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Cutting Edge in Cancer Treatment :
Combination Therapy with Oncolytic Viruses


In Partial Fulfilment of PhD Stem Cell Biology

The Challenges in Cancer Treatment :
Oncotherapy has come a long way since 1930’s when the therapeutic methods began to be formalized
against detected tumors, many in the late stage due to lack of diagnostics or awareness in those times.
The mortality rates have consistently fallen owing to the increased screening , changes in food storage
practices and reduction in infective diseases.

Figure 1 : Cancer Mortality 1930 to 2006

(Ref - 1.The Biology of Cancer. 876 (Taylor and Francis LLC, 2014)

The response of Cancer to conventional therapies developed over the period of 90 years has improved and
has good remission rates. However, treatment for late-stage cancer and the reoccurrence of
tumors,frequently plagued with resistance to the primary treatment are unmet challenges. Treatment-
refractory cancer research has shown need for multi-modal therapy for effective treatment . Novel
strategies like targeted mABs, immunotherapy strategies have shown immense promise in this path.

The role of tumorigenicity of cancer cells as a clonal subset of genetically mutated cells is challenged by
numerous research reports showing existence of a subset of stem-cell like cells in the tumor mass, termed as
Cancer Stem cells (CSCs); which provide resistance to the tumor targeting as they are capable of self-
renewal,drug resistance and are in quinscient stages unless differentiated in cancer cells. The presence of
ABC transporters provides CSCs the opportunity to evade cytotoxic treatment and are hypoxia protected
against reactive oxygen species. induced attacks . Differences in gene expression of different cells within
individual tumors has led to the problems mainly seen post conventional therapies. CSC mutations have also
been reported, which show a worse prognosis for cancers where they have not been targeted. Oncolytic
viruses(OV) are thought to exert their antineoplastic effects via multiple different pathways in the CSC
population, combined with other therapies.

Strategies. Biomedicines 5, 3 (2017

Figure 2 . Role of cancer stem cell (CSCs) in traditional treatment failure. While chemo- and radiation-
therapy can efficiently kill differentiated cells that make the bulk of tumor, they often fail to kill CSCs. Due to
their ability to undergo self-renewal and differentiation, the surviving CSCs can cause disease relapse after
initial remission. Unlike the conventional therapies, OVs have the potential to kill both differentiated cells as
well as CSCs and hence they may cause eradication of the disease. (Ref- Chaurasiya, S., Chen, N. G. &
Warner, S. G. Oncolytic virotherapy versus cancer stem cells: A review of approaches and mechanisms.
Cancers (Basel). 10, 1–19 (2018).)

The table below summarizes research /clinical studies with OV effect on CSC population.

Table 1: Oncolytic Viruses and effect on CSCs

OV CSC Source Susceptible Replicates Comments
to OV? in CSC?
Inhibition of colony
formation in vitro and
CML Ad resistant CML Yes NA
elimination of
xenografts in mice
Prior infection of tumor
cells with virus
prevented engraftment in
AML MYXV AML patients Yes Yes
90% of recipient mice
compared to mock-
infected cells
Effective killing of
CSCs in vitro; prior
Pleural effusion infection of CSCs
Breast Ad from breast Yes NA prevented formation of
cancer patients xenografts; anti-tumor
effect against CSCs
derived tumors in mice

Eradication of CSCs in
Pleural effusion
vitro; anti-tumor effect
Breast Ad from breast Yes NA
against CSCs derived
cancer patients
tumors in mice

Mammospheres Highly toxic to CSCs in
generated from vitro and effective
Breast HSV Yes NA
breast cancer against CSC-derived
cell lines xenografts in mice

Ras expression, a
determinant of reovirus
oncolysis, was similar in
Human breast
Breast Reo Yes NA CSCs and non-CSCs;
cancer patients
similar killing of CSC
and non-CSCs both in
vitro and in xenografts
Highly toxic to Akt
Tumorspheres overexpressing CSCs;
Colon HSV generated from Yes Yes effective against CSC-
HCT8 cells derived xenografts in
CSCs over-expressed Ad
receptor (CAR) and
were highly susceptible
Glioblastoma Ad Yes Yes to the virus in vitro;
significant anti-tumor
effect against CSC-
derived xenograft

