Midori Isobe

Antibody response against NY‐ESO‐1 in CHP‐NY‐ESO‐1 vaccinated patients

NY‐ESO‐1 specific humoral responses are frequently observed in patients with various types of NY‐ESO‐1 antigen expressing tumors. In a large proportion of NY‐ESO‐1 antibody‐positive patients of NY‐ESO‐1‐specific CD8 T‐cells can also be detected suggesting that monitoring of the NY‐ESO‐1 specific humoral immune response may be a relevant and more practical surrogate for estimating the overall immune response against NY‐ESO‐1 in clinical vaccine studies. We have immunized 9 cancer patients with full length NY‐ESO‐1 protein formulated with cholesterol‐bearing hydrophobized pullulan (CHP‐NY‐ESO‐1) and investigated the humoral immune responses against NY‐ESO‐1. Seven patients were NY‐ESO‐1 antibody‐negative and 2 patients were positive prior to vaccination. Vaccination with CHP‐NY‐ESO‐1 resulted in the induction or increase of NY‐ESO‐1 antibody responses in all 9 patients immunized. Epitope analysis revealed 5 regions in the NY‐ESO‐1 protein molecule that were recognized by antibodies induced after vaccination. The 5 regions were also recognized by antibodies present in nonvaccinated, NY‐ESO‐1 antibody‐positive cancer patients. A peptide spanning amino acids 91–108 was recognized in 6 out of 9 vaccinated patients and in 8 out of 9 nonvaccinated, sero‐positive patients, being the most dominant antigenic epitope in NY‐ESO‐1 for antibody recognition in cancer patients. In conclusion, we showed that CHP‐NY‐ESO‐1 protein vaccination had a potent activity for inducing humoral immune responses against NY‐ESO‐1 antigen in cancer patients. The antigenic epitopes recognized by antibodies in the vaccinated patients were similar to those recognized in cancer patients with spontaneous humoral immunity against NY‐ESO‐1.

A phase I study of vaccination with NY-ESO-1f peptide mixed with Picibanil OK-432 and Montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen.

We conducted a phase I clinical trial of a cancer vaccine using a 20‐mer NY‐ESO‐1f peptide (NY‐ESO‐1 91–110) that includes multiple epitopes recognized by antibodies, and CD4 and CD8 T cells. Ten patients were immunized with 600 μg of NY‐ESO‐1f peptide mixed with 0.2 KE Picibanil OK‐432 and 1.25 ml Montanide ISA‐51. Primary end points of the study were safety and immune response. Subcutaneous injection of the NY‐ESO‐1f peptide vaccine was well tolerated. Vaccine‐related adverse events observed were fever (Grade 1), injection‐site reaction (Grade 1 or 2) and induration (Grade 2). Vaccination with the NY‐ESO‐1f peptide resulted in an increase or induction of NY‐ESO‐1 antibody responses in nine of ten patients. The sera reacted with recombinant NY‐ESO‐1 whole protein as well as the NY‐ESO‐1f peptide. An increase in CD4 and CD8 T cell responses was observed in nine of ten patients. Vaccine‐induced CD4 and CD8 T cells responded to NY‐ESO‐1 91–108 in all patients with various HLA types with a less frequent response to neighboring peptides. The findings indicate that the 20‐mer NY‐ESO‐1f peptide includes multiple epitopes recognized by CD4 and CD8 T cells with distinct specificity. Of ten patients, two with lung cancer and one with esophageal cancer showed stable disease. Our study shows that the NY‐ESO‐1f peptide vaccine was well tolerated and elicited humoral, CD4 and CD8 T cell responses in immunized patients.

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.

Induction of immune response against NY-ESO-1 by CHP-NY-ESO-1 vaccination and immune regulation in a melanoma patient.

BackgroundNY-ESO-1 is a cancer/testis antigen highly immunogenic in cancer patients. Cholesterol-bearing hydrophobized pullulan (CHP) is a nanoparticle-forming antigen-delivery vehicle and CHP complexed with NY-ESO-1 protein (CHP-NY-ESO-1) efficiently activates CD4 and CD8 T cells in vitro.AimIn this study we report on a 50-year-old male melanoma patient with multiple skin and organ metastases (T4N3M1c) who was vaccinated with CHP-NY-ESO-1 at biweekly intervals and who had an unusual disease course. We characterized in this patient humoral and cellular immune responses, immune regulatory cells, and cytokine profiles in the peripheral blood and at local tumor sites.ResultsTen days after the second CHP-NY-ESO-1 vaccination (day 25), blisters appeared on the skin at the metastatic lesions associated with inflammatory changes. A skin biopsy showed the presence of many NY-ESO-1-expressing apoptotic melanoma cells as determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) test. However, the tumors continued to grow, and the patient died of pulmonary failure due to multiple metastases on day 48. Serum antibody responses were detected after the second CHP-NY-ESO-1 vaccination and antibody titer increased with subsequent vaccinations. Th1 dependent IgG1 was the predominant immunoglobulin subtype. Both, NY-ESO-1-specific CD4 and CD8 T cell responses were detected in PBMC by IFN-γ secretion assays. After CHP-NY-ESO-1 vaccination a slight decrease in CD4+CD25+Foxp3+ Tregs was observed in PBMC but significantly increased numbers of CD4+CD25+Foxp3+ Tregs and CD68+ immunoregulatory macrophages were detected at the local tumor sites. CD4+CD25+Foxp3+ Tregs were also increased in the blister fluid. Cytokines in the serum suggested a polarization towards a Th1 pattern in the PBMC and those in the blister fluid suggested a Th2-type response at the tumor site.ConclusionsOur observations indicate induction of specific humoral and cellular immune responses against NY-ESO-1 after CHP-NY-ESO-1 vaccination in a melanoma patient. The concomitant appearance of regulatory T cells and of immune regulatory macrophages and cytokines at the local tumor sites in this patient may explain immune escape.

