Reovirus (Reolysin®; Oncolytics Biotech, Calgary, AB, Canada), a proprietary variant of the Respiratory Enteric Orphan Virus, is a benign, double-stranded RNA human virus with oncolytic properties. In a variety of clinical trials, the virus has shown to selectively kill tumor cells with activated signaling through the RAS pathway.

The RAS protein family members belong to a class of protein called small GTPase, and are involved cellular signal transduction [Fig 1]. The RAS-regulated signal pathways controls a number of processes, including actin cytoskeletal integrity, proliferation, differentiation, cell adhesion, apoptosis, and cell migration. RAS and RAS-related proteins are deregulated in about 30% of all human cancers, leading to increased invasion and metastasis, and decreased apoptosis. It’s one of the most common genetic defects that are known to predispose an individual to developing cancer.

Viruses and cancer
Viruses have long been implicated in the development of cancer. More than a century ago, Francis Peyton Rous (1879 – 1970) noted that sarcoma's growing on a domestic chicken could be transferred to other chicken by a virus. While his findings were initially discredited, some influential researchers were impressed enough to nominate Peyton Rous to the Nobel Committee as early as 1926 (he eventually received the Nobel Prize in 1966).  Another important breakthrough came in 1964.  In that year Michael Anthony Epstein, Bert Geoffrey Achong and Yvonne M. Barr isolated the first human cancer virus from lymphoma cells.[1]

In 2002, the estimated total of infection-attributable cancer was 1.9 million cases, or 17.8% of the global cancer burden. The viruses thought to be responsible for this include the bacterium Helicobacter pylori (5.5%; linked to gastric cancers), the human papilloma viruses (5.2%; linked to cervical cancer), hepatitis B and C viruses (4.9%; linked to liver cancer in people with chronic infections), Epstein-Barr virus (1%; linked to Burkitt's lymphoma), human immunodeficiency virus (HIV) and the human herpes virus 8 (0.9%; linked to the development of Kaposi sarcoma).  Other, less important causes of cancer include the schistosomes (0.1%), human T-cell lymphotropic virus type I (0.03%; linked to T-cell leukemia) and the liver flukes (0.02%). [2]

The understanding that viruses were involved in the development of cancers, led to the development of vaccines against some oncoviruses, including hepatitis B and the human papillomaviruses HPV-16 and HPV-18, which cause about 70% of all cases of cervical cancer. [3, 4]. Today, the U.S. Food and Drug Administration (FDA) has approved two vaccines, Gardasil® (MSD/Merck Sharp & Dohme) and Cervarix® (GSK/GlaxoSmithKline) which are designed to protect against some vaginal, vulvar, anal, penile and oropharyngeal cancers.[2, 5]

DNA and RNA viruses
Oncoviruses change cells by integrating their genetic material with the host cell's DNA. In DNA viruses, the genetic material is directly inserted into the host's DNA. On the other hand, RNA viruses must first transcribe RNA to DNA and then insert the genetic material into the host cell's DNA.

A new approach to fight cancer
Viruses are not only involved in the development of cancer, they can also be used to attack and destroy cancer cells via a number of mechanism. The therapeutic use of viruses in cancer therapy is called "oncolytic virotherapy." One approach is based on the virus' ability to act through lysis, just as it would destroy healthy cells when causing an infectious disease. In this appraoch cell lysis spills antigens specific to the tumour into the blood-stream and elicits a systemic immune response against the tumour cells. Another appraoch uses viruses as a vector to express an enzyme in tumour cells followed by the administration of a prodrug that combines with the enzyme to generate cytotoxic compounds that destroy the target cell and surrounding cells.

Safety and regulatory concerns
The deliberate injection of viruses to treat cancer raises a number of important safety and regulatory issues. However, a growing body of evidence from in vitro experiments, preclinical and initial Phase I trials have shown encouraging results with limited toxicity.  Hence, viruses may become a new weapon in the fight against cancer.

In addition to Reolysin, other viruses with the greatest chance of being approved for clinical use include a herpes virus called OncoVEX GM-CSF (BioVex; Woburn, MA, now Amgen, Thousand Oaks, CA) in previously treated patients with Stage III and Stage IV melanoma, a Wyeth vaccinia virus called JX-594 (Jennerex; San Francisco, CA and Transgene; 67405 Illkirch Graffenstaden Cedex, France) in patients with advanced liver cancer, and an adenovirus against solid tumors, including breast and bowel cancer (Institute of Cancer Research/ICR; London, UK).

