Yasuki Hijikata

Absence of LTB4/BLT1 axis facilitates generation of mouse GM-CSF-induced long-lasting antitumor immunologic memory by enhancing innate and adaptive immune systems.

BLT1 is a high-affinity receptor for leukotriene B4 (LTB4) that is a potent lipid chemoattractant for myeloid leukocytes. The role of LTB4/BLT1 axis in tumor immunology, including cytokine-based tumor vaccine, however, remains unknown. We here demonstrated that BLT1-deficient mice rejected subcutaneous tumor challenge of GM-CSF gene-transduced WEHI3B (WGM) leukemia cells (KO/WGM) and elicited robust antitumor responses against second tumor challenge with WEHI3B cells. During GM-CSF-induced tumor regression, the defective LTB4/BLT1 signaling significantly reduced tumor-infiltrating myeloid-derived suppressor cells, increased the maturation status of dendritic cells in tumor tissues, enhanced their CD4(+) T-cell stimulation capacity and migration rate of dendritic cells that had phagocytosed tumor-associated antigens into tumor-draining lymph nodes, suggesting a positive impact on GM-CSF-sensitized innate immunity. Furthermore, KO/WGM mice displayed activated adaptive immunity by attenuating regulatory CD4(+) T subsets and increasing numbers of Th17 and memory CD44(hi)CD4(+) T subsets, both of which elicited superior antitumor effects as evidenced by adoptive cell transfer. In vivo depletion assays also revealed that CD4(+) T cells were the main effectors of the persistent antitumor immunity. Our data collectively underscore a negative role of LTB4/BLT1 signaling in effective generation and maintenance of GM-CSF-induced antitumor memory CD4(+) T cells.

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.

All-trans retinoic acid attacks reverse transcriptase resulting in inhibition of HIV-l replication

Abstract We previously reported that all-trans retinoic acid (ATRA) inhibited growth in HTLV-l-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. Interestingly, ATRA significantly inhibited reverse transcriptase (RT) activity similar to azidothimidine (AZT) in HTLV-l-positive T-cell lines. To clarify whether ATRA has an inhibitory effect on the replication of HIV, we examined HIV proviral DNA in a HIV-l-positive cell line (8E5) using real time PCR. ATRA as well as AZT reduced the proviral DNA load of 8E5 in a dose-dependent manner. These results suggest that there is a common element of ATRA signaling in both HTLV-l and HIV. Furthermore, we examined the effects of ATRA on viral replication in primary lymphocytes of three individuals infected with HIV. ATRA reduced viral replication significantly similar to AZT. These findings suggested that ATRA acts as a RT inhibitor, reducing the HIV-l proviral DNA load. Finally, we conclude that ATRA may be a potential therapeutic agent for HIV infection.

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.

Angiotensin II type I receptor blocker, Losartan, inhibits fibrosis in liver by suppressing TGF-beta1 production

