ONCOLYTIC VIRUSES AS ANTICANCER AGENTS

BY

ADEOYE OPEYEMI ADURAGBEMI

OUTLINE ü INTRODUCTION

ü HISTORICAL BACKGROUND

ü ONCOLYTIC VIRUSES AS AN ANTICANCER AGENT

ü GENOMIC ORGANISATION OF SOME SELECTED ONCOLYTIC VIRUS

ü MECHANISMS OF ANTITUMORAL EFFICACY OF ONCOLYTIC VIRUSES

ü TUMOR SELECTIVITY/SPECIFICITY

ü ONCOLYTIC VIRUSES AND THEIR GENETIC MANIPULATIONS IN CLINICAL

TRIALS

ü ADVANTAGES AND DISADVANTAGES OF ONCOLYTIC VIRUSES

ü BENEFITS AND LIMITATIONS OF GENETIC MANIPULATION WITH POTENTIAL

SOLUTIONS

ü FUTURE PROSPECT OF ONCOLYTIC VIRUSES

ü CONCLUSION AND RECOMMENDATION

ü REFERENCES

INTRODUCTION

v Cancer continues to be a leading cause of death, accounting for 8.2 million deaths worldwide in 2012.

v Cancer mortality with incidence projected to rise from an estimated 12

million cases in 2012 to 22 million within the next twenty years (Bradley et al., 2014 ).

v Recently according to the WHO, over 100,000 Nigerians are diagnosed with cancer annually and about 80,000 die from the disease.

v WHO estimated that 240 Nigerians die per day or 10 per hour die from

cancer disease. v The Nigerian cancer death ratio of 4 in 5 is one of the worst in the whole

world

v Early human trials using viruses in anti-cancer treatments initially yielded excitement as tumor regression was often seen without toxicity.

v However, tumor regression was followed by tumor

progression during the late stages of these trials approaches. v Recently there has been a renewed interest and optimism in

virotherapy with the convergence of ideas from molecular oncology and virology, and the development of recombinant virus technologies.

CONTD…

TABLE 1: CLINICAL VIROTHERAPY: FOUR HISTORICALLY SIGNIFICANT CLINICAL TRIALS

Abbreviations: IA, intra-arterial; IT, intratumoral; IV, intravenous; TC, tissue culture. Source:Kelly andRussell,2007.

Year(s) Virus Disease No. of patients

Administration Outcome Side effect

1949 Hepatitis B virus Hodgkin's disease 22

Parenteral injection of unpurified human serum, tissue extract

4/22 reduction in tumor size, 7/22 improved in clinical aspect of disease; 14/22 developed hepatitis;

Fever, malaise, death (1 confirmed)

1952 Egypt 101 virus (early passage West Nile)

Advanced, unresponsive neoplastic disease

34

IV, intramuscular injection of bacteriologically sterile mouse brain, chick embryo, human tissue

27/34 infected; 14/34 oncotropism; 4/34 (transient) tumor regression

Fever, malaise; mild encephalitis (2 confirmed)

1956

Adenovirus adenoidal-pharyngeal-conjuctival virus (APC)

Cervical carcinoma 30 IT, IA, IV injection of

TC supernatant

26/30 inoculations resulted in localized necrosis

Vaginal hemorrhage; infrequent (3/30) fever, malaise

1974 Mumps virus (wild-type, non-attenuated)

Terminal cancers; gastric, pulmonary, uterine account for more than 50%

90

External post-scarification; IT; IV; oral; rectal; inhalation of purified human saliva or TC supernatant

37/90 complete regression or decrease >50%; 42/90 decrease <50% or growth suppression; 11/90 unresponsive

7/90 adverse reactions: bleeding, fever

ONCOLYTIC VIRUSES

v Literally, Onco- refers to cancer while Lytic- refers to killing v Oncolytic viruses (OVs) are viruses that have the ability to

specifically infect and lyse cancer cells, while leaving normal cells uninfected.

v Examples are üAdenovirus üVaccinia virus üVesicular stomatitis virus (VSV) üSeneca valley virus (SVV) üReovirus üMaraba virus üNewcastle disease virus (NDV)

CHARACTERISTICS OF IDEAL ANTICANCER VIRUS

vSpecific in action

vReplicate only in tumor cells

vHighly lytic

vAble to induce anti-tumor immune response

to the host cells.

