(4)変異型単純ヘルペスウイルス(HSV)を用いた卵巣癌に対する治療戦略 : 新規がんウイルス療法の開発と構築を目指して(<特集>第61回学術講演会シンポジウム3「卵巣癌の新たな治療戦略-基礎から,そして臨床から-」)

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After cytoreductive surgery, advanced-stage epithelialovarian carcinoma (EOC) initially appears chemotherapy sensitive, as response rates to platinum-based therapy exceed 80%. However, longterm survival remains poor as a result of recurrence and emergence of drug resistance, thus, a novel approach to overcome these issues is required. Oncolytic HSV-1 has been developed as a novel anticancer agent. According to the properties and functions of HSV-1 encoded proteins, several genes have been targeted for engineering oncolytic HSV-1. As a result, a variety of strategies have been applied to the engineering of oncolytic HSV-1. To achieve success in cancer therapy for solid tumors, a maximal oncolytic effect is required; however, recombinant HSV-1 that has been adapted to meet neurotoxicity requirements for the treatment of brain tumors may be too highly attenuated for effective use in solid tumors outside the brain. Here, through the preclinical and clinical studies of HF-10 and other replication-competent HSVs, we discussed its potential and perspectives for application in ovarian cancer. 1. Efficacy of replication-competent HSVs for disseminated peritoneal tumors. The survival of mice treated with a high titer hrR3 (5×10^7pfu), which has replaced the gene of UL39 with the lac Z, was significant prolonged as compared with the group given paclitaxel (p<0.0001, log lank test). The lacZ gene product, visualized using X-gal staining, was detected in hrR3 treated tumors in areas also exhibiting apoptotic change. Such viral therapy offered a novel approach to reductions in the dissemination of ovarian cancer. Moreover, we demonstrated that HF10 effectively treated disseminated peritoneal neoplasm in an immunocompetent animal model that all of survived mice acquired resistance to rechallenge with the tumor cells, suggesting the treatment of the disseminated peritoneal tumors with HF10 induced a specific antitumor immune response. The survival time of mice treated with HF10 was longer than that of mice treated with hrR3, indicating that the oncolytic effect of HF10 was more potent than that of hrR3 in this animal model. In the subcutaneous immuno-competent mice model with malignant melanoma cells, HF10 also affected the noninoculated contralateral tumor when injected into the ipsilateral tumor of mice, suggesting that HF10 can induce systemic antitumor immune responses in mice. 2. Biological aspects of HF10. To understand biological properties of HF10, nude mice were subcutaneously infected with 10^6pfu of HF10. The virus disappeared completely by 3 days postinfection and failed to induce any significant histopathological changes on the normal skin, in contrast, KH7, which was isolated from clinical samples, caused severe zoster form on it by 7 days postinfection with the same titer of the virus. In an allograft model of Balb/c nu nu and clone M3 melanoma cells, HF10 or KH7 were injected into the implanted tumors in the skin with 10^6pfu of each virus, respectively. HF10 expanded within all areas of the tumor by 7 days postinfection, however, KH7 scattered over the tumor, resulting in severe dermatitis around the tumor. We speculated this unique manner of HF10 relate to a difference of IFN sensitivity between normal tissues and tumors. Moreover, when we analyzed the level of cytokies in the peritoneal exdate after intraperitoneal injection of HF10, IL-12 was induced 6 times higher than that by hrR3, suggesting HF10 might relate a maturation or induction of dendritic cells (DC). Our sequence analysis also revealed that HF10 lacks the expression of functional UL43, UL49.5, UL55, UL56 and LAT gene products. 3. Enhancement of the efficacy of HF10. We investigated the antitumor effects of a HSV amplicon expressing mouse GM-CSF using HF10 as a helper virus. HF10 packaged mGM-CSF expessing amplicon (mGM-CSF amplicon) was used to infect subcutaneously inoculated murine colorectal tumor cells (CT26 cells) and the antitumor effects were compared to tumors treated with only HF10. The mGM-CSF amplicon efficiently replicated in CT26 cells with similar oncolytic activity to HF10 in vitro. However, when mice subcutaneously inoculated with CT26 cells were intratumally injected with HF10 or mGM-CSF amplicon, greater tumor regression was seen in mGM-CSF amplicon treated animals (p<0.05), resulting in prolonged mouse survival. Immunohistochemical analysis revealed apparently increased inflammatory cell infiltration in the solid tumor in the mGM-CSF amplicon treated animals. These results suggested that expression of GM-CSF enhanced the antitumor effects of HF10. We also investigated the efficacy of gene-directed enzyme prodrug therapy (GDEPT) using a novel system that combines the paclitaxel-2'-ethylcarbonate prodrug (TAX-2'-Et) and an HSV amplicon expressing rabbit-carboxylesterase (CES) with HF10 as a helper virus. This GDEPT system aims to produce high level of CES at the tumor site, resulting in efficient local conversion of the TAX-2'-Et prodrug into the active drug paclitaxel (TAX). We demonstrated that the green fluorescent protein (GFP) gene, as a trace maker, was more efficiently introduced by the HSV amplicon compared to the expression vector, pHGCX, and that the HSV amplicon system expressed an active CES enzyme that could convert TAX-2'-Et to TAX in Cos7 cells. Furthermore, although the cytotoxicity of this amplicon system was not enhanced in virus-sensitive tumor cells, it was significantly enhanced in low virus-sensitive tumor cells in the presence of the prodrug in a concentration-dependent manner, compared to the control virus alone (p<0.05). 4. Perspectives. In order to evade the attack of innate immune responses such as complement, antibodies, and the reticulo-endothelial system, mesenchymal stem cells infected with oncolytic viruses have been considered for cancer gene therapy. This "carrier cell" strategy is expected to allow expansion in a cell-to-cell manner and to yield a higher titer of viruses, compared with the injection of only packaged virions, because carrier cells protect against the attack of the innate immune response and can guarantee sufficient replication of the virus in the infected cells. In fact, SKOV3 ovarian cancer cells cocultured with mesothelial cells infected by HF10 yielded viral titers about 100 times higher than those from SKOV3 cells infected with HF10 alone. To maximize the efficacy of oncolytic therapeutics, the carrier cell strategies will be useful. Recently, the amplicon system has been investigated for oncolytic virotherapy using HF10. HSV-1 amplicon has many advantages for gene delivery, including a broad host range, large transgene capacity, good transduction efficiency, and ease of construction. These properties make amplicons highly suitable for gene transfer in vivo applications. In this system, HF10 functioned not only as a helper virus in the amplicon package system but also as an oncolytic virus. So far, oncolytic HSV-1 has been administered for cancer gene therapy in doses as high as 3×10^9 PFU/injection. It remains to be determined whether the acute immune response to high-dose injection affects the oncolytic effect of the virus. Furthermore, different routes of viral administration will likely influence the efficacy of oncolytic HSV-1; thus, intratumoral, intravenous and intraperitoneal administration should be investigated in preclinical studies and ultimately in clinical trials. Conclusion Oncolytic HSV-1 therapy provides an effective anti-tumor strategy, and HF10 is a powerful weapon against solid malignancies. Further studies are required to elucidate the anti-tumor mechanisms of naturally, non-engineered oncolytic HSV-1 HF10 and the anticancer immune response to oncolytic therapy, in which HF10 has potential as an effective clinical agent for ovarian cancer.
2009-11-01

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