Koji Kurose

Phase Ia Study of FoxP3+ CD4 Treg Depletion by Infusion of a Humanized Anti-CCR4 Antibody, KW-0761, in Cancer Patients

Purpose: FoxP3+ Tregs inhibit immune responses against tumors. KW-0761 is a humanized anti-human CCR4 monoclonal antibody (mAb) that has antibody-dependent cellular cytotoxicity activity. Depletion of CCR4-expressing FoxP3+ CD4 Tregs by KW-0761 infusion was investigated in solid cancer patients. Experimental Design: We conducted a phase Ia clinical trial of KW-0761 infusion in 7 lung and 3 esophageal cancer patients. Toxicity, clinical efficacy, changes in lymphocyte subpopulations, including Tregs, and induction of immune responses were analyzed. Results: The results showed that KW-0761 infusion in a dose range between 0.1 mg/kg and 1.0 mg/kg was safe and well tolerated. No dose-limiting toxicity was observed. Four of 10 patients showed stable disease during treatment and were long survivors. The monitoring of FoxP3+ Tregs in the peripheral blood mononuclear cells during treatment indicated efficient depletion of those cells, even at the lowest dose of 0.1 mg/kg used. The reduction in Th 1 CD4 T cells and CD8 T cells was limited, whereas a significant reduction was observed with Th 2 and Th 17 CD4 T cells. Immune responses to cancer/testis (CT) antigens and an autoantibody response to thyroid peroxidase were observed in some patients. Conclusions: The findings showed Tregs depletion and the possible occurrence of an immune response following KW-0761 infusion. Combined use of KW-0761 to deplete FoxP3+ Tregs with other immunotherapies, such as cancer vaccines or checkpoint inhibitors, is a promising approach to augment immune responses. Clin Cancer Res; 21(19); 4327–36. ©2015 AACR.

Increase in Activated Treg in TIL in Lung Cancer and In Vitro Depletion of Treg by ADCC Using an Antihuman CCR4 mAb (KM2760)

Introduction: Tregs infiltrate tumors and inhibit immune responses against them. Methods: We investigated subpopulations of Foxp3+ CD4 T cells previously defined by Miyara et al. (Immunity 30, 899–911, 2009) in peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) in lung cancer. We also showed that Tregs in healthy donors that express CCR4 could be efficiently eliminated in vitro by cotreatment with antihuman (h) CCR4 mAb (KM2760) and NK cells. Results: In lung cancer, the number of activated/effector Tregs and non-Tregs, but not resting/naive Tregs, was increased in TILs compared with the number of those cells in PBMCs. The non-Treg population contained Th2 and Th17. CCR4 expression on activated/effector Tregs and non-Tregs in TILs was down-regulated compared with that on those cells in PBMCs. Chemokinetic migration of CD25+ CD4 T cells containing the Treg population sorted from the PBMCs of healthy donors to CCL22/MDC was abrogated by pretreatment with anti-hCCR4 mAb (KM2760). The inhibitory activity of CD25+ CD127dim/− CD4 Tregs on the proliferative response of CD4 and CD8 T cells stimulated with anti-CD3/CD28 coated beads was abrogated by adding an anti-hCCR4 mAb (KM2760) and CD56+ NK cells to the culture. Conclusions: The findings suggested the CCR4 on activated/effector Tregs and non-Tregs was functionally involved in the chemokinetic migration and accumulation of those cells to the tumor site. In vitro findings of efficient elimination of Tregs may give the basis for implementation of a clinical trial to investigate Treg depletion by administration of an anti-hCCR4 mAb to solid cancer patients.

Survival of Lung Adenocarcinoma Patients Predicted from Expression of PD-L1, Galectin-9, and XAGE1 (GAGED2a) on Tumor Cells and Tumor-Infiltrating T Cells.