Figure 1: Generalised diagram of the cancer specificty of oncolytic viruses (Mode of action) The diagram above illustrates how oncolytic virus encounter normal cells and cancer cells. Source: Wilkipedia online(modified 2015).

Table 2: Mechanisms of antitumoral activity of oncolytic viruses

Mechanism Examples

1. Direct cell lysis due to viral replication

Adenovirus ,Herpes simplex virus

2. Direct cytotoxicity of viral proteins

Adenovirus E4ORF4,E3KPO11.6kD

3. Induction of antitumoral immunity • Nonspecific (e.g., TNF) Adenovirus (E1A) • Specific (e.g., CTL response) Herpes simplex virus

4.

Sensitization to chemotherapy and radiation therapy Adenovirus (E1A)

5. Transgene expression

Adenovirus (AdTK-RC) Herpes simplex virus (rRp450) Vaccinia virus (GM-CSF)

Source: Kirm, 1996.

GENETIC MANIPULATION OF ONCOLYTIC VIRUSES

v The resistance of cancers to conventional therapies has inspired

the search for novel strategy called gene therapy. v based on manipulation of viral gene / viruses as genetic vectors. v These involve use of replication-competent viruses. v This require understanding genome of the virus to be use(Ring,

2002).

CONTD…

v Knowledge of the pathogenesis of virus diseases and ability to manipulate

specific regions of viral genomes have allowed the construction of viruses

that are attenuated in normal cells but retain their ability to lyse tumor

cells and also will enhance an immune response against it.

v Such manipulations include modifying the ability of the viruses to bind to,

or replicate in a particular cell type while others have involved the

construction of replication-competent viruses encoding suicide proteins or

cytokines (Ring, 2002).

MECHANISMS OF TUMOR SELECTIVITY/SPECIFICITY There are six general mechanistic approaches to tumor-selective

replication described to date. These include: (a) The use of viruses with inherent tumor selectivity (e.g., NDV,

reovirus, VSV(Coffey et al., 1998) (b) Deletion of entire genes e.g. HSV, adenovirus, vaccinia (Mastrangelo, et al., 2000) (c) Replacement of functional gene regions e.g. adenovirus, poliovirus (Gromeier et al., 2000).

CONTD…

(d) Engineering of tumor/tissue–specific promoters e.g. adenovirus, HSV (Miyatake, et al., 1997) (e) Modification of the viral coat to target uptake selectively to tumor cells (e.g., adenovirus, poliovirus) (f) Insertion of genes such as those encoding suicide proteins, tumour suppressor proteins or cytokines into tumour cells by means of a genetic vector.

CRITERIA FOR SELECTING ONCOLYTIC VIRUSES AGAINST TUMORS

vPathogenesis of the oncolytic virus (biological behavior of the virus)

vTumor type vTropism of the virus in relation to location of the

tumor vPresence of viral receptor in the target tumor cells.

GENOMIC ORGANISATION OF ONCOLYTIC VIRUS

Genomic diversities occur among the oncolytic viruses v DNA viruses such as Herpes simplex virus-1, Myxoma virus and

vaccinia virus v RNA viruses such as vaccinia virus, Newcastle disease virus, Seneca

Valley Virus, Reovirus and Maraba virus. v It is worthy knowing that most of the oncolytic viruses are RNA Virus

because of their small virion size.

Figure 2: Morphology and genomic organisation diagram of Adenovirus.

dsDNA, Adenoviridae,nE Icosahedral symmentary, with 20 faces, CAReceptor, Particle-70-90nm in Diameter CV706 (PSA),ONYX-015

Source: Banko and Harrach 2003.

Figure 3:GENOMIC ORGANISATION OF MARABA VIRUS AND VESICULAR STOMATITIS VIRUS

Source: Brun J. et al., 2010

Rhadovirus family Vesiculovirus genus -ssRNA, E unsegmented 3’ NPMGL5’ NonPathogenic in human Sensitive to interfron

3 5 , ,

Figure 4:GENOMIC ORGANISATION OF HERPES SIMPLEX VIRUS

HSV-neurotropic,DNA,Herpesviridae, nonEssential 30kb-15K,NV1020. UL56genes (4,6mp of Us),Us12genes, Repeat unit of a-c

Figure 5:GENOMIC ORGANISATION OF VACCINIA VIRUS

Source: Baroudy and Moss, 1982.

Figure 6: Morphology and genomic organisation diagram of Measles virus.

