Replication-selective Oncolytic Viruses Research Articles

Oncolytic Virotherapy for Thyroid Cancer: Will it Translate to the Clinic?

Observations that viruses can induce tumor regression date back to the beginning of the 20th century [1] and the use of viruses for cancer treatment has been theorized since then ; however only in the late 1990s advancement in viral manipulation technologies together with a better understanding of the molecular alterations in cancer cells allowed the development of oncolytic viruses (virotherapy). So far, dozen of viruses have been tested for their oncolytic activity. Oncolytic viruses (OVs) have several potential benefits as anticancer therapeutics: tumorselective replication, amplification of the input dose, efficacy independent of resistance to current anticancer therapies. OVs do not contribute to the generation of neoplastic cell clones or treatment resistance [2]. To generate viruses able to selectively replicate in neoplastic cells, different viral strain and several strategies have been exploited [3]. Viral strains displaying a natural oncotropism have also been tested [2]. The clinical trials performed in the last two decades have demonstrated the safety of OVs and substantial antineoplastic effects. The best clinically evaluated replication-selective oncolytic viruses to date are the mutant adenoviruses dl1520, ONYX-015 and H101 (Oncorine; Shanghai Sunway Biotech). These mutants bear a E1B55K gene deletion, enabling the mutants to replicate only in cells with functional inactivation of p53. E1B55K viral gene is required for other important functions and its deletion greatly impairs viral replication [4], explaining the modest activity as single agent reported in clinical trials. However, dl1520 and H101 were shown to enhance the effects of chemotherapeutic drugs and on the base of these results, H101 was licensed in China for the treatment of head and neck cancers in combination with cisplatin and 5-FU [5,6]. The majority of thyroid cancers are well differentiated papillary or follicular carcinomas with a good cure rate and an excellent prognosis. In contrast, anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers with a short survival time. Multimodality therapy (surgery, chemotherapy and radiotherapy) has shown a limited success and although ATC accounts for only 2% of thyroid carcinoma, it contributes up to 50% of thyroid cancer deaths [7]. Not surprisingly, the studies with OVs have been focused to the treatment of ATC. The first studies testing selective replicating adenoviruses were performed by our group using dl1520 mutant alone or in combination with paclitaxlel, doxorubicin [8] and radiotherapy [9]. Lovastatin, a cholesterol-lowering drug also acting as an inhibitor of p21 ras activity was used by us in conjunction with dl1520 to improve viral cytotoxicity. The combination significantly reduced the growth of ATC xenografts [10]. It is worth noting that p53 inactivation frequency in ATCs is about 50%, with a strong limitation of the therapeutic range for dl1520. Moreover, the studies were conducted in a variety of long established ATC cells lines, some of them successively identified as derived from other tumors. Despite these limitations, the studies with dl1520 have shown that OVs hold a potential benefit for the treatment of ATC. Abbosh et al. have used an oncolytic adenovirus selectively replicating in cells with an active Wnt/β-catenin pathway showing an oncolytic activity and a prolonged survival of animals bearing ATC xenografts [11], confirming the efficacy of the approach. Due to the attenuated replication potential and the limited fraction of ATCs with a nonfunctional p53 pathway, we evaluated dl922–947, a more effective second generation oncolytic adenovirus. dl922–947 bears a deletion of 24bp in E1A-Conserved Region 2 (CR2) [12].

Antitumor effects of telomerase-specific replication-selective oncolytic viruses for adenoid cystic carcinoma cell lines

We evaluated the antitumor effect of a telomerase-specific replication-selective adenovirus (Telomelysin, OBP-301) for adenoid cystic carcinoma (ACC) in vitro and in vivo. Adenovirus E1 gene expression was controlled by human telomerase reverse transcription (hTERT). Infection of ACC cells by OBP-301 induced high E1A mRNA expression and subsequent oncolytic cell death in a dose-dependent manner. Using OBP-401 (TelomeScan), a genetically engineered adenovirus that carries the GFP gene under the control of the cytomegalovirus (CMV) promoter at the deleted E3 region of OBP-301, ACC cells expressed bright GFP fluorescence as early as 12 h after OBP-401 infection. The fluorescence intensity gradually increased in a time-dependent manner, followed by rapid cell death due to the cytopathic effect of OBP-401, as evidenced by the floating, highly light-refractive cells using phase-contrast microscopy. Effects of intratumorally injected OBP-401 against established Acc2 xenograft tumors were seen in BALB/c nu/nu mice. The levels of GFP expression following ex vivo infection of OBP-401 may be of value as a positive predictive marker for the outcome of telomerase-specific virotherapy. Our data clearly indicated that telomerase-specific oncolytic adenoviruses have significant therapeutic potential against human ACC in vitro and in vivo. These results suggest that treatment with OBP-301 and OBP-401 may improve the quality of life of oral cancer patients.