Effective killing of
Glioblastoma CSCs in vitro and
Glioblastoma HSV specimen from Yes Yes significant anti-tumor
human patients effect against xenografts
in mice

Effective oncolysis of
Glioblastoma CSC in vitro and anti-
Glioblastoma HSV specimen from Yes NA tumor effect against
human patients CSC-derived xenografts
in mice

Highly toxic to CSCs
Liver cancer
Liver Ad Yes NA both in vitro and in
cell lines
xenograft models
CD133-targeted OV
Surgical selectively killed
specimen from CD133+CSCs and
Liver Measles Yes NA
liver cancer prolonged survival of
patients mice bearing orthotopic
TRAIL-encoding OV
was toxic to CSCs in
Tumorspheres vitro and showed
Lung Ad generated from Yes NA significant anti-tumor
A549 cells effect against CSCs
derived xenografts in
The virus first-induced
cell-cycle mobilization
Human castric from G0-G1 to S/G2/M
Gastric Ad cancer cell Yes Yes in CSCs and then killed
lines them; OV also sensitized
those cells to

The ability of OV therapy to co-infect dividing malignant cells and CSCs and its role in reducing disease
relapse could be one of the key parameters of improving Cancer treatment outcomes. (Ref- Aitken, A., Roy,
D. & Bourgeois-Daigneault, M.-C. Taking a Stab at Cancer; Oncolytic Virus-Mediated Anti-Cancer Vaccination
Strategies. Biomedicines 5, 3 (2017).)

Novel Strategy Against Cancer : Immunotherapy
Current Strategies are mainly focused on Chemo and Radio-therapy to create a cytotoxic environment for
rapidly dividing cells. As expected, these strategies are systemic in nature and affect physiologically
multiplying cells which leads to severe side effects in the patients. Approved cytotoxic drugs have low tumor
specificity and high toxicity. (Ref- 1 The Biology of Cancer. 876 (Taylor and Francis LLC, 2014)

The overall contribution of chemotherapy to 5-year ongoing survival(OS) was estimated to be only 2.3% in
Australia and 2.1% in the USA. The numbers seem dismal for the dominant form of therapy for OS and a
need for targeting mechanisms is present.

On the other hand, Immunotherapy aims to re-educate the host adaptive and natural immune system to

 Identify the tumor as malignant
 Create antigens against malignant cells
 Create inflammatory response and activate cell death pathways post recognition

Figure 3 : Comparing Strategies in Cancer Therapy Ref : http://phrma-

The approach is physiological vs chemical or artificial as it attempts to use the body’s own immunity as a
watchdog and reactivate the body’s own defense mechanisms to target Cancer. With the body’s self-
tolerance immunity in play due to presence of antigen recognizing B-cells & T-cells , side effects are low as
systemically normal tissues remain unaffected. An Interferon response , as in OV therapy , is an example of
immunotherapy ; as it activates the body’s innate anti-viral response.

Table 3 : Overview of Chemical & Biological Therapies

(Ref - Schirrmacher, V. From chemotherapy to biological therapy : A review of novel concepts to reduce the
side effects of systemic cancer treatment ( Review ). 407–419 (2019). doi:10.3892/ijo.2018.4661)

 Oncolytic Viruses : A Physiological Immunotherapy
Dr. George Dock. In 1904 reported the case of a patient with leukemia, whose leukocyte count decreased
from 367,070 to 7500 over a period of two weeks, after an attack of possibly viral infection ; influenza . Also,
Levaditi noticed the growth and unusually long survival of the viruses in vaccines administered to mice and
rats with neoplasm . (Ref : Chiocca, E. A. Oncolytic viruses. Nat. Rev. Cancer 2, 938–950 (2002).)

The remission of cancers in patients contracting viral infections, lead to accidental discovery of viral therapy
for cancer. Although live viruses were studied, they are now completely replaced with genetically
reengineered non-pathogenic viruses for delivery in the tumoral or systemic sites for administration. OV
therapy is now an established field and In the last 20 years, nine different virus families , both DNA and RNA
viruses, are in early randomized clinical-phase trials with one FDA approved therapy in the market approved
a monotherapy (Ref- Marelli, G., Howells, A., Lemoine, N. R. & Wang, Y. Oncolytic viral therapy and the
immune system: A double-edged sword against cancer. Front. Immunol. 9, 1–8 (2018).)

