A new peptide vaccine OCV-501: in vitro pharmacology and phase 1 study in patients with acute myeloid leukemia.

Wilms' tumor 1 (WT1) is a promising target of new immunotherapies for acute myeloid leukemia (AML) as well as for other cancers. OCV-501 is a helper peptide derived from the WT1 protein. OCV-501 induced OCV-501-specific Type 1 T-helper (Th1) responses dose-dependently and stimulated helper activity of the specific Th1 cells in peripheral blood mononuclear cells from healthy donors. OCV-501 also enhanced the increase in WT1-killer peptide-specific cytotoxic T lymphocytes. OCV-501 stimulated the OCV-501-specific Th1 clones in an HLA class-II restricted manner and formed a complex with HLA class-II protein. OCV-501-specific Th1 clones demonstrated significant OCV-501-specific cytolytic activity against OCV-501-pulsed B-lymphoblastoid cell line cells. Based on the pre-clinical results, phase 1 clinical trial was conducted. The result of this trial suggested that the subcutaneous administration of OCV-501 once weekly for 4 weeks at doses of 0.3, 1, and 3 mg in older patients with AML during complete remission was safe and well tolerated. The maximum tolerated dose was considered to be ≥3 mg. Of the nine subjects enrolled, neither relapse nor blast cells were observed during the study. Immunological responses were observed in OCV-501-specific delayed-type hypersensitivity test. This trial was registered at http://www.clinicaltrials.gov as NCT 01440920.

Safety and immunogenicity of neoadjuvant treatment using WT1-immunotherapeutic in combination with standard therapy in patients with WT1-positive Stage II/III breast cancer: a randomized Phase I study.

PURPOSE: This Phase I, multicenter, randomized study (ClinicalTrials.gov NCT01220128) evaluated the safety and immunogenicity of recombinant Wilms' tumor 1 (WT1) protein combined with the immunostimulant AS15 (WT1-immunotherapeutic) as neoadjuvant therapy administered concurrently with standard treatments in WT1-positive breast cancer patients. METHODS: Patients were treated in 4 cohorts according to neoadjuvant treatment (A: post-menopausal, hormone receptor [HR]-positive patients receiving aromatase inhibitors; B: patients receiving chemotherapy; C: HER2-overexpressing patients on trastuzumab-chemotherapy combination; D: HR-positive/HER2-negative patients on chemotherapy). Patients (cohorts A-C) were randomized (2:1) to receive 6 or 8 doses of WT1-immunotherapeutic or placebo together with standard neoadjuvant treatment in a double-blind manner; cohort D patients received WT1-immunotherapeutic in an open manner. Safety was assessed throughout the study. WT1-specific antibodies were assessed pre- and post-vaccination. RESULTS: Sixty-two patients were randomized; 60 received ≥ one dose of WT1-immunotherapeutic. Two severe toxicities were reported: diarrhea (cohort C; also reported as a grade 3 serious adverse event) and decreased left ventricular ejection fraction (cohort B; also reported as a grade 2 adverse event). Post-dose 4 of WT1-immunotherapeutic, 10/10 patients from cohort A, 0/8 patients from cohort B, 6/11 patients from cohort C, and 2/3 patients from cohort D were humoral responders. The sponsor elected to close the trial prematurely. CONCLUSIONS: Concurrent administration of WT1-immunotherapeutic and standard neoadjuvant therapy was well tolerated and induced WT1-specific antibodies in patients receiving neoadjuvant aromatase inhibitors. In patients on neoadjuvant chemotherapy or trastuzumab-chemotherapy combination, the humoral response was impaired or blunted, likely due to either co-administration of corticosteroids and/or the chemotherapies themselves.

Wilms tumor 1 peptide vaccination combined with temozolomide against newly diagnosed glioblastoma: safety and impact on immunological response.

To investigate the safety of combined Wilms tumor 1 peptide vaccination and temozolomide treatment of glioblastoma, a phase I clinical trial was designed. Seven patients with histological diagnosis of glioblastoma underwent concurrent radiotherapy and temozolomide therapy. Patients first received Wilms tumor 1 peptide vaccination 1 week after the end of combined concurrent radio/temozolomide therapy, and administration was continued once per week for 7 weeks. Temozolomide maintenance was started and performed for up to 24 cycles, and the observation period for safety encompassed 6 weeks from the first administration of maintenance temozolomide. All patients showed good tolerability during the observation period. Skin disorders, such as grade 1/2 injection-site reactions, were observed in all seven patients. Although grade 3 lymphocytopenia potentially due to concurrent radio/temozolomide therapy was observed in five patients (71.4 %), no other grade 3/4 hematological or neurological toxicities were observed. No autoimmune reactions were observed. All patients are still alive, and six are on Wilms tumor 1 peptide vaccination without progression, yielding a progression-free survival from histological diagnosis of 5.2-49.1 months. Wilms tumor 1 peptide vaccination was stopped in one patient after 12 injections by the patient's request. The safety profile of the combined Wilms tumor 1 peptide vaccination and temozolomide therapy approach for treating glioblastoma was confirmed.

