By Erick Ditmars
Viral oncology is a subsection of Oncology that focuses on treating tumors with viruses and is the most recent and arguably most promising new age tool we have for treating cancer. While this field has gotten a lot of press in recent years the idea of using viruses as oncolytic agents has been around since the early 1920’s, since as early as the mid-1800’s doctors noticed that certain illnesses would cause remission in cancer patients. These patients usually had blood based cancers such as leukemia or lymphoma with significant immune suppression. The most famous report of this type was made by Dock1 in which a 47 year old woman with “Myelogenous leukemia” went into remission after a flu infection. This report was first made in 1896, a whole 37 years before influenza was proven to be caused by a virus. Another more shocking case is that of a 4 year old boy with lymphatic leukemia who contracted chickenpox. His liver and spleen and lymph nodes were all severely swollen and his leukocyte count was greatly elevated (200 cells/ul) after contracting chicken pox his liver, spleen returned to normal size and his white count fell back into normal levels (4.1 cells/ul). However, in both cases the remission was short lived and the cancer soon returned. The first attempt to use viruses in oncology was the treatment of Hodgkin’s lymphoma with hepatitis in the 1950’s. While some did achieve remission for a short time many in the study also contracted Hepatitis B. The study was discontinued. A number of similar experiments were run throughout the 50’s and 60’s with minimal success and viral oncology was largely abandoned. It was almost 50 years later when a breakthrough in viral oncology came in the form of oncolytic adenovirus H101. This virus was approved by the FDA for cancer treatments in 2005 and works by targeting p53 deficient cells (most cancers are p53 deficient). Today there are many different viral pathways that medical research is focusing on in an attempt to make viruses an essential part of the cancer fighting toolbox.
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For a long time after the correlation between viral infection and cancer remission was first discovered researchers wondered why cancer cells made such good hosts for viral cells. It wasn’t until we began to understand the molecular and genetic mechanism behind cancer that that relationship started to become clear.
Cancer cells are the product of small scale evolution which is to say that the accumulation of point mutations and chromosomal shifts result in something called chromosomal instability have resulted in a phenotype drastically different from its ancestor. Typically in cancer cells we see a selection for growth advantages over somatic cells. These increased growth factors include immunity to density and anchorage dependence as well as repression of certain apoptosis measures and the activation of genes that would not normally be transcribed, such as telomerase.
While these genetic and molecular changes result in a massive increase in fitness when compared to somatic cells, often times certain defensive mechanisms are sacrificed. For example in many cancer cells a number of genetic repair enzymes are shut off as well as proteins that defend against mutations in the genome and foreign DNA.
It is these defense deficits seen in so many cancer cells that viruses exploit. Without the ability to defend against foreign DNA many cancer cells are left vulnerable to most viruses. By exploiting the innate viral sensitivity of most cancer cells scientists have been able to engineer viruses that can selectively kill cancer cells and grant immunity towards cancer cells.
Oncolytic viruses are viruses that specifically target and kill cancer cells. This process usually relies on genetically modified viruses that are only able to replicate within cancer cells. This selection is typically accomplished through the knockout of genes that repress cellular apoptosis. These cells then kill cancer cells through the lytic cycle.
Adenoviruses are a class of nonenveloped (Without a lipid bilayer) viruses with a double stranded DNA genome. Adenoviruses enter the cell through endocytosis. Once in the virus enters the cell the capsid breaks apart and ruptures the endosome releasing its DNA into the host cell and eventually attaching to histone proteins to enable transcription and translation of viral genes. For an adenovirus to successfully replicate in a host cell a number of processes must be completed in succinct order. The most important of these events being the repression genes which lead to apoptosis or programmed cell death which if active would hinder the viruses ability to replicate within the cell. The adenovirus accomplishes this through 2 major gene products; E1B-19K and E1B-55K. E1B-19K does two major things in the host cell. The first and most important function of this protein is its ability to bind and sequester BAK. BAK when activated induced apoptosis by binding to BAX and forming pores in the mitochondrial wall. Apoptogenic proteins then leech from the mitochondria ultimately releasing capase and lysing the cell. In addition, this protein works to stabilized viral and cellular DNA.
E1B-55K functions on a p53 dependent pathway. P53 is known as the “Guardian of the Genome” and works to regulate the cell cycle, check for mutations in the genomes and initiate apoptosis. E1B-55K binds to p53 and adds a repression domain to the protein. This results in p53 acting as a repressor instead of an activator for the various apoptotic genes it would normally bind. Additionally this gene results in the modified p53 having a binding affinity 10 times stronger than the original p53. This effectively eliminates all p53 dependent apoptosis pathways allowing adenovirus to replicate without programmed cell death.
In 1996 Frank McCormic had an idea that an adenovirus without its E1B-55K gene could selectively target cancer cells due to their lack of an active p53 gene but still maintain virulence and viral defenses through the E1B-19K gene. McCormic developed the Onyx-15 adenovirus however it never reached Phase III and was therefore never tested on human patients. However, the Sunway group in china took McCormics research and developed the H101 strain which contained a complete E1B-55KB deletion. This strain boasted a 79% response rate when coupled with chemotherapy while chemotherapy alone only resulted in a 40% response rate.