Adenovirus-Mediated Sensitization to the Cytotoxic Drugs Docetaxel and Mitoxantrone Is Dependent on Regulatory Domains in the E1ACR1 Gene-Region

Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922–947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922–947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics.

Enhanced antitumor efficacy of telomerase-specific oncolytic adenovirus with valproic acid against human cancer cells

Replication-selective oncolytic viruses are being developed for human cancer therapy. We previously developed an attenuated adenovirus (OBP-301, Telomelysin), in which the human telomerase reverse transcriptase promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site. OBP-301 can replicate in, and causes selective lysis of, human cancer cells. Valproic acid (VPA), which is an effective antiepileptic drug, is known to inhibit the histone deacetylase activities. We determined whether the antitumor effect of OBP-301 could be enhanced by VPA in human lung cancer cells. In an in vitro cell viability assay, OBP-301 infection killed four human lung cancer cell lines, H1299, H1299-R5 (a subline of H1299 with a low level of the coxsackievirus and adenovirus receptor (CAR) expression), H460, and A549, more efficiently in the presence of VPA than in its absence. VPA treatment increased CAR expression in all the four lung cancer cells. Consistent with their CAR upregulation, the infection efficiency of adenoviruses in the presence of VPA was significantly higher than that in its absence. The molecular mechanism of this combined effect could be explained by an increase in adenovirus infectivity via VPA-mediated upregulation of CAR. These results suggest that treatment with OBP-301 in combination with VPA is a promising strategy for human lung cancer.

Abstract 4973: Telomerase-specific replication-selective oncolytic viruses for adenoid cystic carcinoma cell lines

Abstract There is no effective treatment of adenoid cystic carcinoma (ACC) in oral region, which low sensitivity of chemotherapy and radiation therapy. We previously showed that telomerase-specific replication-competent adenovirus (Telomelysin, OBP-301), in which the human telomerase reverse transcriptase (hTERT) promoter controls the adenoviral E1 gene expression, induces a selective antitumor effect in oral squamous cell carcinoma (OSCC) cells in vitro and in vivo orthotopic graft model. We conducted to evaluate the effectiveness of OBP301 against ACC. First, we could confirm that the expression of hTERT in two types of ACC cell lines (ACC2, ACCM) by Western blotting. Then, we examined the antitumor effect of OBP301 in human ACC cells in vitro. OBP-301 infection induced cell death in human ACC cells in a dose-dependent manner. We also examined the expression levels of coxsackievirus and adenovirus receptor (CAR) on the cell surface of each type of cell by flow cytometric analysis. Apparent amounts of CAR expression were detected on human ACC cells. Relative hTERT mRNA expression in ACC was analyzed by real-time RT-PCR, which showed high expression time dependent. In addition, OBP-401 is a genetically engineered adenovirus that expresses GFP by inserting the GFP gene under the control of the cytomegalovirus promoter at the deleted E3 region of OBP-301. Human ACC cells expressed bright GFP fluorescence as early as 12 h after OBP-401 infection. The fluorescence intensity gradually increased in a dose-dependent manner, followed by rapid cell death due to the cytopathic effect of OBP-401, as evidenced by floating, highly light-refractile cells under phase-contrast photomicrographs. In conclusion, our data clearly indicate that telomerase-specific oncolytic adenoviruses have a significant therapeutic potential against human ACC in vitro. These results suggested that treatment of OBP-301 and OBP-401 are possible to improvement of QOL of oral cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4973. doi:1538-7445.AM2012-4973

Rational strain selection and engineering creates a broad-spectrum, systemically effective oncolytic poxvirus, JX-963

Replication-selective oncolytic viruses (virotherapeutics) are being developed as novel cancer therapies with unique mechanisms of action, but limitations in i.v. delivery to tumors and systemic efficacy have highlighted the need for improved agents for this therapeutic class to realize its potential. Here we describe the rational, stepwise design and evaluation of a systemically effective virotherapeutic (JX-963). We first identified a highly potent poxvirus strain that also trafficked efficiently to human tumors after i.v. administration. This strain was then engineered to target cancer cells with activation of the transcription factor E2F and the EGFR pathway by deletion of the thymidine kinase and vaccinia growth factor genes. For induction of tumor-specific cytotoxic T lymphocytes, we further engineered the virus to express human GM-CSF. JX-963 was more potent than the previously used virotherapeutic Onyx-015 adenovirus and as potent as wild-type vaccinia in all cancer cell lines tested. Significant cancer selectivity of JX-963 was demonstrated in vitro in human tumor cell lines, in vivo in tumor-bearing rabbits, and in primary human surgical samples ex vivo. Intravenous administration led to systemic efficacy against both primary carcinomas and widespread organ-based metastases in immunocompetent mice and rabbits. JX-963 therefore holds promise as a rationally designed, targeted virotherapeutic for the systemic treatment of cancer in humans and warrants clinical testing.

