Documente Academic
Documente Profesional
Documente Cultură
Arjun Kanjarpane
Ms. Mary Jane Sasser
Dr. Sangeetha Underwood
Dr. Samuel Rabkin
Oncolytic Virotherapuetics
IR-3/09GT
2.28.18
Perfecting the Weapon: Novel Approaches to Combat Antiviral Responses and Optimize
In the intense battle against cancer, the second leading cause of death in the United States
(FastStats), a novel weapon has emerged that shows tremendous promise- viral oncotherapeutics.
All foreign organic molecules face severe immune resistance and molecular biology advances
have enabled the development of viral oncotherapeutics and contain the keys for mitigating
antiviral responses for treatment optimization. Three specific anti-immune response processes
While research has shown promise in each of these methods, therapeutic efficacy may be best
cell division takes place, creating masses that have direct and indirect effects on the body
(Cancer). Specific genes called oncogenes lead to cancer formation (Cooper). These are
mutations of genes which normally help cells grow. Mutant oncogenes can stay on beyond
normal conditions and promote cellular replication beyond safe limits and ultimately cause tumor
masses. These organic masses range in size and can be found virtually anywhere in the human
body. The absence of self-programmed cell death or apoptosis gives way to unmodulated cell
division. Direct effects of cancers include limiting blood supply to healthy tissues, diverting
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nutrition, and placing pressure on surrounding tissues to include nerves, vessels, and bones.
Indirect effects include immune and hormonal actions, and corresponding adverse consequences
such as infection, which serve to weaken the human organism further. The collective, cumulative
adverse effects of cancer, promoted by environmental as well as endogenous factors, affecting all
ages, serve to weaken and ultimately cause the body to die. Cancer affects all populations, and
since historical times, the fight to find cures has been a never-ending battle.
There are 1.7 million cases and six hundred thousand deaths in the United States alone
due to cancer (Siegel et al). Cancer affects various body parts, and fatality rates vary based on
morbidity, and genetic factors. Benign cancers cause little morbidity whereas malignant types
result in high mortality and morbidity (Brain). Although the frequency of malignant cancers is
decreasing in the United States, some types remain resistant to usual treatments. On an annual
basis, 26,000 individuals are diagnosed with glioblastoma, a severe and almost uniformly fatal
brain cancer. This cancer’s significantly high mortality and low cure rates have prompted
Since the emergence of cancer in the 15th century, cancer treatments have been
improving. Early treatments were restricted to surgery. Treatments progressed by the turn of the
19th century to chemotherapy, a targeted set of drugs that kill all fast-dividing cells including
tumor, as well as normal cells such as hair, and nail cells as well as beneficial enteric bacteria
(Sudhakar). Side effects of chemotherapeutics were significant (DeVita, Chu). It was seen that
certain cancers such as glioblastoma were resistant to even the highest doses of chemotherapy
(DeVita, Chu). X-rays were discovered in the late 1800s, and in the early 20th Century,
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researchers discovered that radiation therapy improved cancer treatment efficacy (Papac 291).
Multimodal cancer treatment using chemotherapy and radiation, both internal and external, were
the mainstays of medical response in the 1920’s and 1930’s (Papac 291). The serious side effects
of chemotherapy led researchers to study other options for cancer treatment. Immunotherapy
emerged in the 1990s and used elements of the body’s immune system to fight cancer.
Knowledge regarding the existence of the body's “fighting cells,” was well known. How exactly
they worked, and how they communicated with each other and were affected by pathogens, and
tumor cells were and are the core work of molecular biology. Deploying some of the body’s
interleukins, cytokines, endogenous angioinhibitors and antigens, ...” to kill cancer cells
(Sudhaker). The advances in immunology are expanding into the future with other molecular
biological processes and tools being deployed to overcome cancerous tissues. Gene therapy, or
the alteration of genes to minimize the frequency of unmodulated and rogue oncogenes, has
shown significant clinical promise. Genetically engineering the body’s fighting cells, such as
unmodulated T-cell therapy, has also shown considerable promise (Kelly & Russell). Recently,
molecular biology has advanced to include natural biologic weaponry outside the human body,
such as viruses, and these have shown great efficacy. With molecular adaptations to and
attenuation for potency, viruses have quickly overtaken the forefront of cancer research.
Oncolytic viruses are strains of viruses attenuated for potency and whose genetic
structures are modified to infect only tumor cells and ignore healthy cells (Fukuhara et al). Many
oncolytic viruses exist, but the measles, polio, herpes simplex, West Nile, and adenoviruses
dominate the treatment fields (Kelly & Russell ). These viruses each have specific benefits, and
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each includes specific properties, making them more effective towards certain cancers. The most
malignant types of cancers, breast, lung, and melanoma, are all being treated with oncolytic
viratherapeutics (OV), with better than expected outcomes (Sudhakar). Other cancers being
treated with OV include prostate, colorectal, cervical, brain, and neuroendocrine cancers. Viral
oncotherapeutics are currently being optimized to be the most potent weapon against cancer
cells. This can take place by priming the immune system after virtual induction and post-viral
oncolysis to further destroy cancer cells, or by extending viral survival to increase viral
which in turn, is a homeostatic process used to remove damaged organelles and foreign bodies.