Renin-angiotensin system is involved in liver fibrogenesis through activating hepatic stellate cells (HSCs). Losartan, which is an angiotensin II type 1 receptor antagonist, could function as a selective peroxisome proliferator-activated receptor c activator. Here we studied the effect of losartan on liver fibrosis in vivo. In vivo study, we used the Concanavalin A (Con A)-induced mouse liver fibrosis model through T cell activation and upregulation of TGF-β1. The mice were administrated ConA for 4 weeks to induce liver fibrosis, and then co-administrated with losartan. Losartan prevented liver fibrogenesis and downregulated TGF-β1 expression. Also, Losartan inhibited HSCs activation and proliferation. These results suggested that Losartan prevent liver fibrosis through suppressing TGF-β1 expression. Abbreviations: AT1-R, angiotensin II type 1 receptor; ARB: angiotensin II receptor blocker; RAS: renin-angiotensin system; TGF-β1: transforming growth factor β1; NASH: non-alcoholic steatohepatitis Introduction Liver fibrosis is the common histological feature of most chronic liver diseases, and leads to cirrhosis and hepatocellular carcinoma (HCC) [1]. Liver cirrhosis is the end stage of chronic liver diseases. Liver fibrosis is the common pathological basis of numerous chronic liver diseases including viral liver disease, alcoholic, non-alcoholic steatohepatitis (NASH), autoimmune hepatitis, primary biliary cirrhosis, and metabolic disease. Liver cirrhosis is characterized by the increase and excessive deposition of the liver extracellular matrix (ECM) [2-4]. Excessive activation of transforming growth factor-β (TGF-β) increases the synthesis and decreases the degradation of ECM proteins with a gradual destruction of organ tissue and structure [5]. The major source of ECM deposition in the liver is hepatic stellate cells (HSCs). After liver injury, TGF-β1 promotes the activation and proliferation of hepatic HSCs [6]. High levels of TGF-β1 are often found in liver fibrosis and there may be a positive correlation between the elevation of TGF-β1 mRNA level and fibrogenic activity [7]. Thus, synthesis of ECM proteins increases in the liver due to excessive activation of the TGF-β1 signal transduction pathway. Therefore, this pathway has become a potential target for the treatment of the liver fibrosis. The renin-angiotensin system (RAS) plays an important role in controlling liver fibrosis [8]. It is well known that the RAS influences cell differentiation, nutrition, and fibrosis, and it has been reported to play a role in heart and kidney disease [9]. Moreover, recent studies have revealed the RAS plays an important role in the progression of many chronic liver diseases [10]. Furthermore, angiotensin II receptor blocker (ARB) modulation of the RAS to treat liver fibrosis has been reported in animal study [11]. ARBs, such as Losartan are largely regarded as a means to block vasoconstriction and inhibit the cell proliferation and fibrosis that are mediated by the angiotensin II type 1 receptor (AT1-R) [10]. Yang et al. [12] suggested that AT1-R play an important role in the development of fibrosis. An increase in hepatic transforming growth factor β1 (TGF-β1) and pro-inflammatory cytokine levels was attenuated in AT1-R knockout mice compared to WT mice [13]. Furthermore, Bataller et al.[14] demonstrated that increased systemic Angiotensin II augments hepatic fibrosis and promotes inflammation, oxidative stress, and thrombogenic events. Losartan was reported to inhibit CCl4-induced Correspondence to: Hisanobu Ogata, Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan, Tel: +81-92-642-5256; Fax: +81-92-642-5271; E-mail: h-ogata@sentan.med.kyushu-u.ac.jp

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

Abstract LB-370: Genetically engineered oncolytic Edmonston strain of measles strain harboring the wild-type N, P, L genes (MV-NPL) effectively target lung cancer stem cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Increasing evidence suggests that only a small percentage of cancer cells in solid tumors have tumorigenic potential. These highly tumorigenic cells share with normal stem cells the ability to proliferate and give rise to diversified tumor cell types including those with the capacity for self-renewal. These cells are termed cancer stem cells (CSCs) and are believed to contribute to CSCs resistance to conventional therapies. Therefore, specific targeting of CSCs is an attractive and novel therapeutic approach. Measles virus Edmonston strain (MV-Edm) is thought to possess remarkable oncolytic activity that selectively destroys human tumor cells. Recently, we succeeded and reported the development of novel oncolytic measles virus (MV) by genetic engineering of MV-Edmonston strain (MV-Edm), that have demonstrated its remarkable oncolytic activity against wide ranges of human tumor cells, with the N, P, and L genes of wild-type MV (MV-NPL). The oncolytic activities of the MV-NPL on human renal cell carcinoma were superior to those of MV-Edm both in vitro and in vivo studies even in the presence of IFN\[[Unsupported Character - ‐]]α[[Unsupported Character - ]\](Xing, M. et. al. Mol Ther 2010). Here, we demonstrated that lung CSCs, side population fraction of human lung cancer cells of A549 (A549-SP), expressed high level of CD46 required for MV-NPL infection and MV-NPL had a superior oncolytic capacity against both A549-SP and A549-NSP (non SP) cells in a time and dose-dependent manner without damaging normal lung cells. Our results of pan-caspase inhibiton assays showed the partial contribution of caspases-dependent apoptosis in the oncolysis of A549-SP cells by MV-NPL, even though the expression levels of anti-apoptotic protein of both XIAP and Mcl-1 were higher in A549-SP cells than A549-NSP cells. Additionally, we demonstrated that an addition of either PI3K inhibitor or MEK inhibitor further enhanced the oncolytic effects of MV-NPL. Furthermore, our in vivo studies using nude mice bearing A549 xenografts revealed that serial intratumoral MV-NPL administrations exhibited significant antitumor effects without severe side effects. Collectively, these data suggested that genetically engineered MV-NPL could be a promising oncolytic agent to effectively eradicate lung CSCs. 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 LB-370. doi:1538-7445.AM2012-LB-370