Toshihiko Okazaki

Coxsackievirus B3 Is an Oncolytic Virus with Immunostimulatory Properties That Is Active against Lung Adenocarcinoma

Although oncolytic virotherapy is a promising anticancer therapy, antitumor efficacy is hampered by low tumor selectivity. To identify a potent and selective oncolytic virotherapy, we carried out large-scale two-step screening of 28 enteroviral strains and found that coxsackievirus B3 (CVB3) possessed specific oncolytic activity against nine human non-small cell lung cancer (NSCLC) cell lines. CVB3-mediated cytotoxicity was positively correlated with the expression of the viral receptors, coxsackievirus and adenovirus receptor, and decay-accelerating factor, on NSCLC cells. In vitro assays revealed that the CVB3 induced apoptosis and phosphoinositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) survival signaling pathways, leading to cytotoxicity and regulation of CVB3 replication. Intratumoral injections of CVB3 elicited remarkable regression of preestablished NSCLC tumors in vivo. Furthermore, administrations of CVB3 into xenografts on the right flank resulted in significantly durable regression of uninjected xenografts on the left flank, where replication-competent CVB3 was detected. All treatments with CVB3 were well tolerated without treatment-related deaths. In addition, after CVB3 infection, NSCLC cells expressed abundant cell surface calreticulin and secreted ATP as well as translocated extranuclear high-mobility group box 1, which are required for immunogenic cell death. Moreover, intratumoral CVB3 administration markedly recruited natural killer cells and granulocytes, both of which contributed to the antitumor effects as shown by depletion assays, macrophages, and mature dendritic cells into tumor tissues. Together, our findings suggest that CVB3 is a potent and well-tolerated oncolytic agent with immunostimulatory properties active against both localized and metastatic NSCLC.

Enhanced Antitumor Effects of an Engineered Measles Virus Edmonston Strain Expressing the Wild-type N, P, L Genes on Human Renal Cell Carcinoma

Measles virus Edmonston strain (MV-Edm) is thought to have remarkable oncolytic activity that selectively destroys human tumor cells. The P/V/C protein of wild-type MV was shown to resist the antiviral effects of interferon (IFN)-alpha. Here, we engineered new MVs by arming MV-Edm tag strain (a V-defective vaccine-lineage strain, MV-Etag) with the P or N, P, and L genes of wild-type MV (MV-P and MV-NPL, respectively). The oncolytic activities of the MVs were determined in human renal cell carcinoma (RCC) cell lines and primary human RCC cells by the MTT assay. The antitumor efficacy of the MVs was evaluated in A-498 xenografts in nude mice. IFN-alpha effectively inhibited the replication of MV-Etag and MV-P, but not MV-NPL. MV-NPL more efficiently induced cytopathic effects (CPEs) in OS-RC-2 cells, even in the presence of human IFN-alpha. MV-NPL replicated more rapidly than MV-P and MV-Etag in A-498 cells. Apoptosis was induced earlier in A-498 cells by MV-NPL than MV-Etag and MV-P. MV-NPL showed more significant antitumoral effects and had prolonged replication compared to MV-Etag and MV-P. In this study, we demonstrated that the newly engineered MV-NPL has more effective oncolytic activity and may help establish an innovative cancer therapy.

Phase I clinical trial of a five-peptide cancer vaccine combined with cyclophosphamide in advanced solid tumors.

We designed a phase I trial to investigate the safety, immune responses and clinical benefits of a five-peptide cancer vaccine in combination with chemotherapy. Study subjects were patients positive for HLA-A2402 with locally advanced, metastatic, and/or recurrent gastrointestinal, lung or cervical cancer. Eighteen patients including nine cases of colorectal cancer were treated with escalating doses of cyclophosphamide 4days before vaccination. Five HLA-A2402-restricted, tumor-associated antigen (TAA) epitope peptides from KOC1, TTK, URLC10, DEPDC1 and MPHOSPH1 were injected weekly for 4weeks. Treatment was well tolerated without any adverse events above grade 3. Analysis of peripheral blood lymphocytes showed that the number of regulatory T cells dropped from baseline after administration of cyclophosphamide and confirmed that TAA-specific T cell responses were associated significantly with longer overall survival. This phase I clinical trial demonstrated safety and promising immune responses that correlated with vaccine-induced T-cell responses. Therefore, this approach warrants further clinical studies.

