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Background: A Wilms' tumor 1 (WT1) oral vaccine, Bifidobacterium longum (B. longum) 420, in which the bacterium is used as a vector for WT1 protein, triggers immune responses through cellular immunity consisting of cytotoxic T lymphocytes (CTLs) and other immunocompetent cells (e.g., helper T cells). We developed a novel, oral, helper epitope-containing WT1 protein vaccine (B. longum 2656) to examine whether or not B. longum 420/2656 combination further accelerates the CD4 + T cell help-enhanced antitumor activity in a model of murine leukemia., Methods: C1498-murine WT1-a genetically-engineered, murine leukemia cell line to express murine WT1-was used as tumor cell. Female C57BL/6 J mice were allocated to the B. longum 420, 2656, and 420/2656 combination groups. The day of subcutaneous inoculation of tumor cells was considered as day 0, and successful engraftment was verified on day 7. The oral administration of the vaccine by gavage was initiated on day 8. Tumor volume, the frequency and phenotypes of WT1-specific CTLs in CD8 + T cells in peripheral blood (PB) and tumor-infiltrating lymphocytes (TILs), as well as the proportion of interferon-gamma (INF-γ)-producing CD3 + CD4 + T cells pulsed with WT1 35-52 peptide in splenocytes and TILs were determined., Results: Tumor volume was significantly smaller (p < 0.01) in the B. longum 420/2656 combination group than in the B. longum 420 group on day 24. WT1-specific CTL frequency in CD8 + T cells in PB was significantly greater in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 (p < 0.05) and 6 (p < 0.01). The proportion of WT1-specific, effector memory CTLs in PB increased significantly in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 and 6 (p < 0.05 each). WT1-specific CTL frequency in intratumoral CD8 + T cells and the proportion of IFN-γ-producing CD3 + CD4 + T cells in intratumoral CD4 + T cells increased significantly (p < 0.05 each) in the B. longum 420/2656 combination group than in the 420 group., Conclusions: B. longum 420/2656 combination further accelerated antitumor activity that relies on WT1-specific CTLs in the tumor compared with B. longum 420., (© 2023. The Author(s).)

Wilms' tumor 1 (WT1) is a promising tumor-associated antigen for cancer immunotherapy. We developed an oral protein vaccine platform composed of WT1-anchored, genetically engineered Bifidobacterium longum (B. longum) and conducted an in vivo study in mice to examine its anticancer activity. Mice were orally treated with phosphate-buffered saline, wild-type B. longum105-A, B. longum 2012 displaying only galacto-N-biose/lacto-N-biose I-binding protein (GLBP), and WT1 protein- and GLBP-expressing B. longum 420. Tumor size reduced significantly in the B. longum 420 group than in the B. longum 105-A and 2012 groups (P < 0.00 l each), indicating B. longum 420's antitumor activity via WT1-specific immune responses. CD8 + T cells played a major role in the antitumor activity of B. longum 420. The proportion of CD103 + CD11b + CD11c + dendritic cells (DCs) increased in the Peyer's patches (PPs) from mice in the B. longum 420 group, indicating the definite activation of DCs. In the PPs, the number and proportion of CD8 + T cells capable of producing interferon-gamma were significantly greater in the B. longum 420 group than in the B. longum 2012 group (P < 0.05 or < 0.01). The production of WT1-specific IgG antibody was significantly higher in the B. longum 420 group than in the 2012 group (P < 0.05). The B. longum 420 group showed the most intense intratumoral infiltration of CD4 + and CD8 + T cells primed by activated DCs in the PPs of mice in the B. longum 420 group. Our findings provide insights into a novel, intestinal bacterium-based, cancer immunotherapy through intestinal immunity., (© 2022. The Author(s).)

