1. 1
INTRODUCTION
Previous researches in finding effective treatment options of cancer (malignant
tumors) show that some cancers are resistant to known traditional therapies such
as the use of non-replicating Adenoviruses. Thus, patients do not benefit from such
therapies.
Virotherapy entails the use of Oncolytic virus that are either replicative or non-
replicating, to infect cells and terminate target tumor cells. Interesting as this
concept is, it was not efficient. Since then, several modifications have been done to
the “Lead” Adenovirus; Ad 5 by serotype chimerism, arming the virus with
Granulocyte- macrophage colony stimulating factor, GMCSF; anchoring a Ad5/3
chimera with monoclonal antibodies and further combining the chimera with a
tumor specific promoter (E2F). A Quadruple modification can be done to the Ad5/3
Chimera by a 24-base pair deletion in the E1gene and furthering arming it with
GMCSF and a tissue specific promoter.
The latest breakthrough with regards with modifying Adenoviruses is the production
of CGTG- 102 (Ad5/3-Δ24-GMCSF) by ATAP (Advanced Tumor Therapy Program),
(Hemminki et al., 2014). This was taken further by synergistic approaches; this
entails combining chemotherapy drugs such as: Low dose cyclophosphamide or Low
dose pulse temozolomide with CTGT-102. Also modified Adenoviruses can be used
alongside radiotherapy to treat malignant tumors. This “cocktails” have been used
to treat a good number of patients in clinical trials.

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CELL DIVISION
The origin of cancer can be traced to a malfunction in the cell cycle. Thus to get a
full insight of how cancers are formed, an understanding of the cell division is a
good start.
 G0 Phase: Non-dividing stage, cells can remain there forever
 G1 Phase: Growth and development of the cell
 S phase: Chromosomes (DNA) are doubled; proteins and other essential
components needed by the daughter cells are produced.
 G2 Phase: Prepares the Cells for division. At the end of Interphase the cells
are seen in a jellylike form.
 The M Phase also known as the Mitotic Phase, it includes prophase,
metaphase, anaphase and telophase.
 PROPHASE: The chromosomes become less condensed, chromosomes appear
thread like and each chromosome is seen to consist of two sister chromatids
joined at the center, centromeres are formed at opposite ends of the cell.
G2
M
G1 G0
S

3. 3
The end of prophase is marked by the disintegration of the nuclear
membrane
 METAPHASE: Spindle fibers form from the centromeres and are attached to
each sister chromatids and the chromatids align at the equator of the cell
(Mid-point of the cell.
 ANAPHASE: Spindle fibers starts pulling each sister chromatids towards the
ends of the poles; Late anaphase is marked by the arrival of the sister
chromatids at the poles.
 TELOPHASE: The chromatids start condensing again into the jelly form,
nuclear membrane forms around each daughter cell. This is followed by
cytokinesis. Telophase ends with the formation of Plasma membrane around
each daughter cell (Devlin et al., 2010).
HOW ARE CANCERS FORMED?
Cellular homeostasis is defined as a state in which cell division and cell death are in
balance i.e. the number of cells produced and the number of cell death in a given
period of time roughly the same. Cancers are formed when the normal cellular
homeostasis is not obeyed.
Cancer can be defined as an uncontrolled, abnormally fast division of cells leading
to the production of abnormal cells (Devlin et al., 2010).
p53: THE GUARDIAN OF THE GENOME
P53 is a transcription factor that acts in stopping cell cycle when a cellular DNA
damage has been detected. Thus, giving the cells time to repair its DNA if it

