Rapamycin enhances the activity of oncolytic herpes simplex virus against tumor cells that are resistant to virus replication

Oncolytic herpes simplex virus (HSV) is currently in phase III clinical trials for development as a novel therapeutic agent against a broad range of human tumors. Although results have been promising, clinical outcome is likely to be compromised by intrinsic and acquired resistance to HSV replication, leading us to test agents that may overcome this obstacle. We found that, despite showing no effect on HSV replication in tumor cells fully permissive to the virus growth, the mTOR inhibitor rapamycin markedly increased the yield and dissemination of oncolytic HSVs in semipermissive tumor cells. Similar results were obtained in tumor-bearing mice. Co-administration of rapamycin with an HSV-derived oncolytic virus either blocked or reversed the growth of tumor xenografts established from semipermissive human tumor cells, while use of either agent alone produced only transient inhibitory effect. Together, our results suggest that rapamycin could be used to potentiate the activity of oncolytic HSVs against difficult-to-treat human tumors or perhaps to prevent the emergence of resistant tumor cells during virotherapy.     

An armed oncolytic herpes simplex virus expressing thrombospondin‐1 has an enhanced in vivo antitumor effect against human gastric cancer

Advanced gastric cancer is a common disease, but the conventional treatments are unsatisfactory because of the high recurrence rate. One of the promising new therapies is oncolytic virotherapy, using oncolytic herpes simplex viruses (HSVs). Thrombospondin‐1 (TSP‐1) suppresses tumor progression via multiple mechanisms including antiangiogenesis. Our approach to enhance the effects of oncolytic HSVs is to generate an armed oncolytic HSV that combines the direct viral oncolysis with TSP‐1‐mediated function for gastric cancer treatment. Using the bacterial artificial chromosome (BAC) system, a 3rd generation oncolytic HSV (T‐TSP‐1) expressing human TSP‐1 was constructed for human gastric cancer treatment. The enhanced efficacy of T‐TSP‐1 was determined in both human gastric cancer cell lines in vitro and subcutaneous tumor xenografts of human gastric cancer cells in vivo. In addition, we examined the apoptotic effect of T‐TSP‐1 in vitro, and the antiangiogenic effect of T‐TSP‐1 in vivo compared with a non‐armed 3rd generation oncolytic HSV, T‐01. No apparent apoptotic induction by T‐TSP‐1 was observed for human gastric cancer cell lines TMK‐1 cells but for MKN1 cells in vitro. Arming the viruses with TSP‐1 slightly inhibited their replication in some gastric cancer cell lines, but the viral cytotoxicity was not attenuated. In addition, T‐TSP‐1 exhibited enhanced therapeutic efficacy and inhibition of angiogenesis compared with T‐01 in vivo. In this study, we established a novel armed oncolytic HSV, T‐TSP‐1, which enhanced the antitumor efficacy by providing a combination of direct viral oncolysis with antiangiogenesis. Arming oncolytic HSVs may be a useful therapeutic strategy for gastric cancer therapy.     

Multiple strategies to improve the therapeutic efficacy of oncolytic herpes simplex virus in the treatment of glioblastoma

Oncolytic viruses have attracted widespread attention as biological anticancer agents that can selectively kill tumor cells without affecting normal cells. Although progress has been made in therapeutic strategies, the prognosis of patients with glioblastoma (GBM) remains poor and no ideal treatment approach has been developed. Recently, oncolytic herpes simplex virus (oHSV) has been considered a promising novel treatment approach for GBM. However, the therapeutic efficacy of oHSV in GBM, with its intricate pathophysiology, remains unsatisfactory due to several obstacles, such as limited replication and attenuated potency of oHSV owing to deletions or mutations in virulence genes, and ineffective delivery of the therapeutic virus. Multiple strategies have attempted to identify the optimal strategy for the successful clinical application of oHSV. Several preclinical trials have demonstrated that engineering novel oHSVs, developing combination therapies and improving methods for delivering oHSV to tumor cells seem to hold promise for improving the efficacy of this virotherapy.     

