Title: Use of oncolytic viruses as therapeutic strategy for cancer

A dissertation presented by:

Name: Farhath Jabien

Student number: S8706517J

UOB: 09034657

Batch code: BBSD1 1012A

For the BSc (Hons) in Biomedical Sciencesin the

University of BradfordDivision of Biomedical Sciences

School of Life SciencesUniversity of Bradford

Bradford

DATE of SUBMISSION: 28th February 2013

WORD COUNT: 3350

Project Supervisor: Dr.J.Boyne

Abstract:

Cancer is one of the deadly diseases in the world, claiming millions of lives. There

are many reasons behind the complications which cancer putforth to the medical

field.With improvement of knowledge of oncology and biotechnology, researchers

have begun working on creating a novel therapeutic strategy, with minimal side

effects. One of which is the usage of oncolytic viruses against cancer cells. The

oncolytic viruses are able to take advantage of cancer cells strengths and break

through several obstacles to infect and lyse the tumour. Moreover, with own

intelligence and capabilities or with man-made modifications, the ‘foe’ which causes

many deadly diseases or even cancer, can now be trusted as ‘friend’. The once

forsaken research in 1950s, has now steadily gained confidence and funding to carry

out more experiments and even clinical trials. This article aims to introduce the

‘rescuers’ of cancer and what are the mechanisms by which they operate, inclusive

of clinical trials.

Introduction

Cancer is one of the major causes of death worldwide, accounting for about 13% of

all deaths worldwide. (WHO, 2013) The causes of cancer could physical, chemical

or biological carcinogens. Examples of physical carcinogens are excessive, harmful

ultra-violet or ionising radiation. Examples of chemical carcinogens are tobaccoor

coal smoke, alcohols, aflatoxins and asbestos. Examples of biological are parasites,

bacteria and viruses. Chronic illness and genetic inheritance also contribute to tumor

formation.

Amongst various reasons, pathogenic, cancer-causing viruses alone give rise to

estimated 11% of total cases of death due to cancer. (Parkin,D.M.,2002) Some

examples of such viruses are, Epstein Barr virus causes Burkitts Lymphoma, Human

Herpes virus-8 causes Kaposi Sarcoma and cervical cancer is caused by Human

Papilloma virus.

Over the years, the medical field has fought hard against time and complexity of

cancers, to create a conventional, prescribed course of medical treatment. The

treatment consists of chemotherapy, radiotherapy, immuno-therapy, surgery or any

combination. However, the treatment is severely challenged and becomes failure due

to narrow therapeutic index and side effects. Moreover, treatments have very high

probability of incomplete eradication of invasive primary tumor cells or

dissemination of cancer cells. As such, there is a greater need for more effective

modalities.

The improvement of biotechnology and understanding of oncology, scientists are

thriving towards usage of virotherapy and gene therapy. As such the oncolytic

viruses are created using genetic engineering. (figure 1)

Figure 1: overview of mechanisms on oncolytic virus.Nakashima,H. et al.,(2010) Directing systemic oncolytic viral delivery to tumors via carrier cells Cytokine & Growth Factor Reviews .21(2) , 119-126

What are oncolytic viruses?

Oncolytic viruses are either naturally occuring or genetically modified viruses

designed to kill cancer cells, without harming the normal cells. The viral genes act as

tumor destructive agent and the capsids act as nano-sized nucleic acid delivery

vehicle. (Wakimoto, H et al.,2002) The oncolytic viruses operate via several

mechanisms which will be discussed in later sections.

Types of oncolytic viruses:

Enveloped/non-enveloped

Viruses which are classified as enveloped are ones that use the lipid bilayer of the

cancer cells and form a coat around them. An example would be viruses from the

Pox family, such as vaccinia virus or Paramyxovirus family, such as Measles virus.

The advantage of enveloped virus is that the glycoproteins present at the surface of

virus could detect and bind to specific receptors on cancers. Two main

glycoproteins are Hemagglutinin and Fusion proteins. The prior helps in attaching to

receptors and the latter aids in fusion of virus and the host cell like cancer cells.

