Toshifumi Doi

Cytotoxic T lymphocyte-associated antigen 4 inhibition increases the antitumor activity of adoptive T-cell therapy when carried out with naïve rather than differentiated T cells

Although treatment with an antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) combined with multiple therapeutic interventions has been explored, the effect of combination therapy with CTLA-4 inhibition and adoptive T-cell therapy has not been determined. In the present study, our aim was to determine whether CTLA-4 inhibition, combined with adoptive transfer of T cells at different stages of differentiation, exhibits synergistic antitumor effects in a murine colon cancer model. Mice bearing subcutaneous tumors were administered adoptive T-cell transfer of CD62Lhigh or CD62Llow cells combined with an anti-CTLA-4 antibody (α-CTLA-4) or control immunoglobulin G. Subcutaneous tumors were harvested, and the antitumor effects and helper T-cell polarization were analyzed. CTLA-4 inhibition combined with CD62Lhigh cell administration showed the strongest antitumor effect. Combination therapy increased the number of CD3+ cells within the tumor. Moreover, CTLA-4 inhibition induced polarization of T cells infiltrating the tumor toward the T helper 1 lineage, and suppressed the frequency of regulatory T cells within the tumor, particularly in combination with CD62Lhigh T-cell transfer. This is the first report demonstrating that the efficacy of α-CTLA-4 and adoptive T-cell transfer combination therapy depends on the state of differentiation of the transferred T cells. Our data support the notion that a combination of α-CTLA-4 and adoptive T-cell transfer containing an abundance of naïve phenotype cells could potentially exert antitumor effects in a clinical setting.

Transforming growth factor β1-induced collagen production in myofibroblasts is mediated by reactive oxygen species derived from NADPH oxidase 4

Intestinal fibrosis with stricture formation is a severe complication of Crohns disease (CD). Though new therapeutic targets to enable the prevention or treatment of intestinal fibrosis are needed, markers of this condition and the basic mechanisms responsible have not been established. NADPH oxidase (NOX) 4 has already been reported to play a key role in models of fibrogenesis, including that of the lung. However, its importance in intestinal fibrogenesis remains unclear. In this study, we examined the role of NOX4 in collagen production by intestinal myofibroblasts stimulated with transforming growth factor (TGF)-β1. Using LmcMF cells, an intestinal subepithelial myofibroblast (ISEMF) line, we first examined the induction of collagen production by TGF-β1. Subsequently, we investigated the role of NOX4 in TGF-β1-induced collagen I production in these cells using SB525334 (an SMAD2/3 inhibitor), diphenyleneiodonium (an NOX inhibitor), and Nox4 small interfering RNA (siRNA). Production of collagen was assessed with Sirius red staining, and Nox4 expression was measured by quantitative real-time PCR. Reactive oxygen species (ROS) production was determined using DCFDA and fluorescent microscopy. We observed that TGF-β1 induced collagen production via NOX4 activation and ROS generation in LmcMF cells. Nox4 siRNA and inhibitors of TGF-β1 receptor and NOX significantly reduced TGF-β1-induced ROS and collagen production. Thus, in the present study, we revealed that collagen production in ISEMFs is induced via an NOX4-dependent pathway. This work supports a function for NOX4 in intestinal fibrogenesis and identifies it as a potential therapeutic target in recalcitrant fibrotic disorders of CD patients.

Abstract 4009: The expression of PD-L1 on human and murine pancreatic ductal adenocarcinoma is enhanced by anticancer agents via the JAK/STAT pathway

Background: Pancreatic ductal adenocarcinoma(PDA) is the fourth most common cause of cancer-related death in Japan. Recently, new standard chemotherapies for PDA have been developed, but they are still largely unsatisfactory. Therefore, development of new treatment options has been required to improve the outcomes of patients with PDA. To break through this situation, blocking one of inhibitory immune checkpoints, Programmed death-1 (PD-1) /Programmed death-ligand 1 (PD-L1) pathway is considered to be a hopeful candidate for new treatment strategies for PDA. In this context, for the future combination therapy of anticancer agents and immune check point inhibitors, we investigated how anticancer agents influence the expression of PD-L1 on pancreatic cancer cell lines. Additionally we analyzed the molecular mechanism by which PD-L1 expression on the pancreatic cancer cell lines are regulated. Methods: Human PDA cell lines MIA PaCA-2, AsPC-1 and murine PDA cell line Pan02 were used in this study. These cells were adjusted to 1.0 × 105 / ml and incubated with anticancer agents (i.e. gemcitabine, paclitaxel and 5-fluorouracil) at 37°C for 24-72h. Then, the expression level of PD-L1 was determined using qRT-PCR and flow cytometry. For the blocking experiment of the JAK/STAT pathway, AG490 was used as a blocking agent. After 48h incubation of AsPC-1 cells at 37°C, cells were treated with various concentration of AG490 for 1h. Cells were stimulated with 5-fluorouracil, paclitaxel and gemcitabine, and then incubated in the presence or absence of AG490 for an additional 6-48h. The expression of PD-L1 was analyzed using qRT-PCR and flow cytometry. Stat1 and phospho-Stat1 protein were analyzed by western blotting. Results: In AsPC-1, MIA PaCA-2 and Pan02, the expression of PD-L1 was enhanced with all three anticancer agents in a concentration dependent manner. The phosphorylation of STAT1 and the increase of total STAT1 was observed in AsPC-1 when stimulated by each anticancer agents. After the treatment of JAK/STAT inhibitor, the phosphorylation of STAT1 was attenuated, and the PD-L1 upregulation induced by anticancer agents was cancelled in a concentration dependent manner. Conclusion: The stimulation of anticancer agents leads to an enhancement of PD-L1 expression on PDA cell lines. The JAK/STAT pathway is reported to be involved in IFN-γ mediated PD-L1 upregulation in lung cancer and hepatocellular carcinoma cell lines. In the present study, the phosphorylation of Stat1 and the expression of total Stat1 were enhanced after the anticancer agents treatment. Moreover, JAK/STAT inhibitor attenuated anticancer agents-induced PD-L1 expression. Taken together, the JAK/STAT pathway may be responsible for the anticancer agents mediated PD-L1 transcription. Citation Format: Toshifumi Doi, Takeshi Ishikawa, Tomoyo Yasuda, Tetsuya Okayama, Kaname Oka, Naoyuki Sakamoto, Yuji Naito, Yoshito Itoh. The expression of PD-L1 on human and murine pancreatic ductal adenocarcinoma is enhanced by anticancer agents via the JAK/STAT pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4009.