The HLA-A*24:02-restricted peptide vaccine targeting Wilms' tumor 1 (WT1) (WT1 vaccine) is a promising therapeutic strategy for ovarian cancer; however, its efficacy varies among patients. In this study, we analyzed WT1-specific immune responses in patients with advanced or recurrent ovarian cancer that was refractory to standard chemotherapies and their associations with clinical outcomes. In 25 patients, the WT1 vaccine was administered subcutaneously weekly for 3 months and biweekly thereafter until disease progression or severe adverse events. We assessed Wilms' tumor 1-specific cytotoxic T lymphocytes (WT1-CTLs) and Wilms' tumor 1 peptide-specific immunoglobulin G (WT1235-IgG). After vaccination, the percentage of tetramer high-avidity population of WT1-CTLs among CD8+ T lymphocytes (%tet-hi WT1-CTL) and the WT1235-IgG titer increased significantly, although the values were extremely low or below the limit of detection before vaccination (%tet-hi WT1-CTL: 0.003%-0.103%.; WT1235-IgG: <0.05-0.077 U/mL). Patients who had %tet-hi WT1-CTL of ≥0.25% (n=6) or WT1235-IgG of ≥0.10 U/mL (n=12) had a significantly longer progression-free survival than those of patients in the other groups. In addition, an increase in WT1235-IgG corresponded to a significantly longer progression-free survival (P=0.0496). In patients with systemic inflammation, as evidenced by elevated C-reactive protein levels, the induction of tet-hi WT1-CTL or WT1235-IgG was insufficient. Decreased serum albumin levels, multiple tumor lesions, poor performance status, and excess ascites negatively influenced the clinical effectiveness of the WT1 vaccine. In conclusion, the WT1 vaccine induced antigen-specific cellular and humoral immunity in patients with refractory ovarian cancer. Both %tet-hi WT1-CTL and WT1235-IgG levels are prognostic markers for the WT1 vaccine., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Simultaneous induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) is required for an optimal anti-tumor immune response. WT1 332 , a 16-mer WT1-derived helper peptide, induce HTLs in an HLA class II-restricted manner and enhance the induction of WT1-specific CTLs in vitro. However, in vivo immune reaction to WT1 332 vaccination in tumor-bearing patients remained unclear. Here, a striking difference in WT1-specific T cell responses was shown between WT1 CTL + WT1 helper peptide and WT1 CTL peptide vaccines in patients with recurrent glioma. WT1-specific CTLs were more strongly induced in the patients who were immunized with WT1 CTL + WT1 helper peptide vaccine, compared to those who were immunized with WT1 CTL vaccine alone. Importantly, a clear correlation was demonstrated between WT1-specific CTL and WT1 332 -specific HTL responses. Interestingly, two novel distinct populations of WT1-tetramer low WT1-TCR low CD5 low and WT1-tetramer high WT1-TCR high CD5 high CTLs were dominantly detected in WT1 CTL + WT1 helper peptide vaccine. Although natural WT1 peptide-reactive CTLs in the latter population were evidently less than those in the former population, the latter population showed natural WT1 peptide-specific proliferation capacity comparable to the former population, suggesting that the latter population highly expressing CD5, a marker of resistance to activation-induced cell death, should strongly expand and persist for a long time in patients. These results demonstrated the advantage of WT1 helper peptide vaccine for the enhancement of WT1-specific CTL induction by WT1 CTL peptide vaccine.

It has become evident that positron emission tomography/computed tomography (PET-CT) using 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) (FDG PET-CT) can detect anti-tumor immune response induced by various immunotherapies. To evaluate whether FDG PET-CT could detect anti-cancer immune response caused by cancer vaccine therapy, we performed a retrospective analysis of FDG PET-CT imaging of patients who were treated with Wilms Tumor 1 (WT1) vaccine therapy in Osaka University during July 2008 and June 2018. Increased FDG uptakes were detected in WT1-vaccinated skin and their draining lymph nodes during the repeated vaccination. While the FDG uptakes seemed to decrease with time after the cessation of WT1 peptide vaccinations, persistence of FDG uptakes for years in WT1-vaccinated skin were also observed in 2 cases who showed good clinical course. Moreover, the FDG uptakes of patients treated with the combination vaccine of WT1 specific cytotoxic T cell (CTL) and helper peptides were significantly stronger than of those treated with the WT1 CTL peptide alone. Since it is evident that the combination vaccine can induce a more robust anti-tumor immunity than can CTL peptide vaccine alone, the FDG uptakes in WT1-vaccinated skin might reflect the degree of immune response. These results suggest that PET-CT might be a good tool for prediction of anti-tumor immune response induced by WT1 vaccine therapy. Larger scale prospective studies therefore seem to be warranted.

Purpose: The safety and clinical efficacy of WT1 human leukocyte antigen (HLA) class I peptide vaccine have been established, but the safety of a cocktail vaccine of WT1 HLA class I and II peptides has not. To verify its safety, we performed a phase I clinical trial for patients with recurrent malignant gliomas and assessed the immunological responses and survival data., Patients and Methods: Fourteen HLA-A*24:02-positive patients with recurrent malignant glioma (2 with grade 3, 12 with grade 4) were enrolled. Every week, the patients received alternately a vaccine containing 3 mg of WT1 HLA-A*24:02-restricted (HLA class I) peptide and a cocktail vaccine of the HLA class I peptide and one of 0.75, 1.5 or 3 mg of the WT1 HLA class II peptide. For patients who showed no significant adverse effects within 6 weeks, the WT1 vaccine was continued at 2-4-week intervals., Results: Eleven of the 14 patients completed WT1 vaccination for 6 weeks, while 3 patients dropped out earlier due to disease progression. All patients showed grade I level of skin disorders at the injection sites. No grade III/IV toxicity or dose-limiting toxicity was observed for any dose of WT1 HLA class II peptide. Six of the 14 patients had stable disease at 6 weeks. Median OS and 1-year OS rates were 24.7 weeks and 36%, respectively., Conclusion: The safety of a cocktail vaccine of WT1 HLA class I and II peptides for malignant gliomas was verified. This vaccine is, therefore, considered promising for patients with recurrent malignant glioma.