Oncolytic virus therapy: A new era of cancer treatment at dawn

Oncolytic virus therapy is perhaps the next major breakthrough in cancer treatment following the success in immunotherapy using immune checkpoint inhibitors. Oncolytic viruses are defined as genetically engineered or naturally occurring viruses that selectively replicate in and kill cancer cells without harming the normal tissues. T‐Vec (talimogene laherparepvec), a second‐generation oncolytic herpes simplex virus type 1 (HSV‐1) armed with GM‐CSF, was recently approved as the first oncolytic virus drug in the USA and Europe. The phase III trial proved that local intralesional injections with T‐Vec in advanced malignant melanoma patients can not only suppress the growth of injected tumors but also act systemically and prolong overall survival. Other oncolytic viruses that are closing in on drug approval in North America and Europe include vaccinia virus JX‐594 (pexastimogene devacirepvec) for hepatocellular carcinoma, GM‐CSF‐expressing adenovirus CG0070 for bladder cancer, and Reolysin (pelareorep), a wild‐type variant of reovirus, for head and neck cancer. In Japan, a phase II clinical trial of G47?, a third‐generation oncolytic HSV‐1, is ongoing in glioblastoma patients. G47? was recently designated as a “Sakigake” breakthrough therapy drug in Japan. This new system by the Japanese government should provide G47? with priority reviews and a fast‐track drug approval by the regulatory authorities. Whereas numerous oncolytic viruses have been subjected to clinical trials, the common feature that is expected to play a major role in prolonging the survival of cancer patients is an induction of specific antitumor immunity in the course of tumor‐specific viral replication. It appears that it will not be long before oncolytic virus therapy becomes a standard therapeutic option for all cancer patients.     

Therapeutic targeting of chemoresistant and recurrent glioblastoma stem cells with a proapoptotic variant of oncolytic herpes simplex virus

Temozolomide (TMZ) chemotherapy, in combination with maximal safe resection and radiotherapy, is the current standard of care for patients with glioblastoma (GBM). Despite this multimodal approach, GBM inevitably relapses primarily due to resistance to chemo‐radiotherapy, and effective treatment is not available for recurrent disease. In this study we identified TMZ resistant patient‐derived primary and previously treated recurrent GBM stem cells (GSC), and investigated the therapeutic activity of a pro‐apoptotic variant of oHSV (oHSV‐TRAIL) in vitro and in vivo. We show that oHSV‐TRAIL modulates cell survival and MAP Kinase proliferation signaling pathways as well as DNA damage response pathways in both primary and recurrent TMZ‐resistant GSC. Utilizing real time in vivo imaging and correlative immunohistochemistry, we show that oHSV‐TRAIL potently inhibits tumor growth and extends survival of mice bearing TMZ‐insensitive recurrent intracerebral GSC tumors via robust and selective induction of apoptosis‐mediated death in tumor cells, resulting in cures in 40% of the treated mice. In comparison, the anti‐tumor effects in a primary chemoresistant GSC GBM model exhibiting a highly invasive phenotype were significant but less prominent. This work thus demonstrates the ability of oHSV‐TRAIL to overcome the therapeutic resistance and recurrence of GBM, and provides a basis for its testing in a GBM clinical trial.     

Oncolytic viral therapy for breast cancer with herpes simplex virus type 1 mutant HF 10

BACKGROUND AND OBJECTIVES: Many genetically engineered viruses have been evaluated for their potential as therapeutic agents in the treatment of malignant tumors. We applied a spontaneously generated, highly attenuated herpes simplex virus (HSV) type-1 clone, HF10, to the treatment of breast cancer. In this study, we investigated the ability of HF10 to infect and lyse human and murine breast cancer cells in vitro and tested its efficacy in an immuno-competent animal model of breast cancer. METHODS: To assess the therapeutic efficacy of HF10 against subcutaneous tumors in vivo, mouse breast cancer cells were injected into the backs of mice, which were then treated with HF10. Tumor volume and survival rate were used as measures of the antitumor effect in the in vivo experiments. In vitro viral cytotoxity assays and replication assays were also performed in human breast cancer cell lines. RESULTS: In the in vivo study, tumor growth was suppressed and long-term survival rates were prolonged. HF10 was effective in producing cytolytic effects in vitro at various multiplicities of infection (MOI) in all cell lines tested. CONCLUSIONS: HF10 demonstrated antitumor effects in our animal model. The viral growth and oncolytic effect of HF10 in the human breast cancer cell line suggest that HF10 is potentially effective in the clinical treatment of human cancer. These results indicate that replication-competent HSV-1 mutants hold significant promise as cancer therapeutic agents.     