Viruses with deletions in antiapoptotic genes as potential oncolytic agents

Replication-selective oncolytic viruses have emerged as a new treatment platform for cancers. However, selectivity and potency need to be improved before virotherapy can become a standard treatment modality. In addition, mechanisms that can be incorporated to enable targeting a broad range of cancer types are highly desirable. Cancer cells are well known to have multiple blocks in apoptosis pathways. On the other hand, viruses have evolved to express numerous antiapoptotic genes to antagonize apoptosis induced upon infection. Viruses with deletions in antiapoptotic genes can therefore be complemented by antiapoptotic genetic changes in cancer cells for efficient replication and oncolysis. In this review, we summarize the recent development of this concept, the potential obstacles, and future directions for optimization.

Erratum: Replication-selective oncolytic viruses in the treatment of cancer

Correction to: Cancer Gene Therapy (2005) 12, 141–161. doi:10.1038/sj.cgt.7700771 This article was incorrectly published as an Original Article; it should have been published as a Review Article.

Replication-selective oncolytic viruses in the treatment of cancer.

In the search for novel strategies, oncolytic virotherapy has recently emerged as a viable approach to specifically kill tumor cells. Unlike conventional gene therapy, it uses replication competent viruses that are able to spread through tumor tissue by virtue of viral replication and concomitant cell lysis. Recent advances in molecular biology have allowed the design of several genetically modified viruses, such as adenovirus and herpes simplex virus that specifically replicate in, and kill tumor cells. On the other hand, viruses with intrinsic oncolytic capacity are also being evaluated for therapeutic purposes. In this review, an overview is given of the general mechanisms and genetic modifications by which these viruses achieve tumor cell-specific replication and antitumor efficacy. However, although generally the oncolytic efficacy of these approaches has been demonstrated in preclinical studies the therapeutic efficacy in clinical trails is still not optimal. Therefore, strategies are evaluated that could further enhance the oncolytic potential of conditionally replicating viruses. In this respect, the use of tumor-selective viruses in conjunction with other standard therapies seems most promising. However, still several hurdles regarding clinical limitations and safety issues should be overcome before this mode of therapy can become of clinical relevance.

445. CG5757, an Oncolytic Adenovirus for the Treatment of Retinoblstoma (Rb) Pathway-Defective and Telomerase-Positive Cancers

Replication-selective oncolytic viruses hold promise for the treatment of cancer. Among this novel group of therapeutics are oncolytic adenoviruses engineered with tumor-specific transcriptional response elements (TRE) controlling essential genes. These vectors replicate selectively in cancer cells, leading to expression of toxic viral products and oncolysis mediated by viral replication. We and others have compared different transcriptional control strategies by placing one or more tumor-specific TREs upstream of different viral genes including E1A, E1B and E4. Relocation of the viral packaging signal was also investigated. One such oncolytic virus, CG5757, was generated by replacing the E1A and E1B endogenous promoters with promoters derived from the human E2F-1 and telomerase reverse transcriptase (hTERT) genes, respectively. The E2F-1 promoter is activated in Rb-defective tumor types, a pathway mutated in approximately 85% of all cancers. Likewise, telomerase is aberrantly expressed in over 90% of tumors. CG5757 also has a deletion in the coding region of the E1B 19k gene, a Bcl2-like viral antiapoptotic protein, to increase vector cytotoxicity. CG5757 shows strong tumor selectivity. In vitro, expression of E1A and E1B genes was highly restricted to Rb-defective and hTERT-positive cancer cells, including Hep3B (hepatocellular carcinoma), LoVo (colorectal carcinoma), A549 (lung cancer), Panc-1 (pancreatic cancer), 253J B-V (bladder cancer), and Hela (cervical cancer). In normal cells, including a lung fibroblast cell line (WI-38) and several other human primary cell lines (HRE, BSMC, PrEC, HMEC and HMVEC-L), no E1 expression could be detected from infection with CG5757. The transcriptional control of E1 gene expression also correlated with selective viral replication in target cells. CG5757 replicates similarly to wild-type virus in tumor cells, but its replication is, on average, 1,000-times less efficient in normal cells. In a viral cytotoxicity assay, CG5757 destroys tumor cells 100- to 10,000-times more efficiently than normal cells. Comparisons of the cytoxicity of CG5757 in tumor cells versus normal cells (normalized for transduction efficiency with wild-type adenovirus 5) yielded high selectivity indices, some of which were greater than 1000. In vivo, strong antitumor activity was seen using CG5757 in NCR nude mice with subcutaneous lung cancer (A549) and bladder transitional cell carcinoma (253J B-V) xenografts. With respect to the 253J B-V model, four weeks after treatment the average tumor volume in animals treated with four consecutive daily intratumoral injections of CG5757 (4×108 particles/mm3 of tumor) decreased to 72% of baseline while the control group had an increase to 944% of baseline. Furthermore, 50% of treated animals had complete regression of the 253J B-V tumor xenografts. The potential therapeutic efficacy of such dual promoter controlled oncolytic adenoviruses in cancers that are Rb-defective and hTERT-positive has been demonstrated.