Safety and tolerability of allogeneic dendritic cell vaccination with induction of Wilms tumor 1-specific T cells in a pediatric donor and pediatric patient with relapsed leukemia: a case report and review of the literature.

A 15-year-old girl with acute lymphoblastic leukemia received allogeneic dendritic cell vaccination, pulsed with Wilms tumor 1 (WT1) peptide, after her third hematopoietic stem cell transplantation (HSCT). The vaccines were generated from the third HSCT donor, who was her younger sister, age 12 years. The patient received 14 vaccines and had no graft-versus-host disease or systemic adverse effect, aside from grade 2 skin reaction at the injection site. WT1-specific immune responses were detected after vaccination by both WT1-tetramer analysis and enzyme-linked immunosorbent spot assay. This strategy may be safe, tolerable and even feasible for patients with a relapse after HSCT.

A clinical and immunologic phase 2 trial of Wilms tumor gene product 1 (WT1) peptide vaccination in patients with AML and MDS.

This study investigated the immunogenicity of Wilms tumor gene product 1 (WT1)-peptide vaccination in WT1-expressing acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients without curative treatment option. Vaccination consisted of granulocyte-macrophage colony-stimulating factor subcutaneously days 1 to 4, and WT1.126-134 peptide and 1 mg keyhole limpet hemocyanin on day 3. The initial 9 patients received 4 vaccinations biweekly, then monthly, and the subsequent 10 patients received continual biweekly vaccination. Seventeen AML patients and 2 refractory anemia with excess blasts patients received a median of 11 vaccinations. Treatment was well tolerated. Objective responses in AML patients were 10 stable diseases (SDs) including 4 SDs with more than 50% blast reduction and 2 with hematologic improvement. An additional 4 patients had clinical benefit after initial progression, including 1 complete remission and 3 SDs. WT1 mRNA levels decreased at least 3-fold from baseline in 35% of patients. In 8 of 18 patients, WT1-tetramer(+) T cells increased in blood and in 8 of 17 patients in bone marrow, with a median frequency in bone marrow of 0.18% at baseline and 0.41% in week 18. This WT1 vaccination study provides immunologic, molecular, and preliminary evidence of potential clinical efficacy in AML patients, warranting further investigations.

Repeated PR1 and WT1 peptide vaccination in Montanide-adjuvant fails to induce sustained high-avidity, epitope-specific CD8+ T cells in myeloid malignancies.

BACKGROUND: We previously showed that vaccination with one dose of PR1 and WT1 peptides induces transient anti-leukemia immunity. We hypothesized that maintenance of a sustained anti-leukemia response may require frequent boost injections. DESIGN AND METHODS: Eight patients with myeloid malignancies were enrolled in this phase II study, and 6 completed 6 injections of PR1 and WT1 peptides in Montanide-adjuvant with GM-CSF, every two weeks. RESULTS: Both high- and low-avidity PR1 or WT1-specific CD8(+) T cells were detected in all evaluable patients after the first vaccine dose. Repeated vaccination led to selective deletion of high avidity PR1- and WT1-specific CD8(+) T cells and was not associated with significant reduction in WT1-expression. Additional boosting failed to increase vaccine-induced CD8(+) T-cell frequencies further and in all patients the response was lost before the 6(th) dose. PR1- or WT1-specific CD8(+) T cells were not detected in bone marrow samples, excluding their preferential localization to this site. Following a booster injection three months after the 6(th) vaccine dose, no high-avidity PR1 or WT1-specific CD8(+) T cells could be detected, whereas low-avidity T cells were readily expanded. CONCLUSIONS: These data support the immunogenicity of PR1 and WT1 peptide vaccines. However, repeated delivery of peptides with Montanide-adjuvant and GM-CSF leads to rapid loss of high-avidity peptide-specific CD8(+) T cells. These results may offer an explanation for the lack of correlation between immune and clinical responses observed in a number of clinical trials of peptide vaccination. New approaches are needed to induce long-term high-avidity memory responses against leukemia antigens.