Herpesviridae is a class of enveloped viruses with double stranded DNA that cause latent infections that when triggered can become lytic. Herpesvirus enters the cell using viral Glycoproteins attached to the outside of its lipid bilayer that attach to the host cells cell membrane, causing the cell to internalize the herpes virion. Once the viron enters the cell it rapidly decays and viral DNA migrates to the nucleus where transcription and replication of viral genes begins to take place.
Herpesvirus was one of the first classes of viruses that was successfully turned oncogenic with the HSV1716 strain, a mutant form of the herpes simplex virus 1. HSV1716 takes advantage of herpes innate defense systems target cancer cells in a mechanism relatively similar to many other oncolytic viruses like H101. HSV1716 contains a full deletion of the gene ICP34.5 which allows for neurovirulence (viral replication within nerve cells). The ICP34.5 gene functions by counteracting PKR mediated blocks on viral replication and apoptosis. PKR is typically activated by double-stranded RNA, PACT or heparin. Once activated PKR deactivates cellular transcription, halting viral replication. Under extreme viral stress PKR is able to induce apoptosis through the activation of inferon cytokines. PKR is also known to interact with p53 which is also known to be inactivated in cancer cells. A knockout of this gene would cause the virus to target tumor cells due to most cancer cells inactivation of PKR (PKR is known to activate PP2A, a tumor suppression protein).
HSV1716 is currently undergoing a number of clinical trials to determine its usefulness in humans and is currently in phase II trials. Additionally it was reported by Liu that by upregulating US11, GM-CSF and downregulating ICP47 can increase rates of tumor destruction, tumor reduction and antigen presentation as well as enhancing anti-tumor immune response.
Cancer immunotherapy in a broad sense is the use of one’s own immune system to treat cancer. These therapies are wide ranging but typically involve T-cell Costimulation. T-cell immune responses of this type are triggered by cancer antigens and enhanced by immune adjuvants. Through introduction of an attenuated or genetically modified virus carrying either the genetic material for cancer antigens or the cancer antigens themselves, thee bodies defense systems can be triggered mounts a defense that targets cells that express these cancer antigens creating a cancer specific immune response.
Poxviridae is a class of enveloped viruses with a double stranded DNA genome. Poxviruses enter the cell by attaching to glycoaminoglycans on the surface of the host cells lipid bilayer. Once inside the cell the viral capsid falls apart and viral genes are transcribed in the cytoplasm starting the lytic cycle. Poxviruses were the first class of viruses scientists were able to create vaccines for and are among the best understood viruses on earth. Additionally, Poxviruses, across all classes, contain a number of phenotypes that make them ideal candidates for vaccine-mediated cancer immunotherapy.
The first and one of the most important factors in poxviruses ability to vaccinate against cancer is the fact that transcription and translation of poxvirus genes takes place entirely in the cytoplasm. This phenotype means that there is no risk of genetic integration in the host cells thereby avoiding a constant low level immune response for the rest of an individual’s life which could lead to a number of serious, chronic conditions and even, ironically, cancer. Additionally, cytoplasmic transcription and translation means that translation is independent of many host factors allowing for quick and efficient replication in most patients.
Poxvirus is also among the most diverse viruses in the world and is found in many different species. This ultimately means that there are classes of poxvirus that can get its genome into a human cell but is nonpathogenic. Modified vaccina Ankara (MVA) is a perfect example of this. The original vaccina was originally grown in chicken embryos, however after passing the virus through cell culture 570 times the MVA developed a number of mutations in the pathogenic regions of its genome (INF-a, IFN-b and TNF-a). This means, while it infect mammalian cell lines it cannot cause illness, even in the immune compromised, making it the perfect vector for introducing cancer antigens into the body. Additionally, due to its natural diversity these viruses can be engineered to enter virtually all cell types within the body. Finally, because of its large size poxvirus is able to accommodate large genes that code for large proteins including antigens and other immunomodulation molecules. These antigens and immune adjuvants are expressed within the host cell. The adjuvants in conjunction with the antigens cause a large scale immune response against not only the virus but the host cell as well. Because the antigen that triggered this response was originally from a cancer cell memory t cells are formed and the body develops an adaptive immunity to cancer cells resulting in an immune attack on a person’s cancer cells.
Currently a species of poxvirus containing the tumor-associated antigen PSA, i.e. PSA-TRICOM (PROSTVAC-V/F) is currently in phase III clinical trials in metastatic castration-resistant prostate cancer.