Genetic Therapies Inc.: tight-lipped for now

More than a decade ago, tiny biotechnology firm Genetic Therapy, Inc. (GTI), caught the attention of Swiss pharma giant, Sandoz AG. GTI cofounder, Dr. W. French Anderson, had claimed that injecting healthy genes into patients to replace damaged or defective ones could revolutionize the treatment of diseases like cystic fibrosis, hemophilia, and perhaps even cancer. Sandoz merged with Ciby-Geigy to create Novartis in 1996, and soon forged a partnership with GTI and later with SyStemix (Palo Alto, CA), which was pursuing a similar idea. Novartis eventually absorbed both partners in transactions valued at $834 million. But after several gene therapy projects failed in early stages, Novartis’ enthusiasm was tempered, and they reorganized the entire gene therapy unit in the late ‘90s. The company now focuses on oncolytic viruses to treat metastatic cancer. “If they are not leading the way, they are certainly close,” thinks Anderson, who now directs the Gene Therapy Laboratories at the University of Southern California School of Medicine and who consults for GTI. Back in 1990, Anderson conducted an early human gene transfer experiment, in an atmosphere of unbridled enthusiasm. The recipient, four-year-old Ashanti DaSilva, suffered from ADA deficiency. ADA patients are prone to infection, suffering from Severe Combined Immunodeficiency (SCID) or “bubble-boy” disease. Unfortunately, the investigators were unable to demonstrate convincing clinical benefit from the therapy. Indeed, many subsequent attempts at gene therapy failed. GTI's showcase gene therapy effort to treat glioblastoma, a deadly brain tumor, was no exception. GTI's strategy was simple. Mouse cells producing replication-deficient retrovirus carrying the thymidine kinase gene from Herpes Simplex Virus (HSV) were injected into the brain after the tumor mass had been surgically removed. The cells were expected to pump out virus that could infect any remaining cancer cells, rendering them sensitive to the antiviral drug ganciclovir—thereby killing them. Although the initial results were encouraging, when preliminary results of a large, international trial started to roll in, a different story emerged. The survival curves of patients who received the gene therapy were no different from the control group, and the project was cancelled. In a twist of fate, however, the name survived. “A suddenly very successful new leukemia drug needed a name quickly and company officials remembered “gleevec,” the name we had given the glioblastoma vector program,” recalls Anderson. Meanwhile, GTI's early-stage human tests of therapies for cystic fibrosis, breast cancer, and several rare genetic diseases were running into problems as well. One by one, these efforts were halted. The gene therapy program was scaled down, and operations of GTI and SyStemix were combined. “Novartis consolidated all gene therapy research at its facility in Gaithersburg, MD. Gene therapy research is now focused on the development of oncolytic adenoviruses for the treatment of metastatic cancer,” explains Sheldon Jones, vice president of communications at Novartis, and SyStemix works on stem cell technology. Despite the failures, Novartis remains committed to the field. The company just completed a $20 million (US) state-of-the-art facility in Gaithersburg—the Novartis Center of Excellence for gene therapy research. “The program is not as large as it was, but it is still a significant effort,” says Anderson. GTI seems bullish on the clinical potential of their oncolytic viruses. One GTI virus, OVA001, makes use of the E2F-1 promoter to drive the essential E1A gene, restricting viral replication to Rb-defective tumor cells—present in about 85 percent of tumors. Trying to push selectivity even further, GTI researchers have put E4 genes under the control of the human telomerase reverse transcriptase (hTERT) promoter—also reported to be selectively active in tumor cells— in combination with E2F-1 controlled E1A. A third vector uses E2F-1 to drive E1A, and incorporates the gene for macrophage colony-stimulating factor (GM-CSF) with the aim of inducing local inflammatory and systemic anti-tumor responses. The company claims that the vector demonstrates enhanced anti-tumor activity and a higher incidence of complete tumor regression compared to a vector that lacks the cytokine transgene when tested in a xenograft model, although none of these results have appeared in the peer-reviewed literature. Indeed, the company is tight-lipped when prodded for details about the clinical potential of its replication-selective oncolytic viruses. This is perhaps not surprising given the field's early reputation for overstating the technology's potential. “Currently, Genetic Therapy, Inc. does not have any active clinical trials using oncolytic adenoviruses. Several early developmental candidates have been identified, and they are in the preclinical testing phase. We anticipate initiation of a phase I clinical trial in 2004,” stated Jones. Stay tuned.