WT1 peptide vaccinations induce CD4 and CD8 T cell immune responses in patients with mesothelioma and non-small cell lung cancer.

BACKGROUND: The transcription factor, WT1, is highly overexpressed in malignant pleural mesothelioma (MPM) and immunohistochemical stains for WT1 are used routinely to aid in its diagnosis. Using computer prediction analysis we designed analog peptides derived from WT1 sequences by substituting amino acids at key HLA-A0201 binding positions. We tested the safety and immunogenicity of a WT1 vaccine comprised of four class I and class II peptides in patients with thoracic neoplasms expressing WT1. METHODS: Therapy consisted of six subcutaneous vaccinations administered with Montanide adjuvant on weeks 0, 4, 6, 8, 10, and 12, with 6 additional monthly injections for responding patients. Injection sites were pre-stimulated with GM-CSF (70 mcg). Immune responses were evaluated by DTH, CD4 T-cell proliferation, CD8 T-cell interferon gamma release, intracellular cytokine staining, WT1 peptide MHC-tetramer staining, and cytotoxicity against WT1 positive tumor cells. RESULTS: Nine patients with MPM and 3 with NSCLC were vaccinated, with 8 patients receiving at least 6 vaccinations; in total, 10 patients were evaluable for immune response. Six out of nine patients tested demonstrated CD4 T-cell proliferation to WT1 specific peptides, and five of the six HLA-A0201 patients tested mounted a CD8 T-cell response. Stimulated T cells were capable of cytotoxicity against WT-1 positive cells. Vaccination also induced polyfunctional CD8 T cell responses. CONCLUSIONS: This multivalent WT1 peptide analog vaccine induces immune responses in a high proportion of patients with thoracic malignancies with minimal toxicity. A randomized trial testing this vaccine as adjuvant therapy in MPM is planned.

WT1 peptide immunotherapy for cancer in children and young adults.

Wilms tumor gene (WT1) can be overexpressed in childhood cancers. We evaluated the efficacy of WT1 vaccination for five children with solid cancer or acute leukemia. WT1 vaccine was administered to HLA-A*2402-positive patients with WT1-overexpressing residual tumor despite prior conventional treatment. One patient showed complete response and one patient showed stable disease according to the Response Evaluation Criteria in Solid Tumors; the remaining three showed progressive disease. Treatment-related adverse effects were limited to local injection site erythema. These results suggest that WT1 vaccination has therapeutic potential, but any beneficial effect may be insufficient in the presence of gross residual disease.

Vaccination of Urological Cancer Patients With WT1 Peptide-Pulsed Dendritic Cells in Combination With Molecular Targeted Therapy or Conventional Chemotherapy Induces Immunological and Clinical Responses.

The prognosis of metastatic or relapsed renal cell carcinoma (RCC) or bladder cancer (BC) remains poor despite the introduction of immune checkpoint blockade agents. We aimed to investigate the safety and the feasibility of a vaccination with WT1 peptide-loaded dendritic cells (DCs) and OK-432 adjuvant combined with molecular targeted therapy or conventional chemotherapy. Five eligible patients with metastatic or relapsed RCC and five eligible patients with BC were enrolled. No severe adverse events related to a vaccination were observed. Seven patients with RCC or non-muscle invasive BC had durable stable disease and three other patients had disease progression after DC vaccination. DC vaccination augmented WT1 specific immunity and the reduction of regulatory T cells which might be related to clinical outcome. These results indicate that DC-based immunotherapy combined with a molecular targeted therapy or a conventional chemotherapy is safe and feasible for patients in advanced stage of RCC or BC.

Clinical and immunologic responses to very low-dose vaccination with WT1 peptide (5 microg/body) in a patient with chronic myelomonocytic leukemia.

The wild-type Wilms tumor gene, WT1, is overexpressed in myelodysplastic syndrome (MDS) as well as acute myeloid leukemia. In a phase I clinical trial of biweekly vaccination with HLA-A*2402-restricted WT1 peptide for these malignancies, 2 patients with MDS developed severe leukocytopenia in association with a reduction in leukemic blast cells and levels of WT1 messenger RNA (mRNA) after only a single vaccination with 0.3 mg of WT1 peptide. These results indicated that the WT1-specific cytotoxic T-lymphocytes (CTLs) elicited by WT1 vaccination eradicated the WT1-expressing transformed stem or progenitor cells and that MDS patients with little normal hematopoiesis required a new strategy of WT1 vaccination to avoid severe leukocytopenia. We describe the first trial for a 57-year-old male patient with chronic myelomonocytic leukemia who was vaccinated biweekly with a small quantity (5 microg/body) of WT1 peptide. After the start of vaccination, the leukocyte and monocyte counts (13,780/microL and 1930/microL, respectively) gradually decreased to within the normal range in association with a reduction in the WT1 mRNA level. Simultaneously, the percentage of WT1-specific CTLs as measured by the HLA-WT1 tetramer assay increased. This case demonstrates for the first time that vaccination with as little as 5 microg of WT1 peptide can induce WT1-specific immune responses and resultant clinical responses.