Also, it was noticed that enveloped viruses could utilize larger specificity domain

like that of a single-chain antibody. (Cattaneo,R., 2010)

Genome(single/double stranded + DNA/RNA)

There are various types of viral genomes. Some are double stranded DNA like

adenoviruses and Herpes simplex viruses. Some are single stranded DNA viruses

like Parvoviruses. Examples of double stranded RNA viruses are reoviruses and

single stranded RNA are poliovirus (positive –stranded) and paramyxovirus like

measles virus (negative-stranded).

Natural /genetically modified

Naturally occurring viruses mean the virus requires no addition of genes

Generalized mode of actions of oncolytic viruses

In general, there are a few ways by which the oncolytic viruses work. Some of the

oncolytic viruses are designed or chosen to manipulate frequent tumour-specific

mutations, some are engineered to target signalling pathways and transcriptional

mechanisms that are ‘turned-on’ in tumours and some are aimed at antigens that are

overexpressed or unique on tumor cells. Despite the different mode of action, the

ultimate function of the oncolytic viruses is to disrupt the cancer cells’

transcriptional or translational mechanism and cause apoptosis.(Parato,K.A et

al.,2005)

Direct cell lysis:

Direct cell lysis is carried out by naturally occuring or selective-repllicating

viruses.The aim of this mechanism is to replicate and multiply inside the cancer

cell and lyse. Firstly, the viruse would adsorp to a receptor on cancer cell

membrane and creates a path through to penetrate its genetic materials to the host.

The genetic material would intergrate with that of the cancer cell genome. The

virus then uses the cancer cell’s replicating machinery to amplify viral

components. Then maturation and lysis occurs when there are surplus of viral

components, many progeny are created. The new virions then produce lytic

enzymes and burst the cell or activate apoptotic pathways by generating death

proteins to induce cell death. (Nakamouri,M.et al., 2004) (Figure 1)

Figure 1: The lytic cycle of viruses such as HSV-1 or adeno viruses shows the various stages of infection.

Adapted from source: Chiocca, A.E., (2002) Oncolytic viruses .Nature Reviews Cancer( 2), 938-950 

An example of experiment involves Herpes Simplex Virus (HSV) against murine

colorectal carcinoma cells. It was observed that there was a trigger of tumour-

specific immune response and reduction in size. (Nakamouri,M.et al., 2004)

Anti-tumour mechanisms:

1) Targetting tumour environments:

a) Anti-angiogenesis:

Angiogenesis is one is the hallmarks of cancer. (Hanahan.D.,

Weinberg.R.A.,2000).Cancer cells are able to form their own blood vessels by

secreting growth factors like VEGF. Normally, in a mass of tumor, the inner most

cells (core region) are deprived from oxygen and nutrients. Therefore, such survival

technique is used.

Oncolytic viruses are designed to trigger loss of blood flow to the core of tumor,

causing depletion of oxygen supply (hypoxia) and nutrients. Inhibition of blood flow

is done by blocking the synthesis of growth factors by viral genomes. Lack of blood

flow would induce apoptosis in cancer cells, exempting normal functioning cells.

(Caroline et al.,2007) In a research experiment, vaccinia virus was used, with

deletion of thymidine kinase and and its own growth factor, to reduce pathogenicity

to normal cell. The virus was incorporated with soluble vascular endothelial growth

factor (VEGF) receptor –Ig fusion protein (monoclonal antibody). to demonstrate

anti-angiogenic effect (figure 2) (Guse.K et al.,2010)

Figure 2: Diagram of how vaccinia virus perform anti-angiogenesis

Adapted from:Kirn,D.H. and Thorne,S.H., (2011) Targeted and armed oncolytic poxviruses: a novel

multi-mechanistic therapeutic class for cancer.