4. 4
eventually it does not its’ DNA, p53 causes the death of the cell containing
damaged DNA (Apoptosis) and this is supposed to control cancer growth.
Biochemical Basis
Kinases perform a role in monitoring the cell especially the DNA; thus if there is a
DNA damage (due to factors like smoking, diet etc.), kinases are activated and this
activated kinases further activate other kinases or phosphorylate p53 directly
(activation of p53). Either way, an increased concentration of phosphorylated
(active) p53 causes more p21 to be produced (because p53 causes more
transcription of the gene that code for p21, waf 1or cif 1); p21 is a gene product of
waf 1. p21 is a cyclin dependent kinase inhibitor, cdI. Thus, p21 inhibits the G1 and
the G1/S checkpoint. It does this by the activity of G1 and G1/S cdks, these cdks
phosphorylate Rb protein (Retinoblastoma sensitivity protein), when these cdks are
inhibited Rb remains unphosphorylated and retain bound E2F (Transcription factor).
Thus damaged cells having defective DNA are prevented from entering the S phase
(this gives the cell time to repair its DNA if it fails to repair itself, the cells
undergoes apoptosis.
In 50% of cancer cases, p53 is usually mutated or lost. In the other 50%, the p53
protein is defective and the cancer cells contain damaged, unstable chromatin.
STEPS INVOLVED IN CELLULAR REGULATION
 DNA damage triggered by smoking or diet and other environmental factors
 Monitoring kinases are activated
 Activated kinases activate p53

5. 5
 p53 (Transcription factor) promotes transcription of waf 1 gene to produce
p21 protein.
 p21 inhibits G1 and G1/S cdks
 Inhibition of G1/S cdks prevents phosphorylation of Rb protein and
unphosphorylated Rb protein retains bound E2F.
 The detainment of bound E2F prevents damaged DNA cells from entering S
phase
 If p53 is defective, defective cells are allowed to enter S phase and cell
division proceeds and the cellular homeostasis is not accomplished and more
and more defective cells proliferate to give more defective cells (cancerous
cells).
 Hence, p53 is the guardian of the genome
PROPERTIES OF CANCER CELLS
General properties of cancer cells include: Resistance to Apoptosis, Metastasis,
Angiogenesis, and Immortality.
Metastasis can be defined as the ability of a cancer cell to dissociate from the
tumor cells, pass through the basement membrane, through the surrounding
extracellular matrix into a capillary then into the blood and travels to a target site
or tissue. On getting to the target tissue, it extravasate out of the blood vessel and
develops into a secondary tumor. This property of malignant cancer cells is what
actually causes the death of cancer patients.

6. 6
Cancer cells can metastasize because they have lasminin receptors on their plasma
membrane. The lasminin receptors are able to bind lasminin proteins on the
basement membrane (basal lamina).
The lasminin protein becomes activated and it causes the secretion of enzymatic
proteases into the extracellular matrix. Holes are created on the surface of the
basal lamina due to the protease activity, e.g. UPA or Type IV procollagenase; the
tumor cells then pass through the holes and into the extracellular matrix. Cellular
Motility (Metastasis) is possible because the tumor cells can undergo biochemical
changes in their cytoskeleton and membranes.
The reason why cancer cells possess Cellular Immortality is because they
possess telomerase activity. Telomerase is an enzyme that is able to cause
telomerase extension. Telomeres are the terminal ends of a chromatid. Usually,
during cell division, nucleotide fragment are lost, telomerase is able to cause
extension of the chromatid end but with increasing age of the organism, telomerase
activity of cells is usually lost, this makes normal cells go into Senescence and
eventually necrosis due to DNA damage. As the age of an organism or the number
of cell division generations increase, telomerase activity is lost but in 90% of cancer
cases telomerase activity is retained; invariably they continue to proliferate and
divide forever or at least until when steps are taken to kill the cells (Devlin et al.,
2010).
As stated earlier, the cancer cells are able to resist Apoptosis because of the
defects or damage to p53 protein.