The Human Immunodeficiency Virus is a retrovirus that causes progressive immunodeficiency in the form of AIDS. HIV only infects cells that play a role in the human immune response, most famously the CD4+ T cell but also macrophages and dendritic cells. HIV contains 2 RNA “chromosomes” chat code for 9 total proteins. The HIV capsid is surrounded by a lipid bilayer made up of its pervious host cell’s cell membrane. This membrane makes the virus difficult to detect in the body and increases HIVs infectivity. HIV enters its host cell by fusing its cellular membrane to the cellular membrane of its host cell and injecting its viron into the cytoplasm. Once there the capsid falls apart and the viral RNA is turned into double stranded DNA using reverse transcriptase, this segment of DNA is then integrated into the host cells genome where it can sit in perpetuity. HIV immunotherapy focuses on dendritic cells. Dendritic cells are antigen presenting cells that are typically found in regions of the body most likely to be exposed to foreign invaders, i.e. the lungs, skin, nose and stomach. These cells are designed to recognize and present foreign antigens. When a dendritic cell recognizes a foreign antigen it moves to the lymph nodes and attaches to T cells and B cells where the antigen is attached to MHC proteins, triggering a targeted immune response. HIV typically uses these cells as a transportation system, infecting a dendritic cell then lysing it when it moves into the lymph nodes.
Dendritic cells are of special importance to cancer immunotherapy is because they seem to be able to more effectively create a tumor specific immune response when compared with direct presentation of the antigen to a T or B cell. This creates a stronger response to the cancer cells with these antigens and a greater likelihood for remission. HIV presents these antigens when its RNA genome is transcribed inside of the dendritic cell. The HIV viron has been genetically engineered to contain a copy of a cancer antigen that when transcribed can bind to the antigen presenting proteins within the dendritic cell.
HIV is used as the vaccine vector for a number of reasons. First off and most importantly, HIV as discussed earlier, is selective and only infects certain cell types, including the dendritic cell. This means that the vaccine will be more effective per unit than a non-specific virus. Additionally, because HIV is a retrovirus it is able to become what is known as a lentiviral vector. A lentiviral vector is a modified retrovirus that has been given a new series of genes to transcribe within a cell. These vectors have been shown to be very effective in gene therapy as the genes can continue to transcribed and translated long after the original dose. HIV is especially effective as its genes actually integrate into the host’s genome.
There have been a number of cases of modified HIV being used to treat cancer most of which were performed by Dr. Carl H. June. He is currently working on using the virus in treating pancreatic cancer.
Problems Still Facing Viral Oncology
While viruses are certainly promising as a cancer fighting tool there are still a number of problems with the use of viruses in treating cancer. The first and most obvious of these problems is that ultimately viral oncology involves injecting a person with a live virus which does carry with it a risk of contracting a serious disease. While almost all viruses are attenuated significantly or genetically modified to the point where they should be totally non-pathogenic to somatic cells there is still a risk, especially as many cancer patients are immunocompromised. Additionally, because these are ultimately viruses, the patient’s body often mounts a defense against the virus itself resulting in an immune response and the destruction of many circulating viruses within the body before they can infect any cancer cells. The real problems facing viral oncology have to do with the way cancer exists inside of the human body. Most cancers exist in a large mass of cells called a tumor. This tumor is surrounded by connective tissue that differs significantly from the cell membrane. This means that many viruses that enter the cell by binding to specific proteins are not able to penetrate this wall of connective tissue. This ultimately results in an inefficient spread of viral particles and incomplete infection of the cancerous mass. Gorte et al. helped determine that this problem was seen more in large tumors. Additionally, while direct injection of viral particles into the tumor coupled with increased dose does reduce this effect large tumors still end up with and incomplete infection. Another problem with viral oncology stems from the fact that cancerous tumors oftentimes contain a necrotic region within the tumor that maintains an equilibrium with the surface tumor cells. As the tumor continues to expand the necrotic region does as well often times killing the cancer cell before the virus is able to adequately replicate, that coupled with the accelerated division rate seen in cancer cells often means that the virus simply cannot replicate fast enough to keep up with the tumor. Lastly, due to the inherent instability of a cancer cell’s genome often times cancer cells will rapidly develop defenses against a virus. While oncolytic viruses are designed to exploit innate defensive shortcomings in cancer cells most of these shortcomings concern genome regulation and internal defense not external defense. In some trials doctors have observed cancer cells becoming totally resistant to a certain strain of virus by no longer producing surface binding proteins that the virus needs to enter the cell.
As discussed earlier a major problem facing oncolytic viruses today is the inability of the virus to move thorough the connective tissue surrounding the target tumor. Recently, doctors have begun experimenting with pretreatments before dosing someone with a virus, such as hyaluronidase or collagenase, which would work to break up the connective tissue allowing the virus to attach to the cancer cell and infect it.
Doctors are also thinking about employing a “viral cocktail” much like those used in chemotherapy or in treating retroviruses. These would contain a number of different types of viruses that function using different pathways that would, hopefully, overwhelm the cancer making it more difficult for the cells to develop an immunity.
Lastly, because we are able to design these viruses to target very specific surface proteins and shape the treatment to make more infectious for the patients specific type of cancer resistance is something the field is largely able to circumvent. The diversity and malleability of viruses means that doctors have an arsenal of vectors with distinct methods of infection and attack that should be able to overpower even the most aggressive forms of cancer.