Not and easy process
The successful development of oncolytic virotherapy is not easy. The example of Onyx-015 (Onyx Pharmaceuticals, South Francisco, CA), also known as CI-1042 (Warner Lambert, now Pfizer),  a recombinant adenovirus that carries a loss-of-function mutation at the E1B locus that selectively replicates in and kill cells that harbor p53 mutations, shows how complicated the development of a therapeutic anti-cancer virus realy is. [6]  Onyx-015, in combination with the standard chemotherapeutic agents cisplatin and fluorouracil (5-FU), was initially developed as a potential treatment to combat various solid tumors including head and neck, gastrointestinal and pancreatic tumors. The drug candidate was extensively tested in a number of clinical trials. The collected data indicated that Onyx-015 was safe and selective for cancer. However, limited therapeutic effect was demonstrated following injection and systemic spread of the virus was not detected. Further development of Onyx-015 was abandoned in early 2003 when Onyx decided to focus its development efforts solely on sorafenib (Nexavar®; Onyx Pharmaceuticals, Inc., and Bayer HealthCare Pharmaceuticals, Inc.) and discontinue its therapeutic virus program.

The rights of Onyx-015 were sold to Shanghai Sunway Biotech, a Chinese pharmaceutical company, which develops, manufactures, and commercializes cancer biotherapeutics. Following the sale of the intelectual property rights, the Chinese State Food and Drug Administration (SFDA) approved H101 (Oncorine®, Recombinant Human Adenovirus Type 5 Injection), an oncolytic adenovirus similar to Onyx-015 (E1B-55K/E3B-deleted), for use in combination with chemotherapy for the treatment of late-stage refractory nasopharyngeal cancer.[7]

Unfortunately, the monotherapy of H101 has demonstrated limited efficacy in a clinical setting. However, treatment strategies in which H101 is combined with existing chemoterapeutic agents are moving toward potential clinical use. One study combined the oncolytic virus H101 with the alkylating agent dacarbazine (DTIC) to treat uveal melanoma cells in vitro. The initial results demonstrated that the combination exerted a synergistic antitumor effect without enhanced toxicity to normal cells via a type of cell cycle block other than the induction of apoptosis.[8]

Mechanism of action of Reolysin
Reolysin [video] has been formulated to replicate specifically in tumor cells that bear an activated RAS pathway. These cells are deficient in their ability to activate a natural anti-viral response as mediated by protein kinase RNA-activated or protein kinase R (PKR) activity, which is present in normal cells but absent in tumor cells with activated RAS pathways (normal cells do not possess activated RAS pathways). PKR, an enzyme that lies dormant until it detects viruses replicating within a cell,  is one of the immune system’s strongest cellular defenses. Once active, PKR is able to phosphorylate the Eukaryotic translation initiation factor 2A or EIF2A, which inhibits cellular mRNA translation and subsequent viral protein synthesis. Through complex mechanisms, active PKR is also able to induce cellular apoptosis which prevents further viral spread. In this process, as tumor cells die, the progeny viral particles spread to neighboring cells, a process which in turn triggers the continuous cycle of infection, replication and cell death.  This cellular cycle of viral oncolysis is involved in “educating ” the immune system to recognize and kill similar tumor cells, blocking nearly all protein production in the cell to prevent virus replication, stopping only when the virus is eradicated by treatment or cell death.

Clinical trials have been conducted with Reolysin alone and in combination with chemotherapy and radiotherapy for various cancers. Overall, the trial drug has shown to be well-tolerated, with patients exhibiting only mild, flu-like symptoms.

Ongoing trials
Earlier this week, patient enrollment in a Phase I clinical trial using reolysin in combination with FOLFIRI, a drug combination of Folinic Acid (leucovorin) with Fluorouracil (5-FU) and Irinotecan, in patients with oxaliplatin refractory, KRAS mutant, colorectal cancer was completed. The trial, which includes 21 patient in an single arm dose escalation study, is designed to determine a maximum tolerated dose and dose-limiting toxicities for the combination of Reolysin and FOLFIRI. Patients included in this study had histologically confirmed cancer of the colon or rectum with KRAS mutation and measurable disease. They had also progressed on or within one hundred and ninety days after the last dose of an oxaliplatin regimen in the metastatic setting, or where intolerant to oxaliplatin.

Sanjay Goel of the Montefiore Medical Center at the Albert Einstein College of Medicine in New York is the principal investigator for the study.

Patient enrollment in another Phase I clinical trial using intravenously-administered reolysin in combination with cyclophosphamide in patients with advanced malignancies was also completed this week. The primary objective of this open label, dose-escalating, non-randomized, 36-patient study is to determine the Minimum Effective Immunomodulatory Dose (MED) of cyclophosphamide necessary to obtain successful immune modulation. The secondary objectives of this trial includes assessing the safety profile of the combination and gathering any evidence of antitumor activity.