Enhanced tumor immunity of WT1 peptide vaccination by interferon-ß administration.

To induce and activate tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) for cancer immunity, it is important not only to select potent CTL epitopes but also to combine them with appropriate immunopotentiating agents. Here we investigated whether tumor immunity induced by WT1 peptide vaccination could be enhanced by IFN-ß. For the experimental group, C57BL/6 mice were twice pre-treated with WT1 peptide vaccine, implanted with WT1-expressing C1498 cells, and treated four times with WT1 peptide vaccine at one-week intervals. During the vaccination period, IFN-ß was injected three times a week. Mice in control groups were treated with WT1 peptide alone, IFN-ß alone, or PBS alone. The mice in the experimental group rejected tumor cells and survived significantly longer than mice in the control groups. The overall survival on day 75 was 40% for the mice treated with WT1 peptide+IFN-ß, while it was 7, 7, and 0% for those treated with WT1 peptide alone, IFN-ß alone or PBS alone, respectively. Induction of WT1-specific CTLs and enhancement of NK activity were detected in splenocytes from mice in the experimental group. Furthermore, administration of IFN-ß enhanced expression of MHC class I molecules on the implanted tumor cells. In conclusion, our results showed that co-administration of WT1 peptide+IFN-ß enhanced tumor immunity mainly through the induction of WT1-specific CTLs, enhancement of NK activity, and promotion of MHC class I expression on the tumor cells. WT1 peptide vaccination combined with IFN-ß administration can thus be expected to enhance the clinical efficacy of WT1 immunotherapy.

Immunization with WT1-derived peptides by tattooing inhibits the growth of TRAMP-C2 prostate tumor in mice.

The expression of the transcription factor encoded by the Wilms tumor gene 1 (WT1) is associated with a variety of human cancers. WT1 protein has been reported to serve as a target antigen for tumor-specific immune responses. We observed that the immunization of mice with peptide vaccines derived from WT1 in a mixture with the CpG adjuvant (ODN 1826) by tattoo administration was superior to subcutaneous delivery of the peptides in combination with CpG formulated with the mineral oil adjuvant or a DNA vaccine or a recombinant vaccinia virus vaccine expressing the truncated WT1 protein. Tattooing with the WT1122-140 and WT1126-134 peptide elicited the response of WT1-specific interferon-γ-producing T cells. Peptide vaccine administered with a tattoo device had an antitumor effect on the growth of the prostate tumor cell line TRAMP-C2, provided that the transforming growth factor-ß produced by tumor cells was neutralized by anti-TGFß monoclonal antibody. The treatment of the tumor-bearing mice with 5-azadeoxycytidine or poly IC did not work in synergy with the peptide vaccine.

Phase I trial of Wilms' Tumor 1 (WT1) peptide vaccine with GM-CSF or CpG in patients with solid malignancy.

BACKGROUND: The aim of this study was to investigate the safety and efficacy of combinatorial use of granulocyte-macrophage colony-stimulating factor (GM-CSF) and CpG oligodeoxynucleotides (CpG-ODN) as immunoenhancement adjuvants in Wilms' Tumor 1 (WT1) vaccine therapy for patients with solid malignancy. PATIENTS AND METHODS: The patients were placed into treatment groups as follows: WT1 peptide alone, WT1 peptide with GM-CSF (100 µg) and WT1 peptide with CpG-ODN (100 µg). HLA-A *2402 or *0201/*0206-restricted, WT1 peptide emulsified with Montanide ISA51 was injected intradermally every week for eight weeks. Toxicities were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events ver. 3.0. Tumor size, which was measured by computed tomography, was determined every four weeks. The responses were analyzed according to Response Evaluation Criteria in Solid Tumors. RESULTS: The protocol was well tolerated; only local erythema occurred at the WT1 vaccine injection site. The disease control rate of the groups treated with WT1 peptide alone (n=10), with combinatorial use of GM-CSF (n=8) and with combinatorial use of CpG-ODN (n=10), in the initial two months was 20%, 25% and 60%, respectively. CONCLUSION: Addition of GM-CSF or CpG-ODN to the WT1 peptide vaccine for patients with solid malignancy was safe and improved the effectiveness of clinical response.

