Tatsuji Enoki

Oligosaccharides from Agar Inhibit Pro-Inflammatory Mediator Release by Inducing Heme Oxygenase 1

We investigated whether agaro-oligosaccharides have any immunological effects on RAW264.7 mouse macrophages and human monocytes in vitro. We demonstrate that agaro-oligosaccharides suppressed the elevated levels of nitric oxide, prostaglandin E2, and such pro-inflammatory cytokines as tumor necrosis factor-α, interleukin-1β and interleukin-6 in lipopolysaccharide-stimulated monocytes and macrophages. We also demonstrate that those effects of agaro-oligosaccharides on activated monocytes and macrophages may have been caused by heme oxygenase-1 induction. It is therefore proposed that agaro-oligosaccharides might be a good candidate for a functional food to prevent inflammatory diseases.

Angelica keiskei extract improves insulin resistance and hypertriglyceridemia in rats fed a high-fructose drink.

Angelica keiskei is a traditional herb peculiar to Japan and abundantly contains vitamins, dietary fiber and such polyphenols as chalcone. We investigated in the present study the effect of A. keiskei on insulin resistance and hypertriglyceridemia in fructose-drinking rats as a model for the metabolic syndrome. Male Wistar rats were given a 15% fructose solution as drinking water for 11 weeks. Fructose significantly increased the levels of serum insulin and triglyceride (TG) compared with the control level. Treatment with an ethanol extract of A. keiskei (AE) significantly reduced the levels of blood glucose (−16.5%), serum insulin (−47.3%), HOMA-R (−56.4%) and TG (−24.2%). A hepatic gene analysis showed that fructose reduced the expression of the genes related to fatty acid β-oxidation and high-density lipoprotein (HDL) production. Treatment with AE enhanced the expression of the acyl-CoA oxidase 1 (ACO1), medium-chain acyl-CoA dehydrogenase (MCAD), ATP-binding membrane cassette transporter A1 (ABCA1) and apolipoprotein A1 (Apo-A1) genes. These results suggest that AE improved the insulin resistance and hypertriglyceridemia of the fructose-drinking rats.

Synergistic Effect of CH-296 and Interferon Gamma on Cytokine-Induced Killer Cells Expansion for Patients with Advanced-Stage Malignant Solid Tumors

BACKGROUND Cytokine-induced killer cells (CIKs) are heterogenous antitumor effectors including interferon gamma (IFN-γ)-amplified CD3(+)CD56(+) cells. CH-296 has been shown to stimulate T-cell proliferation in the presence of T cell receptor (TCR)-stimulating signals. The purpose of this study was to investigate the synergistic effect of CH-296 and IFN-γ on expansion of CIKs for treating patients with advanced-stage malignant solid tumors. METHODS CIKs were cultured with immobilized CH-296 in the presence (retronectin [RN]-CIKs) or absence of IFN-γ (RN-CIKs/del) for 14 days. Proliferation, apoptosis, phenotype, and cytotoxicity were detected. Twenty (20) patients (18 patients with stage IV solid tumors) received three cycles of RN-CIKs treatment. The clinical responses were evaluated using Karnofsky Performance Status scoring and computed-tomography scanning. RESULTS CH-296 promoted CIKs expansion in a time-dependent manner by inhibiting apoptosis and increasing proliferation. Costimulation of CH-296 and IFN-γ amplified more antitumor effectors of CIKs with activated T-cell phenotype, which displayed potent cytotoxicity and increased cytokines secretion upon antigen priming. Sixteen (16) patients receiving RN-CIKs experienced relief of clinical symptoms. The overall clinical response rate was 65% (13/20) and the mean overall survival was 16.95±6.10 months. No severe adverse events were observed in the clinical trial. CONCLUSIONS CH-296 and IFN-γ synergistically promote antitumor efficiency of CIKs by increasing proliferation, inhibiting apoptosis, and enhancing cytotoxicity.

Six New Chalcones from Angelica keiskei Inducing Adiponectin Production in 3T3-L1 Adipocytes

Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanthoangelol, 4-hydroxyderricin and six new chalcones were isolated in this study from an ethanol extract of A. keiskei by octadecyl silyl (ODS) and silica gel chromatography, and identified by 1D- and 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometric analyses. The chalcones from A. keiskei markedly increased the expression of the adiponectin gene and the production of adiponectin in 3T3-L1 adipocytes. These results suggest that the chalcones from A. keiskei might be useful for preventing the metabolic syndrome.

