
cells in mice, it was observed that GM-CSF and HSP70 plasmid transfected B16 cells grew
significantly slowly, and their growth was more inhibited compared to cells transfected
with plasmids expressing E1a or plasmid pShuttle CMV or non-transfected cells. Flow
cytometry to determine immune cell population in tumors was also conducted. However,
no difference was seen. This could be because of a technical error, and H&E staining or
immune-staining of tumor sections will be conducted in future to determine the difference.
However, to prove the hypothesis high titer virus needs to be generated and in vitro and in
vivo experiments are needed to be performed in future.
50
CHAPTER 6: FUTURE DIRECTIONS
The hypothesis behind this project is that HSP70, which can carry antigen to
antigen presenting cells and which can also act as an adjuvant in giving stimulatory signals
to the antigen presenting cells, can generate more potent immune response against cancer,
when expressed in tumor cells by means of an oncolytic adenovirus, compared to GM-
CSF, which can only recruit antigen presenting cells at tumor site and give them maturation
signals. However, to prove this hypothesis high titer viruses needs to be generated and in-
vitro and in vivo tumor killing assays needs to be performed. We also plan to perform
experiments such as ELISpot, ELISA, T cell cytotoxicity assay to compare the efficiency
of AD-HE and AD-GMCSF in generating an anti-tumor immune response.
In future, after comparing the anti-tumor efficiency of AD-HE and AD-GMCSF we
plan to evaluate the efficacy of AD-HE vaccine in combination with recently approved
blockbuster PD-1/PD-L1 checkpoint therapy inhibitors. Recently, oncolytic viruses
expressing GM-CSF are being planned to be introduced in a clinical trial with PD-1/PD-
L1 checkpoint therapy inhibitors. Figure 15 describes our hypothesis in using AD-HE in
combination with PD-1/PD-L1 checkpoint therapy.
51
Figure 15: Summary of Hypothesis of using AD-HE in combination with anti PD-1/PD-
L1 checkpoint therapy
52
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Abstract (if available)
Abstract
Cancers have been a leading cause of mortality amongst humans for several years. Even persistent innovations in the field of medicine, around the globe, have not yet been able to overcome consistently high mortality rate due to cancers. One reason attributed to being a major hurdle in treating cancers is difficulty in developing tumor specific and selective therapy. Also, severe side effects have made several therapies undesirable. Recent advances made in the field of gene therapy are still difficult to be translated to clinic, again because of inability to achieve safe and tumor specific delivery. Oncolytic viruses have been known since 19th century. Moreover, recent advances in genetic engineering have made it possible to engineer an otherwise pathogenic virus to selectively replicate inside and kill cancer cells. Such a therapy can also help to overcome undesirable side effects associated with traditional therapy. Advances in gene therapy and molecular engineering has made it possible to develop viruses which can not only selectively kill cancer cells, but also express gene of interest inside cancer cells. The goal of this project was to compare efficacy of oncolytic adenovirus expressing HSP70 and another similar virus expressing GM-CSF in inducing anti-tumor and anti-tumor immune response. By western blot analysis and intracellular cytokine staining it was confirmed that AD-GMCF was successfully generated for this purpose. In in vivo tumor growth studies, tumor cells transfected with plasmid expressing GM-CSF and HSP70 grew significantly slowly in mice and their growth was more inhibited in mice compared to the control groups. However, high titer virus needs to be generated in order to compare the efficacy of both viruses in generating anti-tumor and anti-tumor immune response.
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Asset Metadata
Creator
Ghelani, Aazam P.
(author)
Core Title
Oncolytic adenovirus based vaccine in cancer immunotherapy
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Publication Date
09/18/2015
Defense Date
08/28/2015
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
cancer vaccine,GMCSF,HSP70,OAI-PMH Harvest,oncolytic adenovirus,tumor immunotherapy,tumor selective
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application/pdf
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Language
English
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Electronically uploaded by the author
(provenance)
Advisor
Huang, Xue Fen (
committee chair
), Chen, Si Yi (
committee member
), Ouellette, Andre (
committee member
)
Creator Email
aazamghelani@gmail.com,aazampag@usc.edu
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https://doi.org/10.25549/usctheses-c40-185154
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185154
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Ghelani, Aazam P.
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(contributing entity),
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Tags
cancer vaccine
GMCSF
HSP70
oncolytic adenovirus
tumor immunotherapy
tumor selective