Frequency of myeloid dendritic cells can predict the efficacy of Wilms' tumor 1 peptide vaccination.

BACKGROUND: The object of this study was to investigate the clinical predictive capability of peripheral myeloid dendritic cells (DCs) in Wilms' tumor 1 (WT1) vaccine therapy for patients with gynaecological cancer. PATIENTS AND METHODS: Six patients with WT1/human leukocyte antigen (HLA)-A*2402-positive gynaecological cancer were included in this study. The patients received intradermal injections of a modified 9-mer WT1 peptide every week for 12 weeks. Peripheral blood samples were obtained at 0, 4, 8 and 12 weeks after the initial vaccination. Circulating DCs were detected by flow cytometry. RESULTS: The frequencies of CD14(+)CD16(+)CD33(+)CD85(+) myeloid DCs were significantly higher in the therapeutically effective group than in therapeutically inert group (p<0.05). CONCLUSION: These results suggested that myeloid DCs, which should be associated with inducing cytotoxic T-cells, provided additional prognostic information in the use of cancer peptide vaccine.

WT1 peptide vaccine stabilized intractable ovarian cancer patient for one year: a case report.

UNLABELLED: We report on Wilms tumor (WT1) peptide immunotherapy in a patient with intractable ovarian cancer patient over an extended period. CASE REPORT: Immunotherapy using WT1 peptide has been undergoing clinical trials for gynecological cancer. We used WT1 peptide vaccination to treat a 53-year-old woman suffering from ovarian cancer with peritoneal dissemination. After 2 months, her pleural and cardiac effusion had disappeared, and the sum of the longest diameter of the target lesion (in the pelvic mass) was reduced. There was a weak positive correlation between CA125 and mononuclear phagocyte/lymphocyte ratio (Spearman's ϱ=0.275, p=0.015). Intradermally administered WT1 peptide vaccination in a case of intractable ovarian cancer stabilized the disease over the course of one year. However, the immunotherapeutic mechanism of WT1 peptide and immunological escape mechanism for carcinoma cells remain to be elucidated.

Prognostic value of WT1 protein expression level and MIB-1 staining index as predictor of response to WT1 immunotherapy in glioblastoma patients.

The use of Wilms' tumor 1 (WT1) immunotherapy is considered to be an innovative approach for the treatment of malignant gliomas. Because of its novelty, tools that can accurately predict response to this therapy are still lacking. In this article, we investigated the role of WT1 protein expression level (score 1-4) and MIB-1 staining index in predicting survival outcome after therapy in patients with recurrent or progressive glioblastoma multiforme. Tumor samples from 37 patients enrolled in a phase II clinical trial on WT1 immunotherapy were immunohistochemically analyzed for WT1 levels and MIB-1 index. Results showed that median progression-free survival (PFS) was longer in the WT1 high expression group (score 3 and 4) compared with that of the low expression group (score 1 and 2) (20.0 weeks vs. 8.0 weeks; P = 0.022), and that the median overall survival (OS) was likewise longer in the former compared to the latter group (54.4 weeks vs. 28.4 weeks; P = 0.035). Furthermore, within the WT1 high expression group, tumors with intermediate staining intensity (WT1 score 3) have both the longest median PFS and OS, 24.4 weeks and 69.4 weeks, respectively. On the other hand, no significant correlation was noted between MIB-1 staining index and survival. In conclusion, our study has shown that WT1 protein expression level, not MIB-1 staining index, can be used as a prognostic marker to foretell outcome after immunotherapy, and that patients whose tumors have intermediate WT1 expression have the best survival outcome.

WT1 peptide-based immunotherapy for patients with lung cancer: report of two cases.

The Wilms' tumor gene WT1 is overexpressed in various types of solid tumors, including lung and breast cancer and WT1 protein is a tumor antigen for these malignancies. In phase I clinical trials of WT1 peptide-based cancer immunotherapy, two patients with advanced lung cancer were intradermally injected with 0.3 mg of an HLA-A*2402-restricted, 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant. Consecutive WT1 vaccination at 2-week intervals resulted in a reduction in tumor markers such as chorio-embryonic antigen (CEA) and sialyl Lewis (x) (SLX) and by a transient decrease in tumor size. No adverse effects except for local erythema at the injection sites of WT1 vaccine were observed. These results provided us with the first clinical evidence demonstrating that WT1 peptide-based immunotherapy should be a promising treatment for patients with lung cancer.