Moreover, another oncolytic virus,adeno virus has been used to inhibit angiogenesis

is cancer. The adeno virus strain dl922/947 of serotype 5 and which is replicative,

had deletion of CR-2 region in E1A, which binds to cellular retinoblastoma protein

(pRB)*. The deletion of E1A CR-2 region* in the strain was crucial in order to

ensure that viral replication is only in cancerous cells. Concurrently, another strain

of adeno virus Ad-Flk1-Fc*, which was replication-deficient, encoding soluble

VEGF receptor antibody, was used to co infect the tumour cells. The dl922/947

strain became a helper virus and rendered its support by providing E1* functions in

trans to Ad-Flk1-Fc strain, which increased in strength. The end result in vivo was

desirable as anti-tumor and anti-angiogenic effect was observed as Ad Flk-Fc

replicated successfully, intratumoral levels of Flk-Fc proteins increased and

microvessel density declined. (Thorne.S.H., et al., 2006)

b) Anti-tumoral immunity induction

The immune system has two types of defense mechanism. One is non-specific or

innate while the other is adaptive. The innate immune system recognises foreign

particles and release 1st line defense molecules like neutrophils and other pro

inflammatory molecules. Then a complement cascade gets activated. On the other

hand, adaptive immune system holds the postiton to eliminate pathogens in late

phase of infection and creates memory cells after being triggered by presentation of

antigen presenting cells (APC) like macrophage and dendritic cells, from the innate

immune system.

One of the major barriers for oncolytic viruses is to fight against effectively or evade

promptly from the immune cells.under normal circumstances, despite antigens being

present on tumour epitopes, the efficiency of CD8+ T cells* was insignificant, due

to strong immunosuppressive environment around tumours.(Cerullo,V. et al, 2010)

Oncolytic viruses are found to have the capacity to alter tumour microenvironment

via oncolysis with release of molecules like tumour-associated antigen (TAA),

tumour-derived cytokine, viral nucleic acid, viral coat proteins which trigger

immune response. ( Hall,K.et al,2012) Therefore, to activate the immune system to

destroy tumour, a significantly strong firing must be released, to override the

defense set by the tumour,by the oncolytic virus.

In an experiment, HSV-derived strains (Fus-On-H2, Synco-2D, Baco-1) were used

to observe antitumour immunity in neuroblastoma cells which displayed Ras

signalling. It was noted that there was significant in vivo antitumour response and

destruction of cells in in vitro and omission of growth of new tumors at areas further

from viral-introduction site. Moreover, the strains were able to induce a strong

inflammation, leading to possible maturation of dendritic cell, which is needed to act

as antigen-presenting cell to signal cytotoxic T cells. Such weakening of the

“firewall” created by tumour, would be favourable to the host as they would be

infiltration of more immune cells to degrade tumour. (Li,H. et al 2007)

Another example would be the experiment done on adeno virus strain Ad-D24-

GMCSF, which is p16-Rb pathway* selective and had its E1 region removed and

had granulocyte macrophage colony-stimulating factor (GMCSF) incorporated into.

GMCSF aids in attracting natural killer cells to act directly on tumor and dendritic

cells which would recruit cytotoxic T cells, signalling from lymph nodes. The

tumour would be more vulnerable to the immune cells and it could be degraded.

Tumour cells’ immunologic tolerance was weakened as Ad-D24-GMCSF replicates

within tumour and was able to present survivin* as epitope, gaining the attention of

T cells. (Cerullo,V. et al 2010)

There is another method by which the oncolytic viruses ‘cruise’ through the sea of

immune defence molecules. The usage of T-cells as cheperones* to escort viral

vectors into tumours was tested on a research. T-cells have the capability of tumour

homing. Therefore, a replication-defective retroviral vector, which was incorporated

with apoptotic chemokines, was able to reach its target in tumour cells. (Kottke,T.et

al, 2008)

Adding on, another interesting mode of effectively evading immune system attack

and traveling through systemically to tumour site was the the clever tactic of

naturally occuring virus, vaccinia, a member of Pox virus family. It exists as

external-enveloped virion*(EEV), whereby it would be engulfed in lipid bilayer like

its host cell and would be released into the system at early stage of infection. This

was seen as a great advantage because the virus was able to “pretend” it was part of

the tumour as it could use the tumour’s outer lipid membrane as a ‘cloak’ and was

able to infect more tumour cells. Therefore, there was increased biodistribution of

virus within tumor and its microenvironment. Besides intratumoural infection,

intertumoural spread was more prominent. As such, metastatic tumours were could

be reached ( Kirn,D.H.,et al, 2008) (figure 3).

Figure 3: anti-tumoral immunity induction.