Table 4 : Genetic modification of oncolytic viruses.
Oncolytic Virus Genetic Modification Manufacturer
Amgen Inc.,
(talimogene ICP34.5 deletion, ICP47 deletion, GMCSF
Thousand Oaks,
Herpes Simplex laherparepvec, insertion
Virus-1 Imlygic®)
(DNA Virus) HF10 Natural deletion and insertion led to loss of Takara Bio Inc.,
(canerpaturev—C- expression of UL43, Ul49.5, UL55, UL56, Kusatsu, Shiga,
REV) and LAT Japan

Oncolytic Virus Genetic Modification Manufacturer
HSV1716 Virttu Biologics,
ICP34.5 deletion
(Seprehvir®) Glasgow, U.K.
ICP34.5 deletion, ICP6 deletion, and LacZ Medigene,
insertion Planegg, Germany
Daiichi Sankyo
ICP34.5 deletion, ICP6 deletion, ICP47
G47∆ Company, Tokyo,
deletion, and LacZ insertion
Aettis, Inc.,
M032 ICP34.5 deletion and IL12 insertion
Pennsylvania, USA
Orien Gene
ICP34.5 and ICP47 deletion, and GM-CSF
OrienX010 Biotechnology,
Zhejiang, China
Shanghai Sunway
E1B deletion and E3 partial deletion Biotech, Shanghai,
ONYX-015 E1B-55 KDa gene deletion
South San
Francisco, USA
ONCOS-102 (Merged with
(formerly named adeno ∆24-RGD-GM-CSF insertion ONCOS
CGTG-102) therapeutic), Oslo,
VCN Biosciences
pRb-dependent; loaded with genes
VCN-01 SL, Barcelona,
encoding PH20 hyaluronidase

Adenoviruses pRb-dependent; loaded with genes Lokon Pharma,
(DNA Virus) encoding CD40L and 4-1BBL Uppsala, Sweden
pRb-dependent adenovirus with 24-bp
deletion in E1A. The fiber of the capsid has Targovax
been modified with an RGD-4C motif in the (Merged with
ICOVIR-7 HI-loop with a E2F promoter, and E1A Oncos
deletion, the replication is optimized with therapeutics), Oslo,
E2F binding hairpins and contains Kozak Norway
Adeno ∆24-RGD, pRb-dependent
Institut Català
adenovirus, with a deletion in 24bp in EIA
ICOVIR-5 d’Oncologia,
with E2F promoter and contains the Kozak
Barcelona, Spain
sequence at the E1a start codon
Deletion in 24bp in EIA and RGD-motif was
engineered into the fiber H-loop, enabling
DNAtrix, Houston,
DNX-2401 the virus
to use αvβ3 or αvβ5 an integrins to enter

Oncolytic Virus Genetic Modification Manufacturer
Vaccinia Viruses Thymidine kinase deletion and GM-CSF SillaJen, Busan,
(DNA Virus) insertion Korea
(formerly named
Reovirus Pelareorep
Natural virus Biotech® Inc.,
(RNA Virus) (Reolysin®)
Calgary, Canada
hNIS insertion for MV-NIS and
Vyriad, Rochester,
Measles virus Carcinoembryonic antigen (CEA) insertion
Paramyxoviridae USA
for MV-CEA
Newcastle disease Hadassah medical
Natural virus
virus (NDV) organization,
ORYX Medicine,
Parvovirus Parvovirus H-1
Natural virus Vaterstetten,
(RNA Virus) (ParvOryx)
Viralytics, Sydney,
CVA21 (Cavatak) Natural virus
Picornaviruses CD155/Necl5 dependent poliovirus. The
(RNA Virus) internal ribosome entry site (IRES) of the Duke University,
poliovirus replaced with the IRES from Durham, USA
human rhinovirus type 2 (HRV2)
This vector is based on murine leukemia
Retroviral virus (MLV) and carrying the yeast cytosine
Tocagen, San
Replicating TOCA51 deaminase (CD) that convert 5-
Diego, USA
Vectors fluorocytosine (5-FC) into in the presence of
CD, to 5-fluorouracil (5-FU).
The table includes the oncolytic viruses that were mentioned in this review. GM-CSF—granulocyte-
macrophage colony-stimulating factor.
(Ref : Eissa IR, Bustos-Villalobos I, Ichinose T, et al. The Current Status and Future Prospects of Oncolytic
Viruses in Clinical Trials against Melanoma, Glioma, Pancreatic, and Breast Cancers. Cancers (Basel).
2018;10(10):356. Published 2018 Sep 26. doi:10.3390/cancers10100356)