Phase I Clinical Trial of Fibronectin CH296-Stimulated T Cell Therapy in Patients with Advanced Cancer

Background Previous studies have demonstrated that less-differentiated T cells are ideal for adoptive T cell transfer therapy (ACT) and that fibronectin CH296 (FN-CH296) together with anti-CD3 resulted in cultured cells that contain higher amounts of less-differentiated T cells. In this phase I clinical trial, we build on these prior results by assessing the safety and efficacy of FN-CH296 stimulated T cell therapy in patients with advanced cancer. Methods Patients underwent fibronectin CH296-stimulated T cell therapy up to six times every two weeks and the safety and antitumor activity of the ACT were assessed. In order to determine immune function, whole blood cytokine levels and the number of peripheral regulatory T cells were analyzed prior to ACT and during the follow up. Results Transferred cells contained numerous less-differentiated T cells greatly represented by CD27+CD45RA+ or CD28+CD45RA+ cell, which accounted for approximately 65% and 70% of the total, respectively. No ACT related severe or unexpected toxicities were observed. The response rate among patients was 22.2% and the disease control rate was 66.7%. Conclusions The results obtained in this phase I trial, indicate that FN-CH296 stimulated T cell therapy was very well tolerated with a level of efficacy that is quite promising. We also surmise that expanding T cell using CH296 is a method that can be applied to other T- cell-based therapies. Trial Registration UMIN UMIN000001835

Phase I clinical trial of adoptive transfer of expanded natural killer cells in combination with IgG1 antibody in patients with gastric or colorectal cancer: NK therapy combined with antibodies

Natural killer (NK) cells exhibit strong cytotoxic activity against tumor cells without prior sensitization, and have the potential to exert antibody‐dependent cellular cytotoxicity (ADCC). In this clinical trial, we examined the safety and efficacy of the use of NK cells, generated using a novel expansion system, in combination with IgG1 antibodies for the treatment of advanced gastric or colorectal cancers. Treatment consisted of trastuzumab‐ or cetuximab‐based chemotherapy, plus adoptive NK cell therapy. For administration of expanded NK cells, dose escalation with a sequential 3 + 3 design was performed in three steps, at doses of 0.5 × 109, 1.0 × 109, and 2.0 × 109 cells/injection (N = 9). After 3 days of IgG1 antibody administration, patients were infused with expanded NK cells three times at triweekly intervals. NK cell populations expanded with our system were confirmed as being enriched in NK cells (median 92.9%) with high expression of NKG2D (97.6%) and CD16 (69.6%). The combination therapy was very well tolerated with no severe adverse events. Among six evaluable patients, four presented stable disease (SD) and two presented progressive disease. Of the four SD patients, three showed an overall decrease in tumor size after combination therapy. Immune monitoring suggested that combination therapy enhanced whole blood IFN‐γ production and reduced peripheral regulatory T cells (Tregs). In conclusion, this phase I trial provides evidence of good tolerability, induction of Th1 immune responses, and preliminary anti‐tumor activity for this combination therapy, in patients with advanced gastric and colorectal cancer that have received previous therapy.

Stimulation through very late antigen-4 and -5 improves the multifunctionality and memory formation of CD8⁺ T cells.

T cells express multiple integrin molecules. The significance of signaling through these molecules on acquisition of T‐cell effector functions and memory formation capacity remains largely unknown. Moreover, the impact of stimulation through these signals on the generation of T cells for adoptive immunotherapy has not been elucidated. In this study, using a recombinant fragment of fibronectin, CH‐296, we demonstrated that stimulation via very late Ag (VLA)‐4 and VLA‐5 in human and BALB/c mouse CD8+ T cells, in combination with TCR stimulation, enhances effector multifunctionality and in vivo memory formation. Using TCR‐transgenic mouse‐derived CD8+ T cells expressing TCR specific for the syngeneic CMS5 fibrosarcoma‐derived tumor Ag, we showed that stimulation by CH‐296 improved the ability of tumor‐specific CD8+ T cells to inhibit CMS5 tumor growth when adoptively transferred into hosts with progressing tumors. Improved antitumor effects were associated with decreased infiltration of Foxp3+CD4+ Treg cells in tumors. These results suggest that stimulation via VLA‐4 and VLA‐5 modulates the qualities of effector T cells and could potentially increase the efficacy of adoptive therapy against cancer.