Combination therapy of oncolytic herpes simplex virus HF10 and bevacizumab against experimental model of human breast carcinoma xenograft

Breast cancer is one of the most common and feared cancers faced by women. The prognosis of patients with advanced or recurrent breast cancer remains poor despite refinements in multimodality therapies involving chemotherapeutic and hormonal agents. Multimodal therapy with more specific and effective strategy is urgently needed. The oncolytic herpes simplex virus (HSV) has potential to become a new effective treatment option because of its broad host range and tumor selective viral distribution. Bevacizumab is a monoclonal antibody against VEGFA, which inhibits angiogenesis and therefore tumor growth. Our approach to enhance the antitumor effect of the oncolytic HSV is to combine oncolytic HSV HF10 and bevacizumab in the treatment of breast cancer. Our results showed that bevacizumab enhanced viral distribution as well as tumor hypoxia and expanded the population of apoptotic cells and therefore induced a synergistic antitumor effect. HF10 is expected to be a promising agent in combination with bevacizumab in the anticancer treatment.     

Oncolytic virotherapy with SOCS3 enhances viral replicative potency and oncolysis for gastric cancer

Oncolytic virotherapy is an encouraging treatment using herpes simplex virus (HSV) for gastric cancer patients. To treat gastric cancer, we generated and evaluated the efficacy of an attractive type of oncolytic HSV expressing the suppressor of cytokine signaling 3 (SOCS3). We constructed a third-generation type of oncolytic HSV (T-SOCS3) arming with SOCS3 by a bacterial artificial chromosome (BAC) system. We examined the viral replicative intensification and oncolysis of T-SOCS3 for human gastric cancer cell lines ex vivo. T-SOCS3 enhanced its replication and potentiated its cell-killing effect for MKN1 human gastric cancer cell lines, which are resistant to a non-armed third-generation type of oncolytic HSV (T-01) ex vivo. T-SOCS3 also induced the destruction within human gastric cancer specimens. Armed oncolytic HSVs expressing SOCS3 may be an efficacious therapeutic agent for gastric cancer treatment.     

溶瘤麻疹病毒抗瘤治疗“瓶颈”与破解对策

在肿瘤基因治疗领域,具有自我复制、能选择性杀伤肿瘤的溶瘤病毒已成为抗肿瘤治疗的有效武器之一。其中溶瘤麻疹病毒因其拥有极好的安全记录和优秀的抗瘤效果目前已经进入多项临床试验阶段。但是仍存在一些因素会影响溶瘤麻疹病毒成功治疗肿瘤,研究者们正在努力寻找对策以期破解这些瓶颈,使溶瘤麻疹病毒更好的发挥溶瘤作用。     

Human papillomavirus, cytomegalovirus and herpes simplex virus infections for cervical cancer in Taiwan

Previously, we had reviewed 43 cases of invasive cancers, adenosquamous cell carcinoma and adenocarcinoma for HPV type infections. With the same cases we extended the investigation to cytomegalovirus (CMV) and herpes simplex virus (HSV) infections. Results show that the prevalence of CMV and HSV infections from these cases of cervical carcinoma was 67 and 76%, respectively, by polymerase chain reaction. The results of the analysis of the association of HPV, CMV and HSV with various clinical characteristics of cervical cancer patients indicated that the correlation between HSV infections and clinical stages of squamous carcinoma was marginally significant (P=0.068). HSV infections seemed to have a higher association with cell keratinization pattern as compared with the other two viral infections.     

Sensitivity of human pleural mesothelioma to oncolytic measles virus depends on defects of the type I interferon response

Attenuated measles virus (MV) is currently being evaluated as an oncolytic virus in clinical trials and could represent a new therapeutic approach for malignant pleural mesothelioma (MPM). Herein, we screened the sensitivity to MV infection and replication of twenty-two human MPM cell lines and some healthy primary cells. We show that MV replicates in fifteen of the twenty-two MPM cell lines. Despite overexpression of CD46 by a majority of MPM cell lines compared to healthy cells, we found that the sensitivity to MV replication did not correlate with this overexpression. We then evaluated the antiviral type I interferon (IFN) responses of MPM cell lines and healthy cells. We found that healthy cells and the seven insensitive MPM cell lines developed a type I IFN response in presence of the virus, thereby inhibiting replication. In contrast, eleven of the fifteen sensitive MPM cell lines were unable to develop a complete type I IFN response in presence of MV. Finally, we show that addition of type I IFN onto MV sensitive tumor cell lines inhibits replication. These results demonstrate that defects in type I IFN response are frequent in MPM and that MV takes advantage of these defects to exert oncolytic activity.     