To date, only one oncolytic virus—a genetically modified form of a herpesvirus for treating
melanoma; talimogene laherparepvec , abbreviated to T-Vec is has been approved by the Food and Drug
Administration (FDA),& and European Medicines Agency (EMA) for melanoma treatment in 2015. as
monotherapy for patients in the United States with metastatic unresectable melanoma—stage III/IV

Clinical trials of OVs as monotherapies are challenged by dose limitations as some refractory cancers
required doses far above deemed toxic to the human host. Some have displayed effectivity only in case of
failure of conventional therapy and lower effect without prior therapy, displaying the need that it is only in
combinatorics approach will OVs be truly effective in Clinical practice. 1.
(Ref- Wang, S. J. et al. Combined Radiation and Oncolytic Viral Therapy Augments Cytotoxic and
Immunogenic Antitumor Effects Against Melanoma. Int. J. Radiat. Oncol. 102, S153–S154 (2018).) ; National

cancer institute. Oncolytic Virus Therapy: Using Tumor-Targeting Viruses to Treat Cancer. (2018). at

In combination therapy , the safety of several OVs, such as pelareorep (Reolysin®), proved their safety and
efficacy in combination with paclitaxel in breast cancer patients versus outcomes of OVs as monotherapy
against breast cancer not providing a clear therapeutic strategy. Few clinical trials of OVs against pancreatic
cancer have not yet demonstrated efficacy as either monotherapy or as part of combination therapy ,
necessaiting more personalized treatment becoming a necessity for resistant tumors. (Ref- 1: Eissa IR,
Bustos-Villalobos I, Ichinose T, Matsumura S, Naoe Y, Miyajima N,Morimoto D, Mukoyama N, Zhiwen W,
Tanaka M, Hasegawa H, Sumigama S, Aleksic B, Kodera Y, Kasuya H. The Current Status and Future Prospects
of Oncolytic Viruses in Clinical Trials against Melanoma, Glioma, Pancreatic, and Breast Cancers. Cancers
(Basel). 2018 Sep 26;10(10). pii: E356. doi: 10.3390/cancers10100356. Review. PubMed PMID: 30261620;
PubMed Central PMCID: PMC6210336.)

Surprisingly, adverse reactions in newly approved drugs from majority new therapies , chemical or biological,
were severe (grade 1-4) Even within immunotherapeutic approaches, only antitumor vaccines and OVs are
well tolerated with side effects between grades 0 and 2. Thus, it has been suggested to combine vaccines,
OVs and immune checkpoint inhibitors to prime, expand and facilitate effective tumor immunotherapy (Ref-
Aitken, A., Roy, D. & Bourgeois-Daigneault, M.-C. Taking a Stab at Cancer; Oncolytic Virus-Mediated Anti-
Cancer Vaccination Strategies. Biomedicines 5, 3 (2017).)

OV combination therapy in Cancer :
Ovs combination with anti-tumor vaccination, tumor antigens, and immune checkpoints has been previously
suggested in multiple publications ((Ref- Aitken, A., Roy, D. & Bourgeois-Daigneault, M.-C. Taking a Stab at
Cancer; Oncolytic Virus-Mediated Anti-Cancer Vaccination Strategies. Biomedicines 5, 3 (2017).) 2.
Havunen, R. et al. Abscopal Effect in Non-injected Tumors Achieved with Cytokine-Armed Oncolytic
Adenovirus. Mol. Ther. - Oncolytics 11, 109–121 (2018).3. Miest, T. S. & Cattaneo, R. New viruses for cancer
therapy: Meeting clinical needs. Nat. Rev. Microbiol. 12, 23–34 (2014)