The survival of lung adenocarcinoma patients could be predicted with the use of a discriminant function using as parameters tumor cell expression of PD-L1, Galectin-9, and XAGE1 (GAGED2a), and CD4 and CD8 T-cell infiltration. The immune status of tumors varies, and this may affect the overall survival (OS) of patients. We examined tumors from 120 patients with lung adenocarcinomas with a tissue microarray for T-cell infiltration and the expression of PD-L1 and Galectin-9 (both ligands for inhibitory receptors on T cells), and cancer/testis (CT) antigen XAGE1 (GAGED2a; a tumor antigen often found on lung tumors) expression, to determine their relevance to OS. Patients defined as pStage I–IIIA could be grouped, based on the expression profiles of PD-L1, Galectin-9, and XAGE1, into cluster A, who had prolonged survival, and cluster B, who had shorter survival. The difference in survival of the clusters was confirmed separately for pStage I and pStage II–IIIA patients. Cluster A patients who also had CD4 and CD8 T-cell infiltration showed even better survival, as expected. The findings were confirmed by examining an independent validation cohort of 68 pStage I lung adenocarcinoma patients. Our data showed that PD-L1 expression was a positive indicator, whereas Galectin-9 and XAGE1 expression was negative. In vitro analyses suggested that PD-L1 expression was upregulated by IFNγ secreted from activated T cells in the tumor and Galectin-9 expression was counteracting those T cells. Thus, use of these immune markers enables the creation of a discriminant function with which to classify tumors and predict survival. Cancer Immunol Res; 4(12); 1049–60. ©2016 AACR.

Prolongation of overall survival in advanced lung adenocarcinoma patients with the XAGE1 (GAGED2a) antibody.

Purpose: The cancer/testis antigen XAGE1 (GAGED2a) is expressed in approximately 40% of advanced lung adenocarcinomas. We investigated the clinical relevance of the XAGE1 (GAGED2a) immune responses in patients with advanced lung adenocarcinoma. Experimental Design: The XAGE1 (GAGED2a) antigen expression and EGFR mutation were determined with tumor tissues. The XAGE1 (GAGED2a) antibody and T-cell immune responses, as well as immune cell phenotypes, were analyzed with blood samples. Patients with EGFR wild-type (EGFRwt) tumors were treated with conventional platinum-based doublet chemotherapy and patients with EGFR-mutated (EGFRmt) tumors were treated with EGFR-TKI and conventional chemotherapy. The overall survival (OS) rates of the antibody-positive and -negative patients were investigated. Results: The results showed that the OS of antibody-positive patients was prolonged significantly compared with that of antibody-negative patients with either XAGE1 (GAGED2a) antigen-positive EGFRwt (31.5 vs. 15.6 months, P = 0.05) or EGFRmt (34.7 vs. 11.1 months, P = 0.001) tumors. Multivariate analysis showed that the presence of the XAGE1 (GAGED2a) antibody was a strong predictor for prolonged OS in patients with XAGE1 (GAGED2a) antigen-positive tumors and in patients with either EGFRwt or EGFRmt tumors. On the other hand, XAGE1 (GAGED2a) antigen expression was a worse predictor in patients with EGFRmt tumors. Phenotypic and functional analyses of T cells indicated immune activation in the antibody-positive patients. Conclusions: The findings suggest that production of the XAGE1 (GAGED2a) antibody predicts good prognosis for patients with lung adenocarcinoma as an immune biomarker and the protective effect of this naturally occurring immune response supports the concept of immunotherapy. Clin Cancer Res; 20(19); 5052–63. ©2014 AACR.

Step-down of budesonide/formoterol in early stages of asthma treatment leads to insufficient anti-inflammatory effect

Abstract Objective: Administration of the combination of an inhaled corticosteroid (ICS) and a long-acting beta agonist (LABA) is the main treatment strategy for bronchial asthma. The ICS/LABA dosage can be reduced (stepped down) when the patient’s symptoms and lung functions are well-controlled. In this study, we obtained fractional exhaled nitric oxide (FeNO) measurements to clarify whether the anti-inflammatory effect of budesonide/formoterol is shortened by step-down. Methods: Fifty-four patients who visited the Kawasaki Medical School Hospital with newly diagnosed asthma from November 2008 to July 2010 received budesonide/formoterol for 8 weeks or more. In 29 patients, the forced expiratory volume in 1 s% predicted increased to 80% or more, and the Asthma Control Questionnaire (ACQ) score decreased to 0.5 or less within 12 weeks. These 29 patients were randomly divided into two groups: the dosage-continued group (n = 14) and the step-down group (n = 15). Then, the impact of budesonide/formoterol step-down on ACQ score, pulmonary function and FeNO level was compared between the groups. Results: In the step-down group, the dosage was stepped down from 538 mcg/day to 331 mcg/day. In both groups, pulmonary function indicators and symptoms did not change. However, the mean FeNO level decreased significantly in the dosage-continued group (from 50.9 ppb to 45.0 ppb), and increased significantly in the step-down group (from 51.0 ppb to 65.7 ppb). Conclusions: Clinicians should be more careful when stepping down budesonide/formoterol based solely on patients’ symptoms and/or pulmonary function.

NY-ESO-1 Protein Cancer Vaccine With Poly-ICLC and OK-432: Rapid and Strong Induction of NY-ESO-1-specific Immune Responses by Poly-ICLC.