Abstract 634: Impact of tumor inoculation site on the development of cancer cachexia

Background and Aim The incidence of cancer cachexia varies greatly among tumor type. Although certain tumor types are more commonly associated with cachexia, even for the same tumor type, the degree to which patients exhibit cachexia varies. This is due to variations in tumor phenotype and host genotype that contributes to the development of cancer cachexia. The influence of the site of tumor growth on the development of cancer cachexia remains unclear. In this study, we evaluated if differences in tumor microenvironment would affect the development of cancer cachexia in a murine model. Methods The same number of colon26 cells (5×10⁁6 cells/mouse) was inoculated subcutaneously or intraperitoneally in 8-week-old male BALB/c mice. Body weight, food intake, and tumor size were monitored. On day 14 after the tumor inoculation, mice were anesthetized and blood collected, and the weight of peritesticular adipose tissue, gastrocnemius muscle and heart were measured. 23 plasma cytokines were determined by using the Bio-Plex array system and plasma Myostatin and Activin A levels were measured by ELISA. Expression of MuRF-1 and Antrogin-1, which are required for muscle atrophy, in gastrocnemius muscle or heart was assessed by RT-PCR. Results The body weight, adipose tissue and gastrocnemius muscle decreased in tumor-bearing mice. Interestingly, a reduction in heart weight was observed in the intraperitoneal tumor group but not in the subcutaneous group. The weight loss of the body, adipose tissue and gastrocnemius was more severe in the intraperitoneal group than in the subcutaneous group. Atrogin 1 and MuRF1 mRNA expressions in the gastrocnemius muscle increased significantly in both groups of tumor-bearing mice, however, in the myocardium, expression of these mRNAs increased in the intraperitoneal group but not in subcutaneous group. As for plasma cytokine levels, several cytokine levels including IL-6, TNF-α, and activing A of tumor-bearing mice were significantly higher than those of control mice. Eotaxin and G-CSF levels in the intraperitoneal tumor group were higher than in the subcutaneous group. Conclusion Based on these data, we believe that differences in microenvironment where tumor cells develop can affect the progression and phenotype of cancer cachexia through alterations in various circulating factors derived from the tumor microenvironment. Citation Format: Takashi Ando, Takeshi Ishikawa, Tastuzo Matsuyama, Tomoyo Yasuda, Toshifumi Doi, Tetsuya Okayama, Naoyuki Sakamoto, Satoshi Kokura, Yuji Naito, Yoshito Itoh. Impact of tumor inoculation site on the development of cancer cachexia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 634.

Abstract 1326: Elevated levels of plasma VEGF associated with the attenuation of whole blood IFN-γ production and QOL impairment in patients with advanced gastric cancer