OVs can be administered into the tumor itself (i.e. intratumoral administration) , into the tissue environment
(i.e. locoregional administration) or systemically. OVs can be combined with carrier cells, with bispecific
antibodies to improve tumor targeting eg: TAA. (Ref-Aurelian, L. Oncolytic viruses as immunotherapy
progress. Onco. Targets. Ther. 2627–2637 (2016). doi:2016:9 2627–2637)

Early clinical trials of intratumoral T-VEC administration in patients with melanoma combined with ICB
reported augmentation of intratumoral T-cell infiltration and an objective response rate of 62% OV therapy
can be combined not only with ICB but also with hyperthermia, chemotherapy, SMIs, radiotherapy and
adoptive T-cell immunotherapy. (Ref - Schirrmacher, V. From chemotherapy to biological therapy : A review
of novel concepts to reduce the side effects of systemic cancer treatment ( Review ). 407–419 (2019).

Prognosis in patients with cancer is determined by the immune response . In a study of 100 patients with
ovarian carcinoma a significant correlation was determined between survival and the infiltration of tumor
tissue by T lymphocytes. The 5-year survival of patients with tumor-infiltrating T cells (TILs) was 38%,

compared with 4.5% of patients with tumors without TILs (Ref - Zhang L, Conejo-Garcia JR, Katsaros D,
Gimotty PA, Massobrio M, Regnani G, Makrigiannakis A, Gray H, Schlienger K, Liebman MN, et al:
Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med 348: 203-213, 2003.)

Meta Analysis of 74 studies and >48,000 patients, concluded that immunotherapy appears to have a better
safety and tolerability compared with other therapies . ( Ref- Barnes TA, Amir E, Templeton AJ, Gomez-
Garcia S, Navarro B, Seruga B and Ocana A: Efficacy, safety, tolerability and price of newly approved drugs in
solid tumors. Cancer Treat Rev 56: 1-7, 2017)

The most recent update is from the industry where T-Vec is being explored as a neoadjuvant and in
combination with PD-1 inhibitors . eg- Keytruda.( pembrolizumab) and response rate, albeit in 21 patients,
was 62%.

Figure 4 : Illustration of T-Vec+ Keytruda effect on immune cells.

The mechanism of action of anti-PD 1 inhibitors is the conversion of the ‘Cold’ tumor to ‘Hot tumor’ is the
first step in immune response activation.

Figure 5- Schematic Course of Events following Oncolytic Virus and Anti-PD-1 Treatment in Metastatic
Melanoma(A) Patient with advanced-stage melanoma receiving combination therapy consisting of anti-PD-1
intravenously plus intralesional injection of talimogene laherparepvec. (B) Close up of tumor
microenvironment prior to injection with HSV virus: melanoma cells in vascular stroma lacking immune
infiltrate (“cold” tumor).(C) Close up of tumor microenvironment shortly after administration of the viral
vaccine. Infected tumor cells are killed by the virus; the virus spreads to other tumor cells. Innate immune
cells are attracted by local inflammation through (among others) type I IFN release, and dendritic cells (DCs)
are attracted and matured by local secretion of GM-CSF. DCs take up dying tumor cells and present tumor
antigens in the draining lymph node area to tumor-specific T cells. T cells are activated, proliferate, and
home to the tumor site that is injected and to distant non-injected metastases. (D) Close up of tumor
microenvironment with now-dense T cell infiltrate (“hot” tumor). Activated T cells are PD-1 positive, and
upon targeting tumor cells, released IFN-γ induces expression of PD-L1 on tumor cells, which leads to shut
down of T cell immune response. The systemically delivered anti-PD-1 blocks the PD-1/PD-L1 interaction and
reactivates the suppressed T cells, leading to tumor cell killing.

T-VEC is already being used in combination with PD-1 inhibitors for an off-label use. Data have been
published on that, as well, showing that these combinations can have dramatic effects in real-world

Combining mek inhibitors with this combination to give even more impressive regression in mouse model .