Source: Rima et al.,2009

-ssRNA, Paramyxo, Morbillivirus 3 gene code for H ,M,F W.T –CD150 Attenuated MV edm- CD 46

Figure 7:THE GENOMIC STRUCTURE OF POLIOVIRUS TYPE 1

+ssRNA,nE,PvEnterovirus 7500nucleotide Long CD155r (PVR),30nm viral Particles PV1(RIPO).

Source: Goetz and Gromeier, 2010

Poliovirus as an oncolytic agent v Poliovirus is a natural neuropathogen, making it the obvious choice for

selective replication in tumors derived from neuronal cells.

v Poliovirus replicates in the GIT, the virus invades the central nervous

system (CNS) where it targets predominantly motor neurons, thereby

causing paralysis and even death (poliomyelitis).

v Generally, poliovirus replicates efficiently in nearly all tumor cell lines

tested, which has led to the suggestion that it may be suitable for the

treatment of different cancers.

v However, the possibility that poliovirus can cause poliomyelitis calls for

significant neuro attenuation to avoid collateral neurologic complications

in cancer treatment.

v Poliovirus has a positive -strand RNA genome, the

translation of which depends on a tissue-specific internal

ribosome entry site (IRES) within the 5' untranslated

region of the viral genome, which is active in cells of

neuronal origin and allows translation of the viral

genome without a 5’ cap replaced the normal poliovirus

IRES with a rhinovirus IRES, altering tissue specificity. The

resulting PV1(RIPO) virus was able to selectively destroy

maglinant glioma cells, while leaving normal neuronal

cells untouched (Gromeier et al. 2000)

TABLE 3: SUMMARY OF SOME SELECTED ONCOLYTIC VIRUSES AND THEIR GENETIC MANIPULATIONS IN CLINICAL TRIALS

Virus Deletions/Mutations Foreign gene/promoter insertion Tumors targeted in studies

VACCINIA VIRUS JX-594

Deletion of both copies of TK gene

Human GM-CSF and lacZ insertion into the TK gene region

EWS, lymphoma, NB, RMS, Wilms tumor . PhaseI/II

HSV-1 G207

Deletion in both copies of γ134.5 gene and disabling lacZ insertion in UL39

None Phase II Glioma, MDB, OST, RMS, Colorectal cancer

HSV1716 Deletion in both copies of γ134.5 gene

None Non-CNS solid tumors

M002 Deletion in both copies of γ134.5 gene

Murine IL-12 gene insert Glioma, RMS

rQNestin34.5 Deletion in γ134.5 gene and UL39 ICP-34.5 under control of a synthetic nestin promoter

Glioma, NB

rQT3 Deletions in ICP6 and γ134.5 gene

Tissue inhibitor of MMP3, HSV-1 immediate early 4/5 promoter

NB, MPNST

Table continue

HSV-1 VAE

Deletion in both copies of γ134.5 gene

Endostatin–angiostatin fusion gene insert

Glioma

Chase-ABC Deletion of both copies of γ134.5 and in-frame gene-disrupting insertion of GFP within the ICP6 gene

Inserted Chase-ABC cDNA under the viral IE4/5 promoter within the ICP 6 locus

Glioma

Measles virus

Engineered to target CD46 on tumor cells

Phase I Brain,MM CNS tumor

AdenovirusOBP-301

Deletion of native E1 promoter of Ad5

Human hTERT promoter to drive E1A and E1B expression linked to an internal ribosome entry site

Phase III OST

AD5/3-Cox2L-D24

Deletion of Rb binding region from the E1A gene

Inserted cyclooxygenase -2 (COX-2) promoter

NB

ICOVIR-5 Deletion of Rb binding region from the E1A gene

Substitution of the E1A promoter for E2F-responsive elements, RGD-4C peptide motif insertion

NB

Table continues

Abbreviation:Ad5, adenovirus serotype 5; CIK-vvDD, cytokine-induced killer double-deleted vaccinia virus; CNS, central nervous system;

EWS, Ewing's sarcoma; GM-CSF, granulocyte colony-stimulating factor; hTERT, human telomerase reverse transcriptase; IL;interleukin; MDB, medulloblastoma; MMP3, metalloproteinases-3; MPNST; malignant peripheral nerve sheath tumor; MYXV,-myxoma virus; NB, neuroblastoma; OST, osteosarcoma; RB, retinoblastoma; RGD, arginine-glycine-aspartic acid; RMS-rhabdomyosarcoma; SVV, Seneca valley virus; TK, thymidine kinase; VSV, vesicular stomatitis virus

Delta-24-RGD Deletion of Rb binding region from the E1A gene

Inserted RGD into the H1 loop of the fiber protein

Glioma

CRAd-Survivin-pk7

Deletion of native E1 promoter of Ad5, polylysine modification in the fiber knob

Human survivin promoter to drive E1 expression

Glioma

Reovirus type 3 Dearing

None None MDB, RMS, OST, EWS

Seneca Valley Virus SVV-001

None None EWS, glioma, MDB, NB, rhabdoid tumor, RB, Wilms tumor. Phase I

Source: Friedman et al., 2011.