Introduction: Gastric cancer is one of the leading types of cancer and the second leading cause of cancer death in the world. Recently, the foci of treatment shift towards not only survival benefit but also better quality of life (QOL) in patients with advanced gastric cancer. Therefore, the development of an auxiliary tool to assess QOL is likely to be required. On the other hand, immune checkpoint blockade by anti-CTLA-4 antibody and anti-PD-1 antibody led to clinical breakthrough for the treatment of patients with advanced cancer. Early phase clinical trial of pembrolizumab in patients with advanced gastric cancer has already been conducted. Pretreatment circulating VEGF have been recently shown to associate with clinical outcome in patients treated with ipilimumab. Certain circulating cytokine levels have been shown to reflect tumor progression and have a prognostic value. Although, it remains obscure whether circulating cytokine levels affect QOL and immune function. In this study, we examined the correlation of circulating cytokine with QOL as well as parameters of immune function such as whole blood cytokine production and the number of peripheral Tregs in patients with advanced gastric cancer. Methods: Subjects comprised 31 patients with unresectable or recurrent gastric cancer. We evaluated plasma cytokine levels and whole blood cytokine production after phytohemagglutinin (PHA) stimulation using the bioplex array system. We also assessed the number of peripheral Tregs by flow cytometry. Health-related QOL was assessed using the Quality of Life Questionnaire (EORTC QLQ-C30). Results: Significant negative correlation was found between plasma VEGF levels and global health status scores (p = 0.0103) as well as physical functioning scale scores (p = 0.0006) and cognitive functioning scale scores (p = 0.0191). Some symptom scale scores (e.g. fatigue, appetite loss) were correlated with plasma VEGF levels. There was a negative correlation between plasma VEGF levels and whole blood IFN-γ production (p = 0.0002). Significant negative relationship existed between the number of peripheral Tregs and plasma IL-6 levels (p Conclusion: These data indicate that the evaluation of plasma VEGF level is likely to be useful to assess QOL such as global health status, physical functioning and cognitive functioning and elevated levels of plasma VEGF might associated with the attenuation of immune function in patients with advanced gastric cancer. Consequently, clinical utility of anti-VEGF therapy combined with immunotherapy is required to investigate in larger clinical studies in the future. Citation Format: Naoyuki Sakamoto, Takeshi Ishikawa, Tetsuya Okayama, Tomoyo Yasuda, Toshifumi Doi, Hideyuki Konishi, Satoshi Kokura, Mari Tanigawa, Kazuko Uno, Yuji Naito, Yoshito Ito, Toshikazu Yoshikawa. Elevated levels of plasma VEGF associated with the attenuation of whole blood IFN-γ production and QOL impairment in patients with advanced gastric cancer. [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 1326. doi:10.1158/1538-7445.AM2015-1326

Abstract 1330: The influence of anticancer agents and heat treatment on PD-L1 expression on human and murine pancreatic cancer cell lines

Background: Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies. Currently conducted chemotherapy for PDA didn9t get the sufficient clinical results, so we need to get new strategy for PDA therapy immediately. For breaking through this situation, the inhibitors of immune checkpoint PD-1/PD-L1 are hopeful candidates because PD-1/PD-L1 inhibitors relatively got the good results for some advanced malignancies including PDA, but the sole therapy doesn9t result in good outcome. In murine model, the combination therapy of PD-L1 inhibitor and gemcitabine has been reported to show a synergistic effect on PDA compared to each sole therapy. But the mechanism of the synergistic effect is not well understood. On the other hand, we have previously demonstrated the efficacy of the combined therapy of regional heat treatment and gemcitabine in advanced PDA patients, but the effect of heat treatment on immune checkpoint is also unknown. Then, for future combination therapy of anticancer agents, immune check point inhibitor and heat treatment, we analyzed the influence of some anticancer agents and heat treatment on the expression of PD-L1 on pancreatic cancer cell lines. Methods: Human pancreatic cancer cell lines MIA PaCA-2, AsPC-1 and murine pancreatic cancer cell line Pan02 were used in this study. These cells were adjusted to 1.0 × 105 / ml and incubated with anticancer agents such as gemcitabine, paclitaxel and 5-fluorouracil at 37°C for 24 - 72 hours. For checking the influence of anticancer agents on the expression of PD-L1, their expression of PD-L1 was examined by flow cytometry and qRT-PCR. Next, before exposing cancer cells to gemcitabine, MIA PaCA-2 and AsPC-1 were incubated at 43°C as heat treatment. After the heat treatment, PD-L1 expression was analyzed using flow cytometry. Results: In AsPC-1 and Pan02, PD-L1 expression was enhanced with all three anticancer agents in concentration dependency by both flow cytometry and qRT-PCR. In MIA PaCA-2, PD-L1 expression was enhanced with all three anticancer agents by flow cytometry analysis, but the result of qRT-PCR could not show the difference. PD-L1 expression on MIA PaCA-2 and AsPC-1 with heat treatment was suppressed compared to only anticancer agent treatment. Conclusion: The stimulation of anticancer agents tends to lead to an enhancement of PD-L1 expression on both human and murine pancreatic cancer cell lines. The synergistic effect of PD-L1 inhibitor and gemcitabine has the possibility to be brought about by PD-L1 up-regulation. On the other hand, heat treatment has the possibility to suppress PD-L1 expression and this mechanism might lead to cancel out the immune tolerance caused by anticancer agent administration with up-regulation of PD-L1. For future, we need to further examine the most effective timing and method of combined therapy of anticancer agent, PD-L1 inhibitor and heat treatment. Citation Format: Toshifumi Doi, Tetsuya Okayama, Takeshi Ishikawa, Kaname Oka, Naoyuki Sakamoto, Tomoyo Yasuda, Yuji Naito, Yoshito Itoh. The influence of anticancer agents and heat treatment on PD-L1 expression on human and murine pancreatic cancer cell lines. [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 1330. doi:10.1158/1538-7445.AM2015-1330

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