7. 7
The mechanism of induction of Angiogenesis by cancer cells is similar to that of
metastasis. Tumor cells secret chemo attractants such as VEGF (Vascular
Endothelial Cell Growth Factors) and FGF (Fibroblast Growth Factors). These
chemicals are able secrete plasminogen activators and MMPs (Matrix
Metalloprotein) activators. Plasminogen activator is able to break plasminogen
molecule into two equal halves. The C-terminal contains the plasmin protease
activity. The N-terminal contain anti-angiogenic properties, it contains proteins such
as angiostatin and endostatin. The plasmin protease activity in the C-terminal can
promote the proliferation of new blood vessels in secondary metastatic tumor sites.
But due to the presence of angiostatin, angiogenesis might not be possible at
primary tumor sites.
NOTE: A single mutation in a cell cannot cause cancer development.
TREATMENT OPTIONS
Malignant Cancer so far, has been implicated as the second most deadly disease in
the whole world. Thus, several treatment options for combating these diseases has
been in use each with its own advantage and disadvantage. These options include:
 Chemotherapy
 Radiotherapy
 Virotherapy
 Synergistic/ Cocktail Approaches

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The topic of discourse in this dissertation is the current developments in
adenovirus-based gene cancer therapy. This means the recent developments or
research breakthroughs in the use of adenovirus to infect or transduce a genome
into tumor cells to kill or terminate such cells.
CHARACTRISTICS OF ADENOVIRUSES
Out of the 100 known serotypes of Adenoviruses known in existence, only 57 have
been identified in humans (Martin et al., 2007). Of this 57, the most frequently
used and well investigated Serotype is the Ad 5 (Tuuli et al., 2010). Adenoviruses
are the largest of the non-enveloped viruses, as a non-enveloped Icosahedral
Capsid. It is about 90-100nm in size (medium sized). An adenovirus is a single
double stranded DNA genome containing 20 to 22 genes of about 36-38kbps
(Sandra et al., 2012). Although it is known that about 10% of upper respiratory
infection in children is caused by Adenoviruses. Adenovirus was first isolated in
Adenoid tissues, nasopharyngeal tissues (Sandra at al., 2013). Despite the
characteristic of causing respiratory tract infections, it is now being adopted in
treating advanced tumors.
STRUCTURE OF AN ADENOVIRUS

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www.microbewiki.kenyon.edu
The viral capsid consists of 252 proteins, there are three different types of proteins:
Penton (Yellow bead), 12; Hexon (Blue bead), 240; Fiber. Both fiber and penton
base proteins are key to receptor binding and internalization (Martin et al., 2007)
MECHANISM OF ACTION OF AN ADENOVIRUS

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www.microbewiki.kenyon.edu
 Entry and Internalization of the virus cell into the host cells, the
internalization occurs by the interaction of pentose base Arg- Gly- Asp (RGD)
with integrin αv, this leads to its entry into the cell through Clathrin coated
pits (Endocytosis)
 Further entry of the virus into the endosome occurs by the αv integrin
stimulating actin polymerization
 Acidification of the Endosomes causes capsid proteins to dissociate, the virus
is disassembled and its coded information is expressed. The viral genome is
not incorporated into the host genome, thus the risk of mutation is extremely
low. The viral genome remains in the episomal state (Gao et al., 2001)

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www.microbewiki.kenyon.edu
At the onset of gene therapy, non-replicating Adenoviruses were used. It was
observed that the virus could not penetrate solid tumors; thus they were
abandoned for their replicative counterparts. Conditionally replicating Adenoviruses
(CRAds) are now being used they are able to spread beyond the need point i.e. the
point of administration and thus able to target solid tumor cells. Another advantage
of conditionally replicative Adenoviruses is their ability to reach secondary tumor
cells i.e. malignant tumors that have metastasized (Westphal et al., 2013; Pan et
al., 2009).