TLR7 ligand augments GM-CSF-initiated antitumor immunity through activation of plasmacytoid dendritic cells

Narusawa and colleagues found that type 1 IFNs and plasmacytoid dendritic cells in the tumor-draining lymph nodes mediate GM-CSF–induced antitumor immunity in immunocompetent mice, and they report that the synthetic TLR7 ligand imiquimod could overcome tolerance and enhance autologous GM-CSF antitumor effects. Vaccination with irradiated granulocyte macrophage colony-stimulating factor (GM-CSF)–transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity. However, its effectiveness is limited. We therefore attempted to improve the antitumor effect by identifying little-known key pathways in GM-CSF–sensitized dendritic cells (GM-DC) in tumor-draining lymph nodes (TDLN). We initially confirmed that syngeneic mice subcutaneously injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) remarkably rejected the tumor growth. Using cDNA microarrays, we found that expression levels of type I interferon (IFN)–related genes, predominantly expressed in plasmacytoid DCs (pDC), were significantly upregulated in TDLN-derived GM-DCs and focused on pDCs. Indeed, mouse experiments demonstrated that the effective induction of GM-CSF–induced antitumor immunity observed in immunocompetent mice treated with LLC/SeV/GM cells was significantly attenuated when pDC-depleted or IFNα receptor knockout (IFNAR−/−) mice were used. Importantly, in both LLC and CT26 colon cancer–bearing mice, the combinational use of imiquimod with autologous GVAX therapy overcame the refractoriness to GVAX monotherapy accompanied by tolerability. Mechanistically, mice treated with the combined vaccination displayed increased expression levels of CD86, CD9, and Siglec-H, which correlate with an antitumor phenotype, in pDCs, but decreased the ratio of CD4+CD25+FoxP3+ regulatory T cells in TDLNs. Collectively, these findings indicate that the additional use of imiquimod to activate pDCs with type I IFN production, as a positive regulator of T-cell priming, could enhance the immunologic antitumor effects of GVAX therapy, shedding promising light on the understanding and treatment of GM-CSF–based cancer immunotherapy. Cancer Immunol Res; 2(6); 568–80. ©2014 AACR.

A novel, polymer-coated oncolytic measles virus overcomes immune suppression and induces robust antitumor activity

Although various therapies are available to treat cancers, including surgery, chemotherapy, and radiotherapy, cancer has been the leading cause of death in Japan for the last 30 years, and new therapeutic modalities are urgently needed. As a new modality, there has recently been great interest in oncolytic virotherapy, with measles virus being a candidate virus expected to show strong antitumor effects. The efficacy of virotherapy, however, was strongly limited by the host immune response in previous clinical trials. To enhance and prolong the antitumor activity of virotherapy, we combined the use of two newly developed tools: the genetically engineered measles virus (MV-NPL) and the multilayer virus-coating method of layer-by-layer deposition of ionic polymers. We compared the oncolytic effects of this polymer-coated MV-NPL with the naked MV-NPL, both in vitro and in vivo. In the presence of anti-MV neutralizing antibodies, the polymer-coated virus showed more enhanced oncolytic activity than did the naked MV-NPL in vitro. We also examined antitumor activities in virus-treated mice. Complement-dependent cytotoxicity and antitumor activities were higher in mice treated with polymer-coated MV-NPL than in mice treated with the naked virus. This novel, polymer-coated MV-NPL is promising for clinical cancer therapy in the future.

A phase I clinical trial of RNF43 peptide-related immune cell therapy combined with low-dose cyclophosphamide in patients with advanced solid tumors

The objective of this study was to investigate the safety and the tolerability of combined cellular immunotherapy with low-dose cyclophosphamide (CPA) in patients with advanced solid tumors. This study targeted a novel tumor-associated antigen, ring finger protein 43 (RNF43). Eligible patients were resistant to standard therapy, HLA-A*24:02- or A*02:01-positive and exhibiting high RNF43 expression in their tumor cells. They were administered 300 mg/m2 CPA followed by autologous lymphocytes, preliminarily cultured with autologous RNF43 peptide-pulsed dendritic cells (DCs), RNF43 peptide-pulsed DCs and systemic low dose interleukin-2. The primary endpoint was safety whereas the secondary endpoint was immunological and clinical response to treatment. Ten patients, in total, were enrolled in this trial. Primarily, no adverse events greater than Grade 3 were observed. Six out of 10 patients showed stable disease (SD) on day 49, while 4 other patients showed progressive disease. In addition, one patient with SD exhibited a partial response after the second trial. The frequency of regulatory T cells (Tregs) in patients with SD significantly decreased after CPA administration. The ratio of interferon-γ-producing, tumor-reactive CD8+ T cells increased with time in patients with SD. We successfully showed that the combination of immune cell therapy and CPA was safe, might induce tumor-specific immune responses and clinical efficacy, and was accompanied by a decreased ratio of Tregs in patients with RNF43-positive advanced solid tumors.