Abstract 3137: A novel expansion method for functional natural killer cells and its clinical application

Background: Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. But, it is difficult to generate the sufficient scale and purity of NK cells, and reliable methods to produce large number of functional NK cells have not been established yet. We have developed novel clinical-grade NK cells expansion method to produce the high purity, large scale and functional NK cells using a combination of recombinant human fibronectin fragment (RetroNectin®) -induced T-cells (RN-T cells), OK-432 and IL-2. We subsequently conducted a Phase 1 clinical study to evaluate the safety and efficacy of this NK cell therapy. In this paper, we address the characteristics of the NK cells elicited by this method and the results of immune monitoring in the clinical trial. Methods: To confirm the significance of RN-T cells as stimulator, we compared the stimulation effects on NK cells between RN-T cells and aCD3-T cells, which stimulated by anti-CD3 mAb only. Next, we analyzed expanded NK cells from PBMCs obtained from 31 cancer patients to verify the efficacy of this method. In the Phase 1 trial, patients with unresectable digestive cancer were enrolled. They received weekly intravenous administration of autologous NK cells elicited by the novel method three times to assess the safety of the number of adoptive cells at 0.5 × 109(cohort1), 1 × 109(cohort 2) and 2 × 109 cells (cohort 3) per dose. The phenotype and cytotoxicity of expanded NK cells were analyzed. For immune monitoring, cytotoxicity of PBMCs and whole blood cytokine levels were examined following NK cell infusion. Results: The stimulation by RN-T cells could induce preferable NK cell proliferation rather than the one by aCD3-T. As results of 31 cancer patients, 688±76-fold expansion was achieved in this system with PBMCs, and the NK cell populations were highly purified (84.7±3.6%) and highly expressed functional markers such as NKG2D (97.3±0.6%) and CD16 (96.8±0.7%). In the Phase 1 clinical trial, no NK cell infusion related severe or unexpected toxicities were observed. The response rate and the disease control rate in 10 per protocol patients were 0% and 50.0%, respectively. Although no clinical responses were observed, the cytotoxicity of PBMCs against K-562 targets increased in most patients (80%). The average of cytotoxic activity of PBMCs increased more than two times after the NK cell infusion. Conclusion: Although no patients receiving the NK cell therapy alone experienced a clinical response, adoptive immunotherapy of the NK cells elicited by the novel method is expected to exert considerable ADCC activity in vivo because of their high expression of CD16. Now, we are conducting clinical trial in which we explore the combination of this novel NK therapy with IgG1 antibodies such as trastuzumab and cetuximab. We also address the ongoing clinical trial in this paper. Citation Format: Takeshi Ishikawa, Naoyuki Sakamoto, Tetsuya Okayama, Kaname Oka, Satoshi Kokura, Mitsuko Ideno, Akiko Kato, Tatsuji Enoki, Masanari Kitagawa, Junichi Mineno, Tomoyo Yasuda, Toshifumi Doi, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. A novel expansion method for functional natural killer cells and its clinical application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3137. doi:10.1158/1538-7445.AM2015-3137

Abstract CT214: High purity and activity NK cells therapy in patients with advanced gastrointestinal cancer: Phase I study