溶瘤病毒治疗恶性胶质瘤的临床研究进展

恶性胶质瘤是成人原发中枢神经系统最常见的恶性肿瘤,其生物学行为复杂,恶性程度高。目前治疗手段有限,即使通过传统治疗手段,即最大限度安全手术联合放化疗,患者的临床结局仍不尽人意,平均中位生存期仅15个月左右。近年来各种分子靶向及免疫治疗策略的探索不断涌现并取得了一定进展,其中溶瘤病毒(oncolytic viruses,OVs)以其独特的靶向性、安全性及抗肿瘤作用引起了广大研究者的关注和研究。OVs疗法已在多种恶性肿瘤的基础及临床研究中展示出可观的疗效,其在恶性胶质瘤方面的临床探索也在不断深入中,本文主要就OVs治疗恶性胶质瘤的临床研究进展进行综述。     

Considerations for intravascular administration of oncolytic herpes virus for the treatment of multiple liver metastases

Purpose  Oncolytic viral therapy is a newly developed modality for treating tumors. Many clinical trials using oncolytic virus have been performed worldwide, but most of them have used local injection in the tumor. Determination of the effect and safety of intravascular virus injection instead of local injection is necessary for clinical use against multiple liver metastases and systemic metastases. Methods  To evaluate the efficacy and safety of intravascular virus therapy, mice bearing multiple liver metastases were treated by intraportal or intravenous administration of the herpes simplex virus type 1 (HSV-1) mutant, hrR3. Mice treated with hrR3 were killed and organs were harvested for lacZ staining and PCR analysis. Inactivation of oncolytic virus in bloodstream was assessed by neutralization assay in vitro. Infectious activity of hrR3 with vascular endothelial cells was evaluated by replication and cytotoxicity assay. Results  The survival rate of animals treated by hrR3 was significantly improved compared with the untreated group. lacZ staining and PCR analysis demonstrated detectable virus in the tumor but not in normal tissue or other organs except for the adrenal glands. We also showed that vascular endothelial cells allowed virus replication, while normal hepatocytes did not, and human anti-HSV antibody revealed attenuation of the infectious activity of hrR3. Conclusions  Intravascular delivery of hrR3 is effective in treating multiple liver metastases, however, several points must be kept in mind at the time of human clinical trials using intravascular virus administration in order to avoid critical side effects.     

Tumor vascularization is critical for oncolytic vaccinia virus treatment of peritoneal carcinomatosis

Peritoneal carcinomatosis (PC) represents a significant clinical challenge for which there are few treatment options. Oncolytic viruses are ideal candidates for PC treatment because of their high tumor specificity, excellent safety profile and suitability for peritoneal delivery. Here, we described the use of vvDD‐SR‐RFP, a recombinant vaccinia virus, in xenograft and syngeneic models of colorectal PC. Colorectal cancer cell lines were highly susceptible to vvDD‐SR‐RFP replication and cytotoxicity. Intraperitoneal delivery of vvDD‐SR‐RFP on Day 12 to mice with colorectal carcinomatosis significantly improved survival whereas survival was not improved following virus treatment on Day 8, when tumors were smaller. Immunohistochemistry revealed early tumors had a poorly distributed network of blood vessels and lower proliferation index compared to later tumors. Virus infection was also restricted to tumor rims following Day 8 treatment, whereas it was disseminated in tumors treated on Day 12. Additionally, direct infection of tumor endothelium was observed and virus infection correlated with a loss of endothelial staining and induction of cell death. Our results demonstrate that tumor vasculature has a critical role in virus delivery and tumor response. This will have significant implications in the clinical setting, both in understanding timing of therapies and in designing combination treatment strategies.     

A cultured human oligodendroglioma cell line and herpes simplex virus-infected cells share antigenic determinants

Cell cultures derived from 60 different human brain tumors were screened for the presence of HSV infected cell antigens by indirect immunofluorescence using a polyclonal rabbit antiserum reacting with herpes simplex virus (HSV), 3 monoclonal antibodies recognising different HSV specified proteins, and one monoclonal antibody TI81 reacting with a DNA binding protein present in HSV-infected cells. Only one tumor (IN/157), derived from an oligodendroglioma, stained with the polyclonal antiserum. TI81 but none of the other monoclonal antibodies used also specifically reacted with IN/157 cells. High levels of the TI81-defined protein were detected using immunoblotting in HSV-1 infected BHK/21 cells but not in IN/157 cells. T181 may react with either an epitope shared between two different molecules in HSV I infected and IN/157 cells or a cell-specified polypeptide that is upregulated after HSV-1 infection.     