Virotherapy for cancer: Current status, hurdles, and future directions

This special issue on cancer virotherapy will review the emerging field of tumor-targeted viruses. The clinical effectiveness of any cancer treatment depends on both its antitumoral potency and the therapeutic index between cancerous and normal cells. Most current nonsurgical therapies for solid tumors fail on one or both scores, and it is unlikely that changes in dose or frequency, or even the combination of standard cytotoxic chemo- or radiotherapies will satisfy both of these standards. Metastatic cancer is currently treated with agents from four major “platforms”: (1) chemotherapy (e.g., cisplatin, taxanes, etc.), (2) cytokines (e.g., interleukin-2, interferon-alpha), (3) monoclonal antibodies (e.g., rituximab, Herceptin), and (4) targeted small molecules (e.g., Gleeves, Imessa). Novel therapeutic approaches are required that offer greater potency and selectivity and that act by mechanisms that are not subject to cross-resistance when combined with standard therapies. Replication-selective oncolytic viruses fit these criteria. Replication in tumor tissue allows for a massive amplification of the input “dose” (e.g., 1000- to 10,000-fold increases) at the tumor site. Cancer cells are turned into in situ factories producing thousands of copies of the therapeutic agent; these viruses will then kill the “producer” cell and be released to attack neighboring cancer cells. In contrast, replication in normal tissues is abrogated leading to efficient clearance and reduced toxicity. Mechanisms leading to cancer selectivity include the (1) use of inherently selective RNA viruses, some of which take advantage of tumor cell resistance to interferon (e.g., measles virus vaccine strain MV-SPUD, reovirus, Newcastle Disease virus, NDV, vesicular stomatitis virus); (2) deletion of viral genes that are necessary for replication in normal cells but expendable in cancer cells (vaccinia virus, adenovirus, herpes simplex virus, HSV, poliovirus); and (3) tissue-specific transcriptional regulatory elements driving viral genes that are critical for replication (adenovirus, HSV).

The oncolytic virotherapy treatment platform for cancer: unique biological and biosafety points to consider.

The field of replication-selective oncolytic viruses (virotherapy) has exploded over the last 10 years. As with many novel therapeutic approaches, initial overexuberance has been tempered by clinical trial results with first-generation agents. Although a number of significant hurdles to this approach have now been identified, novel solutions have been proposed and improvements are being made at a furious rate. This article seeks to initiate a discussion of these hurdles, approaches to overcome them, and unique safety and regulatory issues to consider.

Gene delivery from replication-selective viruses: arming guided missiles in the war against cancer.

Gene therapy aims to deliver therapeutic genetic material in a safe and efficient manner to a target tissue, where it can accumulate to levels that afford maximal patient benefit. Replication-defective viral vectors have been the workhorse of gene therapy for cancer and other conditions. These vectors allow the efficient delivery of a variety of transgenes to target tissues and have demonstrated clear therapeutic benefit and safety in a variety of preclinical neoplastic animal models. Unfortunately, the tremendous promise of studies using replication-defective viruses in preclinical models has not been translated into similar patient benefits in the clinical setting (1–3). This shortfall may be due in large part to the one-dimensional nature of these approaches, asking for a successful therapeutic outcome against a highly complex biological target like a human tumor through the activity of a single gene. Therapeutic gene delivery needs to develop into a multidimensional system if it is to be successful in treating human cancers. A natural evolution of gene therapy is its incorporation into replication-selective oncolytic viruses, combining the antitumor properties of the viral infection with the action of the therapeutic proteins.