Ryo Sakai

Enhanced antitumor efficacy of telomerase-selective oncolytic adenoviral agent OBP-401 with docetaxel: Preclinical evaluation of chemovirotherapy

Oncolytic adenoviruses are being developed as novel anticancer therapeutics and currently undergoing clinical trials. We previously demonstrated that telomerase‐specific replication‐competent adenovirus (Telomelysin: OBP‐301), in which the human telomerase reverse transcriptase (hTERT) promoter regulates viral replication, efficiently killed human tumor cells. We further constructed OBP‐401 (Telomelysin‐GFP) that expresses the green fluorescent protein (GFP) reporter gene under the control of the cytomegalovirus promoter in the E3 region to monitor viral distribution. Here, we examined the feasibility of a single‐agent therapy with OBP‐401 as well as of combining OBP‐401 with chemotherapeutic agents. Infection of OBP‐401 alone or followed by the treatment of a chemotherapeutic drug, docetaxel (Taxotere), resulted in a profound in vitro cytotoxicity and GFP expression in various human cancer cell lines originating from different organs (lung, colon, esophagus, stomach, liver and prostate), although the magnitude of antitumor effect varied among the cell types. Other chemotherapeutic drugs such as vinorelbine (Navelbine) and SN38 (the potent active metabolite of irinotecan) combined with OBP‐401 also inhibited the growth of human cancer cells. Quantitative real‐time PCR analysis demonstrated that docetaxel did not affect viral replication. For in vivo evaluation, nu/nu mice xenografted with H1299 human lung tumor received intratumoral injection of OBP‐401 and intraperitoneal administration of docetaxel. Analysis of growth of implanted tumors showed a significant, therapeutic synergism, although OBP‐401 alone and docetaxel alone showed modest inhibition of tumor growth. Thus, OBP‐401 in combination with docetaxel efficiently enhances the antitumor efficacy both in vitro and in vivo, and the outcome has important implications for tumor‐specific oncolytic chemovirotherapies for human cancers.

Virus-mediated oncolysis induces danger signal and stimulates cytotoxic T-lymphocyte activity via proteasome activator upregulation.

Dendritic cells (DCs) are the most potent antigen-presenting cells and acquire cellular antigens and danger signals from dying cells to initiate antitumor immune responses via direct cell-to-cell interaction and cytokine production. The optimal forms of tumor cell death for priming DCs for the release of danger signals are not fully understood. OBP-301 (Telomelysin) is a telomerase-specific replication-competent adenovirus that induces selective E1 expression and exclusively kills human cancer cells. Here, we show that OBP-301 replication produced the endogenous danger signaling molecule, uric acid, in infected human tumor cells, which in turn stimulated DCs to produce interferon-γ (IFN-γ) and interleukin 12 (IL-12). Subsequently, IFN-γ release upregulated the endogenous expression of the proteasome activator PA28 in tumor cells and resulted in the induction of cytotoxic T-lymphocytes. Our data suggest that virus-mediated oncolysis might be the effective stimulus for immature DCs to induce specific activity against human cancer cells.

Preclinical Evaluation of Differentially Targeting Dual Virotherapy for Human Solid Cancer

Multimodal approaches combining drugs that differentially function is the most popular regimen for treating human cancer. Understanding the molecular mechanisms underlying the synergistic, potentiative, and antagonistic effects of drug combinations could facilitate the discovery of novel efficacious combinations. We previously showed that telomerase-specific replication-competent adenovirus (Telomelysin, OBP-301), in which the human telomerase reverse transcriptase promoter controls the adenoviral E1 gene expression, induces a selective antitumor effect in human cancer cells. Here, using E1-deleted replication-deficient adenovirus expressing the p53 tumor suppressor gene (Advexin, Ad-p53) and OBP-301, we investigate how these adenoviruses that kill tumor cells with different mechanisms could work in combination on human cancer. We found that E1-deficient Ad-p53 could kill cancer cells more efficiently in the presence of OBP-301 than Ad-p53 alone or OBP-301 alone, because Ad-p53 could become replication-competent by being supplied adenoviral E1 from coinfected OBP-301 in trans. Ad-p53 plus OBP-301 induced high levels of p53 protein expression without p21 induction, resulting in apoptotic cell death documented by active caspase-3 expression with a cytometric bead array and an increased subdiploid apoptotic fraction of the cell cycle. For in vivo evaluation, nude mice xenografted with human lung tumors received intratumoral injection of OBP-301 and/or Ad-p53. Analysis of the growth of implanted tumors showed an enhanced antitumor effect in combination therapy. Our data show that Ad-p53 in combination with OBP-301 induces not only oncolytic but also apoptotic cancer cell death and enhances antitumor activity in vitro and in vivo, providing potential merits as a multimodal treatment for human cancer. Mol Cancer Ther; 9(6); 1884–93. ©2010 AACR.