We conducted a clinical trial of a cancer vaccine using NY-ESO-1 protein with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) and/or OK-432 against solid tumors. A total of 15 patients were sequentially enrolled in 4 cohorts. Patients in cohort 1 received NY-ESO-1 protein; cohort 2a received NY-ESO-1 protein+OK-432; cohort 2b received NY-ESO-1 protein+poly-ICLC; cohort 3 received NY-ESO-1 protein+OK-432+poly-ICLC with Montanide ISA-51. The endpoints of this trial were safety, NY-ESO-1 immune responses, and clinical response. Vaccine-related adverse events observed were fever and injection-site reaction (grade 1). Two patients showed stable disease after vaccination. NY-ESO-1 antibodies were observed in 4 patients at the baseline (sero-positive) and augmented in all patients after vaccination. Eleven patients showed a conversion of negative antibody responses at baseline to positive after vaccination (seroconversion). The seroconversions were observed in all 11 sero-negative patients by the fourth immunization; in particular, it was observed by the second immunization in patients with poly-ICLC, and these induced antibody responses were stronger than those in patients immunized without poly-ICLC. The number of NY-ESO-1–specific interferon (IFN)&ggr;-producing T cells was increased in patients immunized with poly-ICLC and/or OK-432, and furthermore, the increase of IFN&ggr;-producing CD8 T cells in patients immunized with poly-ICLC was significantly higher than that in patients without poly-ICLC. Nonspecific activations of T-cell or antigen presenting cells were not observed. Our present study showed that poly-ICLC is a promising adjuvant for cancer vaccines.

Abstract 4732: Depletion of Tregs from PBMCs by treatment with defucosylated anti-CCR4 mAb.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Objectives: Tregs down-regulate immune responses against various antigens including tumor cells. Impairment of Tregs cause autoimmune responses. CC chemokine receptor 4 (CCR4) is expressed selectively on Foxp3+CD4 T-cells, so that the treatment of PBMCs with KM2760, anti-human CCR4 mAb with a defucosylated Fc region, could deplete CCR4+Tregs by ADCC. In this study, we analyzed depletion of Tregs phenotypically and functionally after treatment of PBMCs with KM2760. Methods and main results: CCR4 expression was analyzed on CD4 T-cells purified from healthy donor PBMCs by FACS. CCR4 positivecells were 21% in CD4 T-cells, and 71% in Foxp3+cells. Depletion of CCR4 positive cells by treatment with KM2760 was examined. PBMCs were incubated with KM2760 (0, 0.01, 0.1, 1, 10 μg/ml) for 4 or 20 hours, and analyzed by FACS. Efficient depletion was observed with higher antibody concentration and longer incubation time. Then we examined inhibition of CFSE dilution of labeled CD4 or CD8 T cells stimulated with anti-CD3 mAb after culture with an equal number of CD4+25+127low Tregs and CD4-8- cells pretreated with or without KM2760. Proliferating responder cells were 68% in culture with CD4+25+127low Tregs pretreated with KM2760, whereas those cells with CD4+25+127low Tregs without pretreatment was 38% in CD4 T-cells, 77%and 40% in CD8 T-cells,respectively. Conclusions: Efficient Treg depletion was observed in vitro by treatment of PBMCs with KM2760. In vivo treatment with the same mAb may induce tumor immune responses by Treg depletion. Citation Format: Koji Kurose, Shingo Eikawa, Yu Mizote, Yoshihiro Ohue, Midori Isobe, Akiko Uenaka, Mikio Oka, Eiichi Nakayama. Depletion of Tregs from PBMCs by treatment with defucosylated anti-CCR4 mAb. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4732. doi:10.1158/1538-7445.AM2013-4732