This process relies on the fusion between autophagosomes which target and store components
for termination, and lysosomes which terminate the cargo held in the vesicles (Ding & Yin).
maintain proper cell function and terminate cells. Induction of mitophagy involves two steps:
“induction of general autophagy and priming of damaged mitochondria for selective autophagic
recognition.” (Ding & Yin). Autophagy is both used by the immune system as an antiviral
mechanism and can also be induced by viruses to lyse infected cells and mitigate immune
Oncolytic viruses induce autophagy and mitophagy, and to test this theory, researchers in
one study used intra-virus autophagic stimulants: ATG7, BECN1, SQSTM1 or RAB7. These
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critical proteins “decreased cell death induced by MV-Edm” (Xia et al). Conversely, the
overexpression of ATG5 or ATG7 enhances virus-induced autophagy which then lyses cells.
When intra-virus autophagic stimulants: ATG7, BECN1, SQSTM1 or RAB7 were impaired
through RNAi mediation, cytokine (virus-replication blockers) production increased, and viral
replication decreased. This led researchers to conclude that autophagic reactions mitigate
Lung cancer cells in two variants, the AF49 and H1299 cells were observed with and
without MV-Edmonston infection. The autophagosome moved towards cells with the
EGFP-MAP1LC3B transgene. The result number of vesicles increased, evidenced by the GFP
being embedded into the transgene, displaying that the overall lysis of the lung cancer cells.
Cells containing EGFP-MAP1LC3B were subject to MV-EDM infection, at 0.5 viral titer and
cultured for 4h. Following this culturation, cells were stained for MV H protein, to highlight the
protein. After 6 hours, increases in viral titer nor a increase in MV H protein were present and
EGFP-MAP1LC3B levels and quantities remained constant. 24 hours after virus infusion, MV H
protein levels rose astronomically along with a subliminal decrease in EGFP-MAP1LC3B levels.
The 9th square merged these factors and provided evidence to a strong viral titer decrease. This
contributed to the overall understanding that autophagy and the late stage, autophagic flux is
induced in greater quantities after viral infection, and this is specific to the Measles Virus-EDM.
Autophagic stimulants, in this study were added via extra tumoral infusion via injection (Xia et
al). In the future, these types of injections are subject to mutation and will complicate the tumor
microenvironment. To operate more seamlessly, MV-EDM should feature RNA coding for
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inherent autophagic stimulants as part of the viral genome. This process now allows the virus to
replicate and while translating, the virus automatically makes autophagic stimulants. However,
autophagy may be of more benefit when stimulated even further through translation.
Viral titer or concentration is another measurement that can be used to explore the
presence and use of autophagy against viral replication and survival time, and therefore assess
efficacy. Downregulation of the BECN1 and ATG7 and RAB7 was associated with a decrease in
viral titer. This observation signifies that not only does autophagy play a part in the replication
abilities of the MV-EDM virus, but this also describes how late and early stages of autophagy
can contribute to the replication effect. Expression levels of antiviral cytokines rose, “35- and
65-fold” when ATG7 was reduced. Correspondingly, over-regulation of the ATG7 protein
greatly decreased expression of cytokines, OAS1, IFNB1 and IFI27 (Xia et al).
as the process of limiting or disabling one or more of the body’s natural defense mechanisms
(Hartono). Immunosuppression first emerged in the 1930s following the need for a modality to
prolong and postpone foreign organ rejection from the host body. The use of immunosuppression
has been implemented in two ways, using T-cell checkpoint blockers or through drug regimens
of preventing a limiting factor using well-known and well-explored technologies. From the start
of a viral infection, the human immune system processes traces of viral matter and through
signaling proteins, summons the body’s fighting cells: T & B cells as well as natural killers.
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Immunosuppressive drugs can block this defense and ultimately limit (to varying degrees) an
that modulate the release of T and B cells after the arrival of signaling proteins. This modulation,
especially downregulation of these proteins, can either delay or advance the initiation of T and B
cell activity. T cells are activated upon the presentation of antigenic peptides which engage with
the T cell antigen receptor. Co-stimulatory T cell receptors, CD28 with associated ligands, CD80
and CD86, are also required for T cell deployment. For example, the immunosuppressant
CTLA-4 works by outmatching CD28 in binding to the associated ligands. The use of CTLA-4
also can delay the time between T cell proliferation and cytokine production (Jacobson).The
downregulation of T cell activation and the delay in T and B cell response can prolong viral
survival be allowing for further replication. Viruses are attacked by the body’s immune response
using the bodies’ ‘fighter’ cells. These T and B cells attack the virus and eventually destroy viral
particles using vesicle-lysosome fusion (Engeland et al). Viral replication increased when the
CTLA-4 protein was used in combination with oncolytic adenovirus. Viral titer increased
profoundly compared to the control model (immunocompetent), the first level of the independent
(Thomas et al).