Source adapted from: Choi, I.K. and Yun,C.O., (2013) Recent developments in oncolytic

adenovirus-based immunotherapeutic agents for use against metastatic cancers Cancer Gene Therapy

(20), 70–76

c) Cytokines and signalling pathways:

IFN pathway:

Cancers often result from defective major signalling pathways which control normal

cellular functions, proliferation, immune response or programmed cell death

(apoptosis). An example of an important cascade is Interferon (IFN) pathway.

Interferons (IFN) are versatile cytokines which activate transcription of genes whose

products are mainly antiviral, anti-proliferative or immunomodulatory in function.

(de Veer.M.J et al., 2001)

For example, a type 1 cytokine, IFN-is produced due to viral infection, as first

line of defense to protect normal neighbouring cells. (Li.Q. and

Tainky.M.A.,2011). The IFN- increases the expression and transcription of IFN-

stimulated anti viral genes (ISG), such as IFN- and double stranded RNA-

dependent protein kinase (PKR). The ISG molecules influence the inhibition of viral

genome replication and protect neighbouring cells. (Saloura.V. et al.,2010)

However in tumour, cancer cells, due to mutation of type 1 IFN pathway, an

oncolytic virus could take advantage of the defect and replicate.

In a research, it was observed that PKR-null mice were highly susceptible to VSV

infection. (de Veer.M.J et al., 2001) The reason being, PKR in normal cell would

phosphorylate translational initiation factor eIf2a, which inhibits cellular translation

and prevents host and viral protein synthesis. Upon omission of PKR, virus could

synthesize proteins and carry out cytolysis. (Saloura.V. et al.,2010)

Moreover, in another experiment, with respect to disrupted IFN signalling pathway,

it was observed that two important Interferon regulatory factors (IRF)* 5 and 7 plays

a crucial role in making a cell susceptible to VSV infection.

In normal cell, IFN pathway has several important functions such as obstructing

viral replication, activating immune cells, increasing or up-regulating antigen

presentation to T cells, keeping uninfected cells safe. However, in cancer cells, the

IFN pathway is unregulated as IRFs are silenced. Thus, anti-proliferation and cancer

surveilance by the immune cells are halted.

d) Apoptosis-induced oncolysis:

The p53 protein is a very important protein that is the name “guardian of the

genome” is given. (Rao, B. et al., 2010) The p53 protein and a transcriptional

factor, plays a crucial role in cell cycle check points and behaves as tumour

suppressor, ensuring the genome is tightly conserved.

Cancer cells have a defect in apoptosis mechanism, which is the reason why there

is an increased cell proliferation and no programmed cell death. However, it was

found that more than 50% of tumours express the wild-type p53. Yet, the cancer

cells inhibit the function of p53 by several mechanisms. (Koo,T. et., 2011) It has

been observed that despite having a defective p53 signalling pathway, the

pathway could be restored. (Chipuk,J.E. et al., 2004)

An experiment was conducted to research if oncolytic viruses could restore wild-

type p53 functions in tumour cells. Tumour cells are found to have

overexpression of Mdm2 molecule which is a proto-oncoprotein and a negative

regulator of p53. As such, p53 would be degraded, allowing cancer cells to

multiply non-stop and escape apoptosis. Thus, using replicative-competent

adennovirus strains (which were modified at either C or N terminus* which had

p53 gene (variant and wild-type) incorporated within, tumours cells were

infected. The outcome showed efficient transcriptional activity of p53 and Mdm2

inhibitory effect on p53 was observed. Therefore, with functional p53, apoptotic

molecules like Bax from Bcl-2 family could be upregulated and apoptosis could

occur. (Koo,T. et., 2011)

Another experiment done on the famous Onyx-015 strain of adenovirus which had

E1B protein* removed. It was observed that irregardless of p53-deficient or p53-

mutated cancer cells, the Onyx-015 could infect and trigger apoptosis to occur.

Clinical trials:

Clinical trials are very important part of a research for a novel therapeutic

modality. The reason being, laboratory results are based on animal models, whose

immune system could be compromised in order to study any pathway and

behaviour of the oncolytic viruses, even when human tumor cell lines are to be

used, most experiments are done in vitro to avoid any unwanted reactions within

a host.