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www.medscape.org
ENSURING THE SAFETY OF ADENOVIRUSES
Having certified the possibility of using Adenovirus to treat cancer, there arose a
question of ensuring the safety of patients that the virus was administered to.
These viruses have to be administered in fairly large doses; therefore, it is possible
that the virus can cause liver toxicity. In fact, there have been reported cases of
liver toxicity in clinical trials (Raper et al., 2010). The problem of liver toxicity was
circumvented by a procedure known as Transductional Targeting. This involves
targeting the tumor tissue cells while detargeting the liver; thus the chance of the
Adenovirus causing Necrosis of Hepatic cells is reduced to the minimum (Tuuli et
al., 2010).
SEROTYPE CHIMERISM
With the question of safety answered, the next line of research was improving on
the efficacy of the Adenoviruses in tumor cell infectivity. One of the drawbacks with
the use of Adenoviruses was that CAR (Coxsackie Adenovirus Receptor) is
down-regulated in advanced tumors. During the mechanism of action/ infection of

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tumor cells, the Adenovirus has to attach its fiber knob to the Adhesion molecule
(CAR) on the target cells; without this initial step, infection is impossible. Thus, the
down-regulation of CAR on cancer cells was a major challenge.
This challenge was overcomed by a technique called Serotype Chimerism. It
involves the replacement of Fiber Knob 5 with Fiber Knob 3; Ad 3 does not depend
or make use of CAR, instead it uses another Adhesion molecule (Receptor) such as:
Desmoglein-2, as a means of initial infection. Fortunately, Desmoglein-2 is not
down-regulated in advanced tumors. This Serotype chimerism was able to avert the
problem of down-regulation of CAR on target cells. Ad 5/3 Chimeras were
produced.
www.microbewiki.kenyon.org
NOTE: Refer to table 1.1 in the appendix for a list of Ad 5/3 Chimera CAR.

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NATURAL ANTIBODIES (NAbs) AND IMMUNE RESPONSES
Another challenge that arose during the cause of employing Oncolytic
immunotherapy was the problem of neutralizing antibodies (NAbs). Certain titer of
NAbs is usually isolated from children, neonates and even adults. Prior exposure to
this Adenovirus caused an immune response to the virus (Ad 5) which is the natural
antibodies observed. NAbs binds to the capsid proteins thus blocking Ad
internalization into target tumor cells, NAbs further induces Kupffer cells and
dendritic cells (Immune-stimulatory) thus causing natural immunological responses.
NAbs and natural immune responses are the prominent factors that determines the
efficacy or efficiency of Adeno- Virotherapy (Dhar et al., 2009). In addition, NAbs
develop against capsid proteins within weeks after the first administration thus
making Re-administration difficult. It was further discovered that the major fraction
contributing to the total NAbs titer was not due to the fiber capsid protein (Vogels
et al., 2003) but rather the hexons (Sumida et al., 2005). Since the fiber protein
was the most abundant capsid protein, the use of anti-fiber antibodies (modifying
the Ad 5 fiber) showed an RGD modification in the HI- loop of the Ad5 fiber.
Hemminki reported that it would be possible for the Ad 5 fiber to partially escape
the NAbs in the ascites fluid (Hemminki et al., 2001). This increased the efficacy of
the use of Adenoviruses as vesicles in gene therapy (as a cancer treatment option).

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CAPSID MODIFICATIONS
With the safety of administering Adenoviruses to cancer patients certified, the next
point of discourse or attention was focused on increasing the efficacy or infectivity
of these Adenoviruses, this was done by modification of their capsid. Amongst these
modifications there is:
“PROMOTER BASHING” AND DELETION MUTANTS
Tumor specific promoters (TSP) were used in conjunction with Deletion mutants of
Adenoviruses having a 24-base pair deletion in its genome. (E1 is the first gene to
be activated during Adenovirus replication). Tumor specific promoters used are
such that they had very little or no expression in normal cells but very high
expression in the tumor cell. A good example of such a TSP is Secretory
Leukoprotease Inhibitor (SLPI). SLPI, a 11.7 KD serine protease inhibitor that is
upregulated up to 60- fold in ovarian, breast and lung carcinomas.
Thus, the chances of replication of SLPI armed adenoviruses replicating in normal
cells was reduced to a minimum (Hough et al., 2001), SLPI was observed to be
expressed minimally in normal liver (Abe et al., 1997). Ad5/3-D24-SLPI CRAds
showed a high efficacy in lysing tumor cells compared with ordinary unarmed
Adenovirus. MSLN (Mesothelin) is expressed in ovarian cancer cells but not found in
normal cells except mesothelial cells; even when they are shed into the blood
stream, it is usually in very small safe amounts (Chang et al., 1996).
Another promoter that is being used is the Cyclooxygenase-2 (COX-2) in infecting
ovarian cancer cells. Ad5/3-RGD-4C-COX-2 CRAds with a RGD-4C motif in its’ Ad5