Thalidomide suppressed the growth of 4T1 cells into solid tumors in Balb/c mice in a combination therapy with the oncolytic fusogenic HSV-1 OncdSyn

Purpose  The anti-tumor properties of thalidomide or in combination with an oncolytic herpes virus (OncdSyn) was investigated in a mouse model of human breast cancer. Methods  To determine if thalidomide could act alone, 4T1 cells were injected into Balb/c mice. Tumors were sized, and the mice were fed chow or chow-containing thalidomide. After 4 days the tumor volumes were compared. To determine if thalidomide could act with the virus, tumors of mice were injected with phosphate buffered saline (PBS), or fed thalidomide with injections of PBS, or fed thalidomide with injections of OncdSyn, or received injections of OncdSyn. Results  Thalidomide alone suppressed tumor growth. The most significant treatment occurred in thalidomide-fed-OncdSyn-injected mice. Compared to PBS controls, there was a significant difference in the number of metastatic nodes in the lungs. Conclusions  Thalidomide alone delayed tumor growth, but the combination of thalidomide with OncdSyn appeared to produce the best results. A. Baghian tragically deceased in June 2008.     

The potential application of a transcriptionally regulated oncolytic herpes simplex virus for human cancer therapy

Background:

Emerging studies have shown the potential benefit of arming oncolytic viruses with therapeutic genes. However, most of these therapeutic genes are placed under the regulation of ubiquitous viral promoters. Our goal is to generate a safer yet potent oncolytic herpes simplex virus type-1 (HSV-1) for cancer therapy.

Methods:

Using bacterial artificial chromosome (BAC) recombineering, a cell cycle-regulatable luciferase transgene cassette was replaced with the infected cell protein 6 (ICP6) coding region (encoded for UL39 or large subunit of ribonucleotide reductase) of the HSV-1 genome. These recombinant viruses, YE-PC8, were further tested for its proliferation-dependent luciferase gene expression.

Results:

The ability of YE-PC8 to confer proliferation-dependent transgene expression was demonstrated by injecting similar amount of viruses into the tumour-bearing region of the brain and the contralateral normal brain parenchyma of the same mouse. The results showed enhanced levels of luciferase activities in the tumour region but not in the normal brain parenchyma. Similar findings were observed in YE-PC8-infected short-term human brain patient-derived glioma cells compared with normal human astrocytes. intratumoural injection of YE-PC8 viruses resulted in 77% and 80% of tumour regression in human glioma and human hepatocellular carcinoma xenografts, respectively.

Conclusion:

YE-PC8 viruses confer tumour selectivity in proliferating cells and may be developed further as a feasible approach to treat human cancers.     

Apoptin enhances the oncolytic properties of vaccinia virus and modifies mechanisms of tumor regression

A recombinant vaccinia virus VVdGF-ApoS24/2 expressing apoptin selectively kills human cancer cells in vitro [Kochneva et al., 2013]. We compared the oncolytic activity of this recombinant with that of the parental strain L-IVP using a model of human A431 carcinoma xenografts in nude mice. Single intratumoral injections (2×10⁷ PFU/mouse) of the viruses produced a dramatic decrease in tumor volumes, which was higher after injection of apoptin-producing virus. The tumor dried out after the injection of recombinant while injection of L-IVP strain resulted in formation of cavities filled with cell debris and liquid. Both viruses rapidly spread in xenografts and replicate exclusively in tumor cells causing their destruction within 8 days. Both viruses induced insignificant level of apoptosis in tumors. Unlike the previously described nuclear localization of apoptin in cancer cells the apoptin produced by recombinant virus was localized to the cytoplasm. The apoptin did not induce a typical apoptosis, but it rather influenced pathway of cell death and thereby caused tumor shrinkage. The replacement of destroyed cells by filamentous material is the main feature of tumor regression caused by the VVdGF-ApoS24/2 virus. The study points the presence of complicated mechanisms of apoptin effects at the background of vaccinia virus replication.