Combination of oncolytic adenovirotherapy and Bax gene therapy in human cancer xenografted models. Potential merits and hurdles for combination therapy.

Cancer gene therapy and oncolytic virotherapy have been studied extensively. However, their clinical application is hampered by their weak anticancer activity. We previously constructed a replicating adenovirus (OBP‐301, Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of the adenoviral E1 genes, and causes selective lysis of human cancer cells. We hypothesized that combination adenoviral therapy containing OBP‐301 and a nonreplicating adenovirus expressing the proapoptotic Bax gene could overcome the weakness and augment the anticancer efficacy of each modality. Combination treatment resulted in marked Bax protein expression and enhanced efficacy in in vitro cell viability assay, when compared with either single treatment. However, combination treatment was not as effective in suppressing both subcutaneous and pleural disseminated tumors compared with OBP‐301 treatment alone. Further investigation revealed that combination treatment resulted in suppressed E1A protein expression associated with reduced viral replication. Our results suggest that Bax gene therapy in combination with oncolytic adenovirotherapy potentially augments their antitumor activity, but further improvements may be required to maximize the combinatorial effect in vivo, for the Bax gene expression to avoid interference with production of the oncolytic virus.

A Novel Antiangiogenic Effect for Telomerase-Specific Virotherapy through Host Immune System

Soluble factors in the tumor microenvironment may influence the process of angiogenesis; a process essential for the growth and progression of malignant tumors. In this study, we describe a novel antiangiogenic effect of conditional replication-selective adenovirus through the stimulation of host immune reaction. An attenuated adenovirus (OBP-301, Telomelysin), in which the human telomerase reverse transcriptase promoter element drives expression of E1 genes, could replicate in and cause selective lysis of cancer cells. Mixed lymphocyte-tumor cell culture demonstrated that OBP-301-infected cancer cells stimulated PBMC to produce IFN-γ into the supernatants. When the supernatants were subjected to the assay of in vitro angiogenesis, the tube formation of HUVECs was inhibited more efficiently than recombinant IFN-γ. Moreover, in vivo angiogenic assay using a membrane-diffusion chamber system s.c. transplanted in nu/nu mice showed that tumor cell-induced neovascularization was markedly reduced when the chambers contained the mixed lymphocyte-tumor cell culture supernatants. The growth of s.c. murine colon tumors in syngenic mice was significantly inhibited due to the reduced vascularity by intratumoral injection of OBP-301. The antitumor as well as antiangiogenic effects, however, were less apparent in SCID mice due to the lack of host immune responses. Our data suggest that OBP-301 seems to have antiangiogenic properties through the stimulation of host immune cells to produce endogenous antiangiogenic factors such as IFN-γ.

303. Enhanced Antitumor Efficacy of Telomerase-Selective Oncolytic Adenoviral Agent OBP-401 with Docetaxel: Preclinical Evaluation of Chemovirotherapy

Top of pageAbstract Oncolytic adenoviruses are being developed as novel anticancer therapeutics and currently undergoing clinical trials. We previously demonstrated that telomerase-specific replication-competent adenovirus (Telomelysin: OBP-301), in which the hTERT promoter regulates viral replication, efficiently killed human tumor cells. We further constructed OBP-401 (Telomelysin-GFP) that expresses the green fluorescent protein (GFP) reporter gene under the control of the cytomegalovirus promoter in the E3 region to monitor viral distribution. Here, we examined the feasibility of a single-agent therapy with OBP-401 as well as of combining OBP-401 with chemotherapeutic agents. Infection of OBP-401 alone or followed by the treatment of a chemotherapeutic drug, docetaxel (Taxotere), resulted in a profound in vitro cytotoxicity and GFP expression in various human cancer cell lines originating from different organs (lung, colon, esophagus, stomach, liver, and prostate), although the magnitude of antitumor effect varied among the cell types. Other chemotherapeutic drugs such as vinorelbine (Navelbine) and SN38 (the potent active metabolite of irinotecan) combined with OBP-401 also inhibited the growth of human cancer cells. Quantitative real-time PCR analysis demonstrated that docetaxel did not affect viral replication. For in vivo evaluation, nu/nu mice xenografted with H1299 human lung tumor received intratumoral injection of OBP-401 and intraperitoneal administration of docetaxel. Analysis of growth of implanted tumors showed a significant, therapeutic synergism, although OBP-401 alone and docetaxel alone showed modest inhibition of tumor growth. Thus, OBP-401 in combination with docetaxel efficiently enhances the antitumor efficacy both in vitro and in vivo, the outcome has important implications for tumor- specific oncolytic chemovirotherapies for human cancers.