PURPOSE: Our group exploited the novel technique of cultivating high purity and activity NK cells compared with traditional technique from peripheral blood of cancer patients. This phase I study investigated the safety and maximum-tolerated dose of the infusion NK cell number in patients with untreatable advanced gastrointestinal cancer. PATIENTS AND METHODS: Patients with gastrointestinal cancer who previously failed standard chemotherapy regimens were treated with three doses of NK cells infusion. We set three dose levels of NK cells infusion. The numbers of infusion NK cells were 5 X 108 cells as cohort 1, 1 X 109 cells as cohort 2, 2 X 109 cells as cohort 3 and each patient was infused NK cells three times per 1 or 2 weeks. The culture method of NK cells described as below; firstly CH296 (FN-CH296, RetroNctin®) induced T cells (RN-T) were prepared in advance by previously reported method, and processed to use as stimulator cells. NK cells were expanded from peripheral blood mononuclear cells by stimulating with modified RN-T, OK-432 and IL-2, then cultured for 21-22 days. We also established large-scale culture system using gas-permeable culture bag for clinical application. RESULTS: 14 patients were totally enrolled in this study. Of 14 patients, 7 patients were enrolled in cohort 1 study. Of 7 patients, 4 patients couldn9t complete NK cells infusion because of some reasons such as bad NK cell proliferation, less purity of active NK cells and worsening of primary cancer. 3 patients could complete in cohort 1. 4 patients were enrolled in cohort 2 study. Of 4 patients, 1 patient couldn9t complete the NK cell infusion because of worsening of primary cancer. 3 patients could complete and 1 patient experienced grade 2 pleural effusion accumulation. In cohort 3 study, 3 patients were enrolled. All of 3 patients could complete NK cells infusion, and only 1 patient experienced grade 1 low grade fever. As clinical response, 1 partial response (PR) at cohort 2 and 6 stable diseases (SD) at 4 patients in cohort 1, 1 patient in cohort2 and 1 patient in cohort 3 were observed. In addition, we checked the immune monitoring in detail for all patients. CONCLUSIONS: High purity and activity NK cells therapy was safety and maximum-tolerated dose was 2 X 109 cells. The evaluation is needed to further refine the efficacy and the toxicity of the combination therapy, chemotherapy, IgG1 molecular target drugs and this therapy for using this therapy in clinical in the future. Citation Format: Tetsuya Okayama, Satoshi Kokura, Takeshi ishikawa, Naoyuki Sakamoto, Mitsuko Ideno, Fumiyo Sakai, Akiko Kato, Tatsuji Enoki, Junichi Mineno, Hideyuki Konishi, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. High purity and activity NK cells therapy in patients with advanced gastrointestinal cancer: Phase I study. [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 CT214. doi:10.1158/1538-7445.AM2014-CT214

Abstract 2791: Advantages and clinical application of fibronectin CH296-stimulated T cells in cancer immunotherapy

Background: In adoptive T-cell therapy (ACT), the differentiation state of transferred T cells is considered to be crucial to the success of ACT-based approaches. It has been reported that less-differentiated T cells, which have a higher proliferative potential and less prone to apoptosis than more differentiated cells, are ideal for ACT transfer therapy. In this study, we compared the co-stimulation effects of fibronectin CH296 (FN-CH296, RetroNectin®) with that of anti-28Ab or anti-4-1 BB Ab on T-cell expansion and its phenotype. Furthermore, we examined persistence of T cells expanded by these methods in NOG® mice. In addition, we conducted phase 1 clinical study to evaluate the safety and efficacy of FN-CH296 stimulated T cell therapy in patients with advanced cancer. Methods: PBMCs were activated by various stimulation methods and cultured in gas-permeable culture bag CultiLife TM 215 and CultiLife TM Eva for 10-14 days. After that, each expanded cells were analyzed for its phenotypes, in vivo persistence and the ability of accumulation in lymph node. In clinical study, patients underwent FN-CH296 stimulated T cell therapy up to six times every two weeks and safety and antitumor activity of the ACT were assessed. In order to determine immune function, whole blood cytokine levels were analyzed prior to ACT and during the follow up. Results: Co-stimulation by FN-CH296 with anti-CD3Ab led to higher T-cell expansion and proportion of CCR7 + CD45RA + T cells than that by the others (anti-28Ab/anti-4-1BB Ab). In the experiment for engraftment of expanded T cells in NOG mice, human CD3 + cells were detected in all groups, but the proportion of CCR7 + CD45RA + and CD8 + T cells was significantly higher specifically in the FN-CH296 co-stimulation condition. In addition, higher accumulation of human CD3 + cells in lymph node of NOG® mice was observed when stimulated by the FN-CH296 co-stimulation condition. In phase 1 clinical trial, nine patients were enrolled and infused 1, 3, or 9×10 9 of T cells to each cohort group (3 patients in each group). As a result, there was no ACT-related serious adverse event in all doses and one patient achieved CR, one achieved PR, four had SD, and three had PD. The number of less-differentiated cells that was infused showed a strong positive correlation with the change in whole blood IFN-γ level after ACT treatment. Conclusion: These results indicated that FN-CH296 stimulated T cells therapy was very well tolerated with a level of efficacy that is promising and the FN-CH296 stimulation method for ex vivo T-cell expansion is useful as basic technology for adoptive T-cell therapy, as it can be expanded preferentially less-differentiated T cells. Citation Format: Takeshi Ishikawa, Satoshi Kokura, Tetsuya Okayama, Naoyuki Sakamoto, Mitsuko Ideno, Nobuko Muraki, Akiko Kato, Tatsuji Enoki, Junichi Mineno, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. Advantages and clinical application of fibronectin CH296-stimulated T cells in cancer immunotherapy. [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 2791. doi:10.1158/1538-7445.AM2014-2791