Figure 6 : Combining an oncolytic virus with PD-1 and MEK inhibitors appears to boost the immune response
to melanoma. (National Cancer Institute)

The addition of Imlygic to Novartis’ MEK inhibitor Mekinist and an FDA-approved anti-PD-1 monoclonal
antibody led to regression of tumors in nearly 100% of animals in a study with mouse models of melanoma
The lead author comments “All three agents we used are already FDA approved, so our study provides
justification for using them in combination. A clinical trial to examine this three-drug regimen should be a

For patients with BRAF-positive(a difficult to treat mutation) tumors, dabrafenib (Tafinlar) and trametinib
(Mekinist) have also been approved as adjuvant treatment
melanoma/ )

BRAF mechanism is shown here ,

Figure 7 :BRAF mutation effect on Cancer tumorogenity

Ref :

After testing the combination in mice, the team found that its anti-tumor activity is dependent on
competent CD8+ T cells and certain dendritic cells. What’s more important, the combination of T-Vec and
the MEK inhibitor boosted the expression of PD-1 and PD-L1, "checkpoints" that prevent the immune system
from recognizing and attacking cancer.

Many other combinations studied exist and we await FDA approval of combined therapies for effective
Clinical use.

Latest Developments :

Modified Maraba Virus : Researchers tested the Maraba virus, which was originally isolated from a species of sand fly in
Brazil, able to sensitize cells of triple-negative breast cancer. (Ref - Neoadjuvant oncolytic virotherapy before surgery
sensitizes triple-negative breast cancer to immune checkpoint therapy , Marie-Claude Bourgeois-Daigneault et al . Science
Translational Medicine 03 Jan 2018:
Vol. 10, Issue 422, eaao1641 DOI: 10.1126/scitranslmed.aao1641 )

Credit: The Ottowa Hospital

Figure 8 : Maraba Virus
“From the first patients, we observed very clear signs that the virus elicited antitumor immune responses,” Dr. Gromeier
recalled. Some patients had swelling in the brain and An immunotherapy approach using the Maraba virus (above) and
checkpoint inhibitors cured aggressive breast cancer in mice. “profound changes in the tumor that took months to develop,
which is consistent with an immune event,” he Explained

Modified Poliovirus :

Based on the results of the clinical studies, FDA in 2016 granted “breakthrough status” to Duke’s poliovirus
therapy, which allows officials at FDA to accelerate the agency’s review of the therapy for approval.

Figure 9 : Poliovirus

(Ref - Sci Transl Med. 2017 Sep 20;9(408). pii: eaan4220. doi: 10.1126/scitranslmed.aan4220. Cancer immunotherapy with
recombinant poliovirus induces IFN-dominant activation of dendritic cells and tumor antigen-specific CTLs. Brown MC(1), Holl
EK(2), Boczkowski D(2), Dobrikova E(1), Mosaheb M(1)(3), Chandramohan V(4), Bigner DD(4), Gromeier M(5)(3), Nair SK(6)(4)) )

Conclusions & Future Directions :

In 2018, the Nobel Prize for Physiology or Medicine was awarded to James P. Allison and Tasuko Honjo for
breakthroughs in cancer immunotherapy. The group of JP Allison discovered the TCR and went on to
develop the field of checkpoint blockade this led to the breakthrough drug ipilimumab. The group of Honjo
discovered PD1 and the mechanism underlying PD1 checkpoint protein blockade (Ref-Schirrmacher, V.
From chemotherapy to biological therapy : A review of novel concepts to reduce the side effects of systemic
cancer treatment ( Review ). 407–419 (2019). doi:10.3892/ijo.2018.4661)

These approaches are complementary to OV therapy mechanism of action creating cascade in host immunity
recognition of malignant cells. With the approaches mentioned above and the proliferative clinical trials in
the space and the focus on developing biomarkers to further enhance patient selection for multi factorial
therapies and algorithm development to the time and personalized recommendations on duration and
dosage of these therapies, we are entering the era of immunotargeting Cancer tumors effectively using
Oncolytic viruses and using the immune signals to deploy other strategies effectively to lead to effective
remission and stable disease.

The clearance of multimodal therapies through the FDA process is awaited for Real world clinical data

(Ref- “Oncolytic Virus Therapy: Using Tumor-Targeting Viruses to Treat Cancer was originally published by
the National Cancer Institute.”)

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