Patients eligible for this trial include those who have been diagnosed with advanced or metastatic solid tumors, including pancreatic, lung and ovarian cancers that are refractory to standard therapy, or for which no standard curative therapy exists.

The principal investigators for this study are James Spicer, M.D. of King's College, London, Johann de Bono, M.D. and Kevin Harrington M.D. of the Royal Marsden NHS Foundation Trust and the Institute of Cancer Research, London, and Professor Hardev Pandha of the Royal Surrey County Hospital NHS Trust, Surrey and Mount Alvernia Hospitals.

References:
[1] Epstein MA, Achong BG, Barr YM. Virus particles in clutured lymphoblasts from Burkitt's Lymphoma. Lancet. 1964 Mar 28;1(7335):702-3 [PubMed Abstract]
[2] Parkin DM. The global health burden of infection-associated cancers in the year 2002.Int J Cancer. 2006 Jun 15;118(12):3030-44. [PubMed Abstract]
[3] Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet 2007; 370(9590):890–907. [PubMed Abstract]
[4] Muñoz N, Bosch FX, Castellsagué X, et al. Against which human papillomavirus types shall we vaccinate and screen? The international perspective. International Journal of Cancer 2004; 111(2):278–285. [PubMed Abstract]
[5] Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer. Clinical Science 2006; 110(5):525–541. [PubMed Abstract]
[6] Cohen EE, Rudin CM. ONYX-015. Onyx Pharmaceuticals.Curr Opin Investig Drug. 2001 Dec;2(12):1770-5.[PubMed Abstract]
[7] Lu W, Zheng S, Li XF, Huang JJ, Zheng X, Li Z Intra-tumor injection of H101, a recombinant adenovirus, in combination with chemotherapy in patients with advanced cancers: a pilot phase II clinical trial. World J Gastroenterol. 2004 Dec 15;10(24):3634-8 [PubMed Abstract]
[8] Cun B, Song X, Jia R, Zhao X, Wang H, Ge S, Fan X. Combination of oncolytic adenovirus and dacarbazine attenuates antitumor ability against uveal melanoma cells via cell cycle block. Cancer Biol Ther. 2012 Jan 15;13(2):77-84. doi: 10.4161/cbt.13.2.18436. [PubMed Abstract]

For more information:
- Reolysin: Mechanism of Action
- A systematic approach to improve productivity and increase regulatory compliance for a vaccine (SAFC; Vaccine Manufacturing, Case Study)
- Experience peace of mind in your vaccine manufacturing process (SAFC)
- Oncolytics Biotech and NCIC CTG Sign Agreement for Randomized Phase II Study in Colorectal Cancer, Onco'Zine; May 3, 2012
- REO 017 Meets Primary Endpoint for First Part of U.S. Phase II Pancreatic Cancer Clinical Trial, Onco'Zine; February 14, 2011
- Randomized Phase II Ovarian Cancer Study With Reolysin® To Be Conducted By The Gynecologic Oncology Group (GOG), Onco'Zine; September 2, 2010

Selected Clinical trials:
- NCT01274624 - Study of REOLYSIN® in Combination With FOLFIRI in Patients With Oxaliplatin Refractory/Intolerant KRAS Mutant Colorectal Cancer
- NCT01240538 - Viral Therapy in Treating Young Patients With Relapsed or Refractory Solid Tumors [ADVL1014]
- NCT00861627 - Phase II Study of REOLYSIN® in Combination With Paclitaxel and Carboplatin for Non-Small Cell Lung Cancer With KRAS or EGFR Activation.
- NCT00289016- A Study of OncoVEXGM-CSF in Stage IIIc and Stage IV Malignant Melanoma
- NCT01380600 - Safety Study of Recombinant Vaccinia Virus Administered Intravenously in Patients With Metastatic, Refractory Colorectal Carcinoma
- NCT01394939 - Recombinant Vaccinia Virus Administered Intravenously Followed by Intratumoral Boosts in Patients With Metastatic, Refractory Colorectal Carcinoma

Disclaimer
Reolysin is not licensed for use by the  U.S. Food and Drug Administration (FDA) or European Medicines Agency (EMA) and is only available in clinical trials. The trial drug is currently being manufactured under a commercial supply agreement between Oncolytics Biotecn and SAFC, a Division of Sigma-Aldrich Corporation.

Fig 1: Overview of signal transduction pathways involved in apoptosis.

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