Yoshihiro Ohue

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.

Comparison of T-cell interferon-γ release assays for Mycobacterium tuberculosis-specific antigens in patients with active and latent tuberculosis.

Through the use of QuantiFERON-TB Gold, a commercial IFN-γ assay, we compared differences in quantitative T-cell responses to Mycobacterium tuberculosis (MTB)-specific antigens [QuantiFERON TB-2G (QFT-2G)] between patients with active tuberculosis (TB) disease and those with latent TB infection (LTBI). The patient group consisted of 180 patients with active TB disease (culture-positive for MTB) and 50 screening contacts with LTBI-positive response to the QFT-2G test. We prospectively performed a tuberculin skin test (TST) and a QFT-2G test for all subjects. The median IFN-γ levels upon the application of both antigens, ESAT-6 and CFP-10, were significantly higher in patients with active TB disease than in those with LTBI. A combined positive response to both antigens occurred at a higher rate in patients with active TB disease than in those with LTBI. There were no significant relationships between the quantitative responses of IFN-γ to both antigens and the maximum induration on TST in both patient groups. We demonstrated significant differences in the quantitative responses of IFN-γ to MTB between patients with active TB disease and those with LTBI in this study. However, there was an overlap in the IFN-γ levels between active TB disease and LTBI groups. Therefore, it would be difficult to use the QFT-2G test to completely discriminate active TB disease from LTBI.

Spontaneous antibody, and CD4 and CD8 T-cell responses against XAGE-1b (GAGED2a) in non-small cell lung cancer patients

The spontaneous immune responses against XAGE‐1b (GAGED2a) were analyzed in non‐small cell lung cancer (NSCLC) patients. An antibody response against XAGE‐1b (GAGED2a) was observed in 10% (20/200) of NSCLC patients and in 19% (13/69) of stage IIIB/IV lung adenocarcinoma patients. A CD4 T‐cell response was detected in 88% (14/16) and a CD8 T‐cell response in 67% (6/9) in the XAGE‐1b (GAGED2a) antibody‐positive patients examined. Frequent antibody responses and CD4 and CD8 T‐cell responses in XAGE‐1b (GAGED2a) antibody‐positive patients indicate the strong immunogenicity of the XAGE‐1b (GAGED2a) antigen in NSCLC patients. We established T‐cell clones from PBMCs of antibody‐positive patients and determined the DRB1*04:05‐restricted XAGE‐1b (GAGED2a) 18–31 peptide (14‐mer) as a CD4 T cell epitope and the A*02:06‐restricted XAGE‐1b (GAGED2a) 21‐29 peptide (9‐mer) as a CD8 T cell epitope. As for peptide recognition, CD4 and CD8 T‐cell clones responded to naturally processed antigen. The CD4 T‐cell clone recognized DCs pulsed with the synthetic protein or a lysate from XAGE‐1b‐transfected 293T cells. The CD8 T‐cell clone showed cytotoxicity against a tumor expressing XAGE‐1b (GAGED2a) and the appropriate HLA class I allele. These findings establish XAGE‐1b (GAGED2a) as a promising target for a lung cancer vaccine.

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.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20‐mer NY‐ESO‐1f peptide vaccine was evaluated in a lung cancer patient TK‐f01, immunized with the peptide with Picibanil OK‐432 and Montanide ISA‐51. We showed that internalization of the peptide was necessary to present CD8 T‐cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T‐cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01‐restricted CD8 T‐cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02‐restricted CD8 T‐cell clones were defined and peptide/HLA tetramers were produced. NY‐ESO‐1 91‐101 on A*24:02, NY‐ESO‐1 92‐102 on B*35:01, NY‐ESO‐1 96‐104 on B*52:01 and NY‐ESO‐1 96‐104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T‐cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20‐mer NY‐ESO‐1f peptide harboring multiple CD8 T‐cell epitopes as an NY‐ESO‐1 vaccine. Characterization of CD8 T‐cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T‐cell responses comprised the dominant response.

Nutritional deficits in elderly smokers with respiratory symptoms that do not fulfill the criteria for COPD

Background and objective Whereas nutrition deficits are recognized as an expression of systemic inflammation in the elderly with diagnosed chronic obstructive pulmonary disease (COPD), if they occur in symptomatic elderly smokers, unfulfilled COPD criteria are not confirmed. Methods Respiratory function, anthropometry assessment, and diet intake evaluation of 13 COPD patients (COPD group), ten symptomatic elderly smokers (SYSM group), and 27 healthy volunteers (control group) were compared. All were 70 years old or older. Results The SYSM group had lower body weight, body mass index, percentage ideal body weight, body fat percentage, arm muscle circumference, tricep skin fold thickness, serum albumin, prealbumin, and transferrin than the control group and were similar to the COPD group (P < 0.05 each and nonsignificant each). Resting energy expenditure was no different among the groups. Intake of energy, vitamins (A, B1, B2, and C), calcium, iron, fiber, and sodium was also lower in the SYSM group than in the control group (P < 0.05 all) and was similar to the COPD group. Conclusion Elderly smokers who are symptomatic but who do not fulfill the COPD diagnostic criteria have nutritional deficits related to insufficient energy intake that are similar to those seen in COPD 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.

Antibody response to cancer/testis (CT) antigens: A prognostic marker in cancer patients.

Immune responses to tumor antigens have been reported in cancer patients. However, the relevance of such spontaneous immune responses to the clinical course has not been studied extensively. We showed that the overall survival of patients with antibodies against NY-ESO-1 or XAGE1 (GAGED2a) antigen was prolonged in gastric or lung cancer patients, respectively.

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.