Table 4: Specific Advantages and Disadvantages of different oncolytic viruses

Virus Virus type

Insertion size

Advantage Disadvantages

HSV-1 (152 kb)

DNA 40-50kb Neurotropic Ability to infect a wide variety of tumors Large amount of nonessential genes can be replaced with foreign DNA to enhance cytotoxicity Clinically available antiviral agents

Systemic delivery may be limited by preexisting immunity and hepatic adsorption

Adenovirus (36 kb)

DNA 7.5kb Ability to infect a wide variety of tumors with modifications of the fiber knob Large amount of nonessential genes can be replaced with foreign DNA to enhance cytotoxicity

CAR variability in human cancers Systemic delivery may be limited by preexisting immunity, hepatic adsorption and toxicity. Small insert capacity

Reovirus RNA ? Wild-type virus causes mild to no disease Systemic delivery possible

Activated Ras or Ras effectors necessary Inability to enhance infection with foreign DNA

Maraba virus

RNA ? Ability to boost adaptive antitumor immunity. Does not cause human disease

Limited preclinical data. unknown mechanism of action

VV (187 kb)

DNA 25kb Ability to infect a wide variety of tumors Large amount of nonessential genes can be replaced with foreign DNA to enhance cytotoxicity

Inefficient systemic delivery

NDV RNA ? Targets cancer cells with loss of interferon responsiveness. Ability to express foreign DNA to enhance cytotoxicity Used safely in children with recurrent gliomas.

Immune-mediated clearance of virus. Transgene insertion reduces viral replication.

MYXV DNA ? Targets cancer cells with altered Akt signaling Does not cause human disease

Limited preclinical data in pediatric cancers

VSV RNA ? Targets cancer cells with loss of interferon responsiveness

Limited preclinical data in pediatric cancers Uncertain tumor selective oncolytic effect

SVV RNA ? Virus does not cause human disease Mechanism of infection unclear Inability to enhance infection with foreign DNA

Source: Friedman et al., 2011

GENERAL BENEFITS AND LIMITATIONS OF ONCOLYTIC VIRUSES

Benefits 1. Effective tumor removal 2. Limited or no side-effects 3. Selectivity 4. Cost-efficiency

Limitations 1. Revertant of mutant virus (accidental emergence of a new viral pathogen) 2. Deletion of viral genes 3. Anti Immune response of the host. 4. According to Russell, 1994, the limitation to Oncolytic viruses are the safety

concerns arising from the use of it, for human cancer therapy, these can be divided into two areas:

v Risk to the patient v Risk to the population (Serious epidemics)

Possible solutions vUse of a competent replicative virus v further attenuation of the virus v Suppressing the host immune response or enabling the virus

to evade immune recognition vUse non-transmissible virus and preferably derived from a

virus to which the population is generally immune.

vuse of an ideal oncolytic virus which should be selective for the tumor, nonpathogenic for normal host tissues, non-persistent and genetically stable (Russell, 1994).

PROSPECT OF ONCOLYTIC VIRUSES v Despite the fact that Oncolytic viruses may be effective

against cancer cells without any supplements; the combination of chemotherapy or radiation therapy with oncolytic virus may be highly efficient in cancer destruction.

v Oncolytic viruses can be used as vector for the delivery of

vaccine. v There is now a compelling need to develop new classes of

anticancer agents with little or no side effect.

Conclusion and Recommendation

v Cancer virotherapy is a rapidly maturing field that will be an integral part of future cancer therapies.

vWith the advent of genetic engineering a wide range of viruses are

being manipulated and evaluated into various types of cancers. vMany clinical trials around the world have had good results with

high success rates using oncolytic virotherapy, and many more clinical trials are in progress with new viral vectors for the treatment of intractable cancers.

v Nigeria should not be left behind! It is thus recommended that our

research centers should be actively involved in clinical trials of these anticancer agents.

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