Analysis of peripheral and local anti-tumor immune response in esophageal cancer patients after NY-ESO-1 protein vaccination.

NY‐ESO‐1 antigen is a prototype of a class of cancer/testis antigens. We carried out a clinical trial using NY‐ESO‐1 whole protein as a cancer vaccine for 13 advanced cancer patients. We have recently reported that vaccine elicited humoral and cellular immune responses in 9 cancer patients including 4 esophageal cancer patients, and clinical responses were also observed in 4 of 5 evaluable patients. In this study, we analyzed the responses in 8 esophageal cancer patients including 4 newly enrolled patients. Patients were injected subcutaneously at biweekly intervals with NY‐ESO‐1 recombinant protein formulated with cholesterol‐bearing hydrophobized pullulan. Induction of antibody, and CD4 and CD8 T‐cell responses were observed in 7, 7 and 6 patients, respectively, out of 8 patients. 1 PR, 2 SD and 2 mixed clinical responses were observed in 6 evaluable patients. No significant adverse events were observed. Furthermore, we analyzed NY‐ESO‐1 and MHC class I expression and the infiltration of immune cells into tumor samples obtained before and after vaccination from 4 patients by immunohistochemistry. The results showed 2 patients with disappearance of CD4 and CD8 T‐cell infiltration, 1 patient with increase in the number of CD68+ macrophages and 1 patient with tumor antigen loss in the progressive tumors following vaccinations. The induction of NY‐ESO‐1 immunity and some preferable clinical outcomes were observed in esophageal cancer patients by vaccination with NY‐ESO‐1. However, the tumors grew eventually by various mechanisms after vaccination.

Heteroclitic serological response in esophageal and prostate cancer patients after NY-ESO-1 protein vaccination.

NY‐ESO‐1 is a prototypic cancer/testis antigen. In a recent phase I clinical trial, we vaccinated 13 patients bearing NY‐ESO‐1‐expressing tumors with a complex of cholesterol‐bearing hydrophobized pullulan (CHP) and NY‐ESO‐1 protein (CHP‐NY‐ESO‐1) and showed efficient induction of NY‐ESO‐1 antibody, and CD4 and CD8 T cell responses using peripheral blood from the patients. In our study, we analyzed heteroclitic serological responses in those patients after vaccination. Serological response against 11 tumor antigens including MAGE‐A1, MAGE‐A3, MAGE‐A4, CT7/MAGEC1, CT10/MAGEC2, CT45, CT46/HORMAD1, SOX2, SSX2, XAGE1B and p53 was examined by enzyme‐linked immunosorbent assay (ELISA) using sera from ten vaccinated patients. Expression of tumor antigens was determined by reverse transcription‐polymerase chain reaction or immunohistochemistry. Eight of nine patients who showed antibody responses against NY‐ESO‐1 also showed an antibody response against at least 1 of these 11 tumor antigens after vaccination. In one patient, seven tumor antigens were recognized. Specificity analysis of the antibody response by ELISA using control recombinant proteins and synthetic peptides and by Western blot showed that the response was not against His6‐tag and/or bacterial products included in a preparation of CHP‐NY‐ESO‐1 used for vaccination. Thus, heteroclitic serological responses appear to be indicative of the overall immune response against the tumor, and their analysis could be useful for immune monitoring in cancer vaccine.

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.

Vaccination with NY-ESO-1 overlapping peptides mixed with Picibanil OK-432 and montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen.

We conducted a clinical trial of an NY-ESO-1 cancer vaccine using 4 synthetic overlapping long peptides (OLP; peptides #1, 79–108; #2, 100–129; #3, 121–150; and #4, 142–173) that include a highly immunogenic region of the NY-ESO-1 molecule. Nine patients were immunized with 0.25 mg each of three 30-mer and a 32-mer long NY-ESO-1 OLP mixed with 0.2 KE Picibanil OK-432 and 1.25 mL Montanide ISA-51. The primary endpoints of this study were safety and NY-ESO-1 immune responses. Five to 18 injections of the NY-ESO-1 OLP vaccine were well tolerated. Vaccine-related adverse events observed were fever and injection site reaction (grade 1 and 2). Two patients showed stable disease after vaccination. An NY-ESO-1-specific humoral immune response was observed in all patients and an antibody against peptide #3 (121–150) was detected firstly and strongly after vaccination. NY-ESO-1 CD4 and CD8 T-cell responses were elicited in these patients and their epitopes were identified. Using a multifunctional cytokine assay, the number of single or double cytokine-producing cells was increased in NY-ESO-1-specific CD4 and CD8 T cells after vaccination. Multiple cytokine-producing cells were observed in PD-1 (−) and PD-1 (+) CD4 T cells. In conclusion, our study indicated that the NY-ESO-1 OLP vaccine mixed with Picibanil OK-432 and Montanide ISA-51 was well tolerated and elicited NY-ESO-1-specific humoral and CD4 and CD8 T-cell responses in immunized 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.