There are 4 main phases under clinical trial. Under phase 1, maximal dose and

pharmacokinetics are the focus. In phase 2, the dose-response, type of patients,

frequency of dose and efficacy is observed. Phase 3 is more on submission of

paper works and formalities, to obtain permission from Drug regulation

authorities of the country. Phase 4 would be last one, where product can be

marketed. A clinical trial has to follow many rules and strict regulation as to

comply to medical ethics. There are some key factors to abide to during clinical

trials.

Volunteers:

The sample size of the number of test subjects to be used and drawing out

eligibility of the test subjects is very important (Geletneky et al.2012).The

volunteers should not have any other medical problems that would affect the

administration of the viruses and they should be well versed with the aim and

procedures of the trials. They must be withdrawn upon any major complications

and adverse effects.

Standards:

Most clinical trials compare data by following the response evaluation criteria

soild tumour (RECIST). The sum of biggest diameter of the target tumour is

measured. Also, partial response, complete response, progressive disease, stable

disease, immune cell counts are all factors that would determine if the therapy is

effective. (Van der Veldt, A.A.M. et al., 2010)(Li, J.C. et al., 2008)

Aim:

a) Primary:

Firstly, the safety and tolerability of the investigational medicinal product (IMP)

is important. Researchers need to find out the maximum tolerance dose for each

oncolytic virus therapy used. Then, the maximum tolerance dose is calculated. It

relates to determining whether prolonged use of the therapy would cause any

adverse effects to the mass. Normally, an estimated maximum dose is given to

reduce number of test subjects and cost of administration.

b) Secondary:

The antitumour efficacy is monitored via tests like tumor markers, Computered

Topography (CT) scan, cytokines assay. Also, the survival of the test subject and

the period the subject is free from progression or recurrance is recorded.

Till date, there are several clinical trials after the re surfacing of research of

oncolytic viruses.

Phase I:

The first trial of ParvOryx, a Parvovirus H-1strain, was carried out on patients

with recurrent Glioblastoma Multiforme (GBM) which is high-grade gliomas.

The oncolytic virus was administered intravenously to study the systemic delivery

and gather data to serve as platform for future trials. (Geletneky et al.2012)

In another trial, JX-594, a pox virus strain with Thymidine kinase gene removed

was used against Hepatitis B virus which causes Hepato cellular carcinoma

(advanced refractory stage). The pox virus is relatively well-tolerated as there is

previous exposure to most people who have encountered ‘chicken pox’ by

varicella zoster virus. The dose was injected intratumorally. The host cells

retained thymidine kinase gene to allow replication of the oncolytic virus. The

outcome of the result was satisfactory as distal tumours were also targetted

despite presence of large number of antibodies.

PhaseII:

An example of phase II trial consists of replication-competent adenovirus strain,

Onyx-015. It was tested against squamous cell carcinoma of head and neck

(SCCHN). It was observed the virus remains in the blood stream for only a short

time and some discomforts of pain at site of injection and mild fever was felt by

the test subjects. (Nemuaitis,J. et al., 2001)

Phase III:

Reolysin is commercial product of Oncolytics Biotech, is a name for reovirus

strain, in combination with two drugs, Paclitaxel and Carboplatin. Being able to

clear phase II would mean that the therapy was efficient in reducing tumour size

and maximal dose has been determined. (Net resources international, 2012)

Adding on to the list of victory, OncoVex GM-CSFby BioVex company, a strain of

HSV-1 with genetic modification, has also progressed to phase III. In phase II,

the tumour size reduced to more than 50% (melanoma). OncoVexGM-CSF was able

to infect cancer cells and replicate and lyse, recruiting immune cells to clear out

the debris. As a strong oncolytic virus, it has shown remarkable results in breast ,

pancreatic, head and neck cancer and melanoma. With no modifications and tests,

the strain stands a great chance to tackle more cancer types. (BioVex Inc, 2012)

Conclusion

There are several types of oncolytic viruses with various types of mechanisms by

which they function. However, there are several drawbacks and contradictions and

problems laying which each experiment done. For instance, there could transient

viremia, where by the oncolytic virus is removed from blood stream quickly before

it could exert its impact or there could incomplete incorporation of viral genome

with the cancer cell, failing to turn on apoptosis . Despite these drawbacks, there is

hope that oncolytic viruses would be a novel therapeutic strategy earlier than once

expected.

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