Efficacy in the Syrian Hamster Model”, presents the advantage that immunosuppressant drugs
can have on the overall therapeutic efficacy of an oncolytic virus, as measured with tumor
adenoviruses VX-007 and AD5 were measured with the immunosuppressive and alone (without)
on hamster adenocarcinoma models. The experiment was measured over the course of five
independent variable of the study observed the activity of the tumor alone (vehicle), CP alone,
the viruses alone, and the viruses with CP in a tumor microenvironment. The gross tumor
volume (µl), with the vehicle alone at the end of 40 days measured volumes upward of 5,200μl
but did not see any signs of decreased volume. The CP alone did not have a statistically
significant effect on the growth of the tumor and is evidenced by the similar pattern it takes when
growing over the course of 40 days. The overall volume decreased very slightly, about 50μl
compared to the vehicle alone, but this is still statistically insignificant. The infusion of the
oncolytic viruses VRX-OO7 (an adenovirus with an overexpression of the Ad death protein) and
AD5 (an un-attenuated adenovirus serotype) into the environment without CP yielded
satisfactory results with both viruses reaching 2000μl from a previous 8,000μl 40 days post
infection (from a starting 1000μl at day 0). This was significant oncolysis as compared to the
vehicle, but growth was further reduced after CP was added, with tumor volume reaching below
1000μl 40 days post infection with the wild Ad5 virus being slightly more efficacious as it
reached 970μl compared to VX-007, which reached 1,000μl.. With the vehicle alone, with a
7,000μl volume, the oncolytic viruses brought the overall tumor volume lower than the starting
point, and lower than the viruses alone, proving the modality as beneficial to overall therapeutic
efficacy. Specifically, the first two weeks saw both immunosuppressant and immunocompetent
hamsters began to display tumor regrowth, which is represented on the first graph by the incline
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in tumor volume around day 14 and 15. The overall conclusion represents the ability for
immunocompetent environments to host viruses for longer periods of time leading to longer
Studies have shown that checkpoint inhibition results in more tumor lysis. Murine
models are commonly used as subjects for in vivo experiments due to their many similarities
with human models. Murine models with inserted B16 cells (since CD46 lacks in mouse models)
displayed that oncolytic viruses with modified checkpoint blockades (CTLA-4) were able to lyse
more cells and have greater viral titer than a non-modified virus. This observation allows for
(Engeland et al).
an additional emphasis due to its ability to tilt the fight against the immune system in the viruses’
favor. This, in turn, creates greater therapeutic efficacy (Gang et al). Cellular mitophagy, in
conclusion, uses a viruses’ natural ability to limit antiviral response and downregulate cytokine
synthesis while using this to the virus’s benefit. Greater replication can allow the virus to reach,
infect and lyse more tumor cells and achieve greater therapeutic efficacy. The overexpression of
mitophagy and autophagy-inducing stimulants can provide benefits in viral survival, and these
modalities can serve as crucial virus-based factors which can abrogate immune response.
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technologies have existed since the first organ transplants and have worked for most patient
populations (Hartono et al). As in all in vivo experiments, multiple events affect the outcome of a
given modulating factor. Interventions whose outcomes and interactions are measured can allow
for greater confidence in clinical trials and eventually, lead to a full-fledged treatment option.
understood and translated into amino acids, which eventually form proteins (Molecular).
Translation is a step in protein synthesis where genetic material is carried by mRNA and is
decoded to produce amino acids needed to create polypeptide chains and eventually proteins.
This process occurs in the ribosome and is present in all cells. This translation process also
occurs in tumor cells. Novel molecular biology solutions allow for the modulation of this process
Modulating tumor translation affects the speed at which it can divide and make more
cells. Translational control of the virus dictates the rate at which new proteins are produced and
thus the rate at which new cells are produced. Viral replication throughout the tumor intensifies
as the tumor itself multiplies at a faster rate. Transitional control of the tumor tissue also allows
for the programmed safety mechanism to protect healthy cells from unexpected viral replication:
if the virus spreads at an unexpectedly high accelerated rate, the “translational inhibition
(modification) suppresses measles virus potency.” In another study, using IGF-I stimulation of
mesothelioma cells activated the eIf4F complex which, “thereby triggers cap-dependent
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translation,” and, “stimulation of host [tumor] cell translation…,” and this resulted in enhanced
measles activity which therefore increases strength and potency toward tumor cells and immune
cells alike. The assessment of the impact that this stimulation had on MV activity was
symbolized by cell viability which, 24 hours later was “significantly reduced” in IFG-I models
treated with MV than in non-transitionally stimulated MV-infected models (Jacobson et al). The
increased potency of the virus because of stimulated transitional control describes how
Virus infusion and control relies heavily on translatory measures, as by itself, viruses are
not a stable platform to induce protein synthesis needed for viral survival and transmission.