631. Novel Polymer-Coated Stealth Oncolytic Measles Virus Overcame Immune Suppression and Induced Stronger Antitumor Activity

Background: Although there have recently been much progress in cancer treatment including surgery, chemotherapy and radiotherapy, cancers have long been the leading cause of death in Japan in the last 30 years and the development of new therapeutic modalities is imminently required. As a new modality, there has recently been great interest in oncolytic virotherapy. Particularly, measles virus is one of the candidate viruses expected for strong antitumor effects. The efficacy of virotherapy, however, was strongly limited by the immune response of the host in previous clinical trials.Methods: To enhance and prolong the antitumor activity of the virotherapy in vivo, we combined newly developed tools of the genetically engineered measles virus (MV-NPL) and multilayer virus coating method of layer-by-layer deposition of ionic polymers. We compared the oncolytic effects of this polymer-coated MV-NPL with naked MV-NPL in vitro and in vivo.Results: In the presence of anti-MV neutralizing antibodies, the polymer-coated virus showed more enhanced oncolytic activity than naked MV-NPL in vitro. We also examined the antitumor immunity in virus treated mice. Complement-dependent cytotoxicity activities in mice treated with polymer-coated MV-NPL were higher than those with naked virus and stronger antitumor activity was observed in these mice.Conclusion: This novel polymer-coated MV-NPL may be promising for future clinical cancer therapy.

Abstract LB-172: A phase I clinical trial of RNF43 peptide-specific immune cell therapy combined with low-dose cyclophosphamide for patients with advanced solid tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Accumulated clinical studies have validated the efficacy of immunotherapies for patients with solid tumors. Although effective anti-tumor immune responses have been introduced in many clinical trial cases, several immunosuppressive factors such as regulatory T cells (Tregs) have been considered to inhibit induction of anti-tumor immunity. We have developed new approach of immunotherapy in order to augment anti-tumor effect by expanding tumor-specific cytotoxic T cells (CTL), and eliminate Tregs by utilizing chemotherapy. We have recently reported the efficacy of multiplex vaccination with HLA-A*2402 restricted tumor-associated antigens peptides (TAA), preceded by the administration of low-dose cyclophosphamide (CPM) in order to eliminate Tregs. This time, we have been conducting a phase I clinical trial to study the utilization of CTL after CPM administration followed by a consequent administration of dendritic cells (DC) and IL-2. This study targets the novel HLA-A*0201 or HLA-A*2402-restricted TAA, RING finger protein 43 (RNF43), and is designed as a dose-escalation study of CTL. The purpose of this trial is to evaluate the safety and tolerability of the proposed method as primary endpoint and the efficacy including over all survival and immune responses as secondary endpoint. 11 patients with advanced solid tumors refractory to standard therapy were enrolled in this trial. They were HLA-A*2402 or A*0201 positive and exhibiting RNF43 expression levels in their tumor cells more than those in RNF43-positive control cell (HCT15). Primarily, severe adverse event greater than Grade 3 was not observed in our patients. Clinical responses of stable disease (SD) were observed in 8 out of 11 patients. Among them, 2 patients showed a decrease in the tumor markers with stabilized tumor sizes during the observed period, and 1 patient showed a mixed response. On the other hand, 3 other patients showed progressive disease (PD). The number of Tregs significantly decreased after the administration of CPM (p=0.033), and the higher decreasing rate of Tregs was related to the good clinical response. In CD107a/b mobilization assay, the ratio of RNF43-specific CD8 T cells in SD increased with time, conversely that in PD decreased (p=0.005). Also, in Intracellular Staining Assay, the ratio of IFN-g produced by RNF43-specific CD8 T cells was significantly correlated with clinical benefit (p=0.02), while TNF-α and IL-2 were not. Consequently, the combination of immunotherapy and CPM may induce tumor specific immune cells accompanied by the decreased number of Tregs. In conclusion, this trial is tolerable and may be clinically efficient against advanced solid tumors. Further immunological assessment should be conducted in order to explore the valid biomarkers to predict clinical efficacy before treatment. Citation Format: Yasuki Hijikata, Toshihiko Okazaki, Hiroyuki Inoue, Yoshihiro Tanaka, Kenzaburo Tani, Shinichi Kobayashi, koji Yoshida. A phase I clinical trial of RNF43 peptide-specific immune cell therapy combined with low-dose cyclophosphamide for patients with advanced solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-172. doi:10.1158/1538-7445.AM2014-LB-172