16. 16
fiber knob region showed increased infectivity and safety of hepatocytes; thus,
ensuring transduction targeting. This process of promoter bashing and deletion
mutants when combined with Ad5/3 chimeras yields a triple modified Adenovirus
(Pesonen et al., 2010) which is not only safe i.e. reduced risk of liver toxicity but is
also effective in infecting malignant tumors.
Further modification was done to the triple modified Adenovirus to produce a better
infecting vehicle by arming Ad5/3 chimera with a tissue promoter E2F and the 24-
base pair deletion with GMCSF (Granulocyte Macrophage Colony Stimulating
Factor); thus, producing a quadruple modified Adenovirus, Ad5/3- E2F1- Delta 24-
GMCSF (CTGT-602) (Ranki et al., 2012). CTGT-602 was very safe and had a high
infectivity. Since then several patients have been treated. GMCSF had one
drawback which was its ability to suppress myeloid derived suppressor cells. Hence,
CD 40 Ligand (CD40L) is another arming “equipment” coming in handy which can
cause cell death (necrosis) of tumor cells (Koski et al., 2012).
Also, Anti CTLA4 antibodies are also used to arm the Ad 5/3 serotype chimera and
it serves two functions of releasing the break on immunosuppressive as well as
enhancing antitumor efficacy while reducing any possible toxic effect on other
systems in the body (Dias et al., 2012).
A research carried out in 2012 hypothesized the probability of using sodium iodide
symporter hNIS to “load” tumor cells with radio iodide. This idea was copied from
the use of radio iodide to cure (when loaded into) thyroid cancers. The use of hNIS
was used practically but still a low concentration of radio iodide was found to

17. 17
accumulate in the tumor cells. The reason for this small concentration was the
small window period between transgene expression and tumor cell lysis; thus, radio
iodide loading was ruled out (Rajecki et al., 2012).
According to Liikanen in 2013, ATAP incorporated low-dose Cyclophosphamide and
low-dose pulse Temozolomide (Chemotherapeutic agents) with the use on
Adenoviruses bringing about a synergistic effect in the treatment of malignant
tumors. Low-dose Cyclophosphamide is a known inducer of autophagy (Cerullo et
al., 2011) and via some mechanism which is not yet completely understood; they
help to enhance cell death (oncolysis), thus reducing the tumor cell mass (Liikanen
et al., 2013).
FUTURE PERSPECTIVES AND RESISTANCE TO ONCOLYTIC
IMMUNOTHERAPY
Researches carried out in 2013 revealed that there was a resistance to Adeno -
therapy (study done in murine model); Mouse model showed resistance to
Adenovirus. It was further discovered that the resistance was due to interferon
response to tumor stroma and not from the tumor cells. This response made
tumors become refractory (Moerdyk-Schauwecker et al., 2013; Liikanen et al.,
2011), anti- interferon armed Oncolytic viruses should be a good future
perspective. Another treatment option for refractory cancers is to treat the cancer
in its early stage by combining Oncolytic Virotherapy with other standard therapies
such as chemotherapies and radiotherapy rather than the customary Phase 1
advanced disease population (Nokisalmi et al., 2010; Rajecki et al., 2009)