Abstract A101: Role of TIM-3/Galectin-9 pathway in lung cancer

Purpose: T-cell inhibitory receptors and their ligands, and their roles in the tumor immune response have been extensively studied recently. Cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death 1 (PD-1) or T cell immunoglobulin and mucin domain 3 (TIM-3) have been identified on the CD4 and CD8 T-cell surface, and it has been shown that the pathways of CTLA-4 and its ligand CD80 or CD86 (B7-2), PD-1 and its ligand PD-L1 or PD-L2 (B7-DC), and TIM-3 and its ligand Galectin-9 axes contributed to effector T-cell dysfunction in the tumor immune microenvironment. Those T-cell inhibitory receptors and their ligands also play important roles in the immune response in lung adenocarcinoma. In this study, we investigated inhibitory roles of TIM-3/Galectin-9 pathway in lung adenocarcinoma. Experimental Design: TILs or PBMCs were obtained from lung cancer patients. Expression of inhibitory molecules on those cells was analyzed by flow cytometry using FACS. PD-L1 and Galectin-9 expression in lung cancer was analyzed using tissue microarray by immunohistochemistry (IHC). To investigate soluble Galectin-9 released from the tumor cells, an EGFR-mutated tumor cell line, PC-9 and control EGFR wild type cell line, OU-LC-SK were treated with Afatinib at a concentration of 10 nM for 3 days. Then, the amount of Galectin-9 in culture supernatant was measured by ELISA. To investigate T-cell apoptosis induction by Galectin-9, established XAGE1-specifiic CD8 cloned T-cells were incubated with Galectin-9 protein for 8 hrs. Results: First, we examined expression of PD-1, TIM-3, BTLA and LAG-3 on CD4 and CD8 T-cells obtained from PBMCs and TILs in 11 lung cancer patients. Augmented expression in TILs compared to PBMCs was observed on PD-1 and TIM-3, but not on BTLA or LAG-3 in CD4 and CD8 T-cells. Next, we analyzed expression of the T-cell inhibitory receptor ligands PD-L1 and Galectin-9 in lung cancer by IHC. With adenocarcinoma, the frequencies of high PD-L1 and Galectin-9 expression in the cell membrane or cytoplasm were 49% and 31%, respectively. On the other hand, with squamous cell carcinoma, the frequencies were 32% and 16%, respectively. Furthermore, correlated expression of PD-L1, Galectin-9 and CD3 (T-cell infiltration) was observed at the periphery of the tumor nest. Those findings suggest the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment in lung cancer. To investigate soluble Galectin-9 released from the tumor cells, EGFR-mutated and -wild type tumor cell lines were treated with EGFR-TKI. Galectin-9 was detected in the medium of EGFR-mutated lung cancer cell lines following treatment with Afatinib. Moreover, apoptosis was induced in TIM-3-positive CD8 T-cell clones following interaction with Galectin-9 protein and this was inhibited by the addition of anti-Galectin-9 or an anti-TIM-3 antibody. The findings suggested that a significant amount of Galectin-9 could be released and induced T-cell apoptosis in tumor microenvironment. Conclusions: The results suggested the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment in lung cancer and that release of soluble Galectin-9 from tumor cells could negatively regulate T-cell function. Citation Format: Yoshihiro Ohue, Koji Kurose, Yumi Nishio, Midori Isobe, Mikio Oka, Eiichi Nakayama. Role of TIM-3/Galectin-9 pathway in lung cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A101.

Stability of sealed-bag samples for off-line measurement of fractional exhaled nitric oxide

BACKGROUND Fractional exhaled nitric oxide (FeNO) measurement is an index of airway eosinophilic inflammation and is primarily employed to diagnose bronchial asthma and assess airway inflammation. It can be measured with on- and off-line methods. In the latter, sample storage is possible; this is useful in Japan, because the number of facilities in which this instrument has been installed is limited. OBJECTIVE We investigated the maximum period in which the samples continued to show stable values, as well as the influences of the storage environment. METHODS Exhaled air samples were collected from 19 bronchial asthma patients (male/female: 8/11; mean age: 54.9 ± 15.6 years), and divided into 2 groups: a group with an FeNO level of less than 30 ppb and that with a level of 30 ppb or more. They were stored at 4 and 25°C to examine serial changes. RESULTS The off-line-measured FeNO value was 33.7 ± 17.1 ppb, significantly lower than the on-line measured value (71.4 ± 47.7 ppb) (P < .01). In a group with an FeNO level of less than 30 ppb, temperature had no influence. However, in the other group, the period was prolonged to 72 hours when the samples were stored at 4°C. The samples were stable at 4°C for 24 hours, regardless of the initial measurements. CONCLUSION The duration of sample storage can be prolonged at 4°C. The standard FeNO measurement method is the on-line method, but the alternative use of the off-line method may contribute to the diagnosis and treatment of the patients.

Nivolumab-induced polyarthritis

A 48-year-old Japanese woman with non-small cell lung adenocarcinoma (cT3N2M1b) received nivolumab (an immune checkpoint inhibitor) at a dosage of 3 mg/kg every 2 weeks. Treatment with nivolumab was effective. The primary tumour as well as metastases to the lymph nodes and spine almost completely disappeared, as shown on positron emission tomography-CT before treatment (figure 1A) and 4 months after treatment (figure 1B). However, she developed sustained pain and swelling in the shoulders and knees bilaterally after only one infusion of …