Measuring the effect of transitional control in a tumor of viral control measures the rate at which
proteins are produced within the tumor, and inferentially the virus. This rate, is increased,
increases both the rate of tumor multiplication and virus multiple. To measure the effect of
translational control on virus survivability and overall efficacy, a translational inhibitor called
eIF4E targeted antisense oligonucleotide (ASO) was used to treat H514, H2461, H2596
mesothelioma cancer cells with MV-CEA, a measles virus which over expresses the
carcinoembryonic antigen. A mismatch control ASO was used for control purposes. 48 hours
later, cell viability in H256 cells decreased from 1.1 to 0.5, more than 200% reduction. Levels of
CEA were present in all cells inside the tumor environment, however cells infected by MV-CEA
and not mmAS, 4EASO displayed 300ng/ml of CEA compared to the other two CEA acquiring
methods in H513 cells, while the CEA levels in H2373 cells was closer to 200 in MV-CEA cells.
These results display that the mismatch control item had an inhibitory effect upon the H513 cell
line. 75% of cells tested displayed unviability while 25% displayed continued viability with
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continued use of the MV-CEA virus as well as 3EASO. Transitional stimulants such as IGF-I,
after 24 hours in use with MV-CEA drastically lowered CEA levels, reaching a staggering 60
When using biological weapons such as viruses, that have tendencies to mutate or
genetically reshuffle, safety systems including ones to drastically limit virus potency are vital.
For example, the backward compatibility of transitional suppressants (as compared to transitional
stimulants) that can to test the effect of 4E-BP1A37/A46 to suppress measles virus (MV)
activity. In testing, multiple melanoma cells were treated with MV-GFP, along with this
suppressant, and this resulted in less cytotoxicity. On the contrary, the transitional stimulant
Overall, the ability to have controlling factors outside of virus-modulatory items allows
for direct control of the strength of the MV-virus. This can allow for greater control of viral
efficacy for safety-related and other strategic objectives. The proven effects of translational
stimulants and their effects on viral strength and titer are therapeutic hallmarks in the cancer
antiviral response. Due to its novel approach to limiting viral strength through tumor
reproduction, blunting antiviral efficacy through increased viral titer secondary to translational
Prior treatments for malignant tumors have existed from the early 18th century. While the
earliest treatments included surgery to remove cancerous material, these treatments had low
success rates due to serious complications to include infection. Chemotherapy has worked for
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many, but the resulting side effects and concomitant treatment failures continue to suggest that
more optimal treatments should be researched. The use of novel treatments, to include biologic
agents such as viruses and the body’s immune system, holds promise that a more synchronous
6097-6101). Furthermore, the level of modularity, eons of viral development and adaptation,
suggest that viruses are a perfect therapeutic candidate for tumor elimination (Vile). Historical
and recent evidence supports the fact that viruses have the molecular development to be the
perfect tool to replicate, infect and destroy cells. Furthermore, virology research to include
modulation, modification, and adaptability is strong, as is work into associated viral processes.
The use of multimodal approaches to combat various issues in medicine is vast. Multiple
antibiotics treat tuberculosis, and the combination of surgery, chemotherapy, radiation for cancer
is also very effective.. Viral-oncotherapy has also seen multimodal approaches in using the virus
to first, directly lyse tumor cells and then initiate a subsequent major immune response to destroy
enhanced numbers of tumor cells. The use of unmodulated T cell blockade research enhances
this therapeutic approach (Vile). However, there is limited research into the deployment of
multimodal approaches to combat antiviral immune responses. Further research into the effect
had by combining approaches should yield answers that help optimize therapeutic efficacy and
The human body initiates severe and efficacious responses to combat foreign attacks.
Many of these attacks attempt to destroy foreign bodies such as bacteria or viruses. To place a
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virus in an environment fraught with survival challenges, certain modifications must be used to
help aid viral survival, and therefore, subsequent oncolytic rate and efficacy. Combining
modalities to help double or triple individual efficacy, holds promise in the innovative field of
viral oncotherapeutics.
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Appendix A, Definitions:
Oncolytic Virus: A genetically modified virus which is attenuated for potency and is used to kill
CTLA-4: A immunosuppressant drug used frequently to suppress the immune system to allow
Tumor Translation: The process of the nucleus decoded chromosomes and then produced
Appendix B: Data
Cellular Mitophagy
Translation
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Immunosuppresion
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