18. 18
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22. 22
APPENDIX
TABLE 1.1: TABLE SHOWING THE VARIOUS ALTERNATIVE CAR FOR Ad5/3
CHIMERA
Viruses Subgrou
p
Capsid
Modificatio
n
Receptor/
Specific homing
Results Ref.
Ad5/7 C/B1 Ad7 fiber CD 46 Infection of CAR deficient DC Gall et al.,
1996.
Ad5/35 C/B1 Ad35 knob CD 46 Infection of CAR-deficient
CD34+ hematopoietic stem cells
Shayakhmeto
v
et al., 2000
Ad5/35S C/B1 Ad35 fiber CD 46 Hepatocyte transduction was
independent of the interaction
with CAR and reduced 10 fold
with short shaft.
Shayakhmeto
v
et al., 2004.
Ad5/35L C/B1 Ad35 knob CD 46 Increased transduction of
patient-derived glioma cells with
serotype 35,16, 50 and 11
chimeras.
Brouwer et
al., 2007.
Ad5/9S C/C Ad9 fiber CAR
Ad5/9L C/C Ad9 fiber CAR
Ad5.Fib12 C/A Ad12 fiber CD 46

23. 23
Ad5.Fib16 C/B1(I) Ad16 fiber CD 46
Ad5.Fib35 C/B1(I) Ad35 fiber CD 46
Ad5.Fib50 C/B1(I) Ad50 fiber CD 46
Ad5.Fib7 C/B1(II) Ad7 fiber Receptor x
Ad5.Fib11 C/B1(III
)
Ad11p fiber Receptor x and
CD46
Ad5.Fib10 C/D Ad10 fiber CD46
Ad5.Fib17 C/D Ad17 fiber Sialic acid
Ad5.Fib24 C/D Ad24 fiber Sialic acid
Ad5.Fib30 C/D Ad30 fiber Sialic acid
Ad5.Fib33 C/D Ad33 fiber Sialic acid
Ad5.Fib37 C/D Ad37 fiber Sialic acid
Ad5.Fib38 C/D Ad38 fiber Sialic acid
Ad5.Fib47 C/D Ad47 fiber Sialic acid

24. 24
Ad5.Fib40
S
C/F Ad40 short
Fiber
Sialic acid
Unknown
ColoAd1
(Oncolytic)
B1/B2
(II)/(III
)
Major
capsid
Proteins
from
Ad11p
Receptor x and
CD46
Directed evolution resulted in an
Ad11p virus with a nearly
complete E3 region deletion,
smaller deletion in E4 and a
chimeric Ad3/Ad11p E2B region.
Over 2 log increase in potency
and selectivity when compared
to ONYX-015 on colon cancer
cell lines and in vivo.
Kuhn et al.,
2008.
Ad5/3luc1 C/B1 Ad5 fiber/
Ad3 knob
Receptor x Enhanced gene transfer to
various cancer cell lines and
primary tumor tissues.
Guse et al.,
2008
Ad 5/3-
Δ24
(Oncolytic)
C/B1 Ad5 fiber/
Ad3 knob
Receptor x Enhanced cell killing of cancer
cell lines and Xenograft tumors.
Ranki et al.,
2007
Ad5/19p-
HIT
C/D Ad19p
fiber,
HIT peptide
Sialic acid,
Phage
displayed-
Selected for
Enhanced gene transfer to renal
cancer cell lines and xenograft
renal cancer tumors in vivo,
decreased gene transfer to liver
Diaconu et
al., 2009.

25. 25
hom-ing to
Kidneys.
Ad5/3-
Δ24-
GMCSF
C/B1 Ad5 fiber/
Ad3 knob
Receptor x Enhanced cell killing of cancer
cell lines and syngeneic hamster
tumors. Objective clinical benefit
in 8/12 patients with
progressing chemotherapy
refractory solid tumors as
evaluated by radiology with
RECIST criteria
Koski et al.,
2010.
(Gotten from Hemminki et al., 2010).