The identification of human Wilms' tumor gene 1 (WT1) protein-derived cytotoxic T lymphocyte (CTL) epitopes and the in vivo efficacy of WT1 peptide-based immunotherapy in a mouse model were reported in 2000. This successful basic research led to clinical studies of a WT1 peptide vaccine, and a positive impact on clinical response was first demonstrated in 2003 in the form of a reduction in blast cells of vaccine-treated patients with myelodysplastic syndromes (MDS). Since then, data on WT1 peptide vaccine-treated patients with immunological and/or clinical response have been accumulated. MDS and acute myeloid leukemia were the major target diseases to provide proof of concept for the therapeutic potential of the WT1 peptide vaccine. WT1 vaccination-induced clinical responses or usefulness were also shown for chronic myeloid leukemia, multiple myeloma, and acute lymphoblastic leukemia, as well as various types of solid cancers. Non-Hodgkin's lymphoma and myeloproliferative neoplasms may also be target diseases because of their WT1 expression. Of note, recent clinical studies have demonstrated that patients with hematological malignancies who have minimal residual disease after chemotherapy or allogeneic hematopoietic stem cell transplantation may be cured by WT1 peptide vaccination. Further enhancement of the efficacy and usefulness of the WT1 peptide vaccine is expected., (© 2017 S. Karger GmbH, Freiburg.)

We previously evaluated Wilms' tumor gene 1 (WT1) peptide vaccination in a large number of patients with leukemia or solid tumors and have reported that HLA-A*24:02 restricted, 9-mer WT1-235 peptide (CYTWNQMNL) vaccine induces cellular immune responses and elicits WT1-235-specific cytotoxic T lymphocytes (CTLs). However, whether this vaccine induces humoral immune responses to produce WT1 antibody remains unknown. Thus, we measured IgG antibody levels against the WT1-235 peptide (WT1-235 IgG antibody) in patients with glioblastoma multiforme (GBM) receiving the WT1 peptide vaccine. The WT1-235 IgG antibody, which was undetectable before vaccination, became detectable in 30 (50.8%) of a total of 59 patients during 3 months of WT1 peptide vaccination. The dominant WT1-235 IgG antibody subclass was Th1-type, IgG1 and IgG3 . WT1-235 IgG antibody production was significantly and positively correlated with both progression-free survival (PFS) and overall survival (OS). Importantly, the combination of WT1-235 IgG antibody production and positive delayed type-hypersensitivity (DTH) to the WT1-235 peptide was a better prognostic marker for long-term OS than either parameter alone. These results suggested that WT1-235 peptide vaccination induces not only WT1-235-specific CTLs as previously described but also WT1-235-specific humoral immune responses associated with antitumor cellular immune response. Our results indicate that the WT1 IgG antibody against the WT1 peptide may be a useful predictive marker, with better predictive performance in combination with DTH to WT1 peptide, and provide a new insight into the antitumor immune response induction in WT1 peptide vaccine-treated patients., (© 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Advances in cancer immunotherapy in the pediatric field are needed in order to improve the prognosis of children with malignancies. We conducted a prospective phase I/II study of WT1 peptide vaccination for children with relapsed or refractory malignancies., Methods: The main eligibility criteria were affected tissues or leukemic cells expressing the WT1 gene, and patients (and donors for allogeneic hematopoietic stem cell transplantation) having HLA-A*24:02. Vaccination using the WT1 peptide (CYTWNQMNL), which was modified for higher affinity to this HLA-type molecule with the adjuvant Montanide ISA51, was performed weekly 12 times., Results: Twenty-six patients were enrolled and 13 (50.0%) completed the vaccination 12 times. Evidence for the induction of WT1-specific cytotoxic T-lymphocyte (CTL) responses without severe systemic side effects was obtained. Two out of 12 patients with bulky disease exhibited a transient clinical effect (one mixed response and one stable disease), three out of six patients with minimal residual disease achieved transient molecular remission, and five out of eight patients without a detectable level of the molecular marker, but with a high risk of relapse, had the best outcome of long-term continuous complete remission., Conclusions: WT1 vaccination is a safe immunotherapy and induced WT1-specific CTL responses in children; however, as a single agent, vaccination only provided patients in remission, but with a high risk of relapse, with "long-term benefits" in the context of its use for relapse prevention. WT1 peptide-based treatments in combination with other modalities, such as anti-tumor drugs or immunomodulating agents, need to be planned., (© 2015 Wiley Periodicals, Inc.)

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.

Allogeneic hematopoietic stem cell transplantation (HSCT) has largely improved the prognosis of leukemia patients. However, relapse is still a major concern. One promising option for the prevention of relapse is vaccination therapy. The post allogeneic HSCT period provides a unique platform for vaccination, because tumor burden is minimal, lymphopenic condition allows for rapid expansion of cytotoxic T cells (CTLs), donor-derived CTLs are not exhausted and inflammatory condition is caused by allo reactions. Tumor cells, dendritic cells and peptides have been used as vaccines targeting leukemia-associated antigens or minor histocompatibility antigens. Clinical trials with several types of vaccines for post-HSCT patients showed that the vaccination induced immunological response and might benefit patients with minimal residual disease, while their effect in patients with advanced disease were limited. To enhance the effect, vaccination in combination with other immune-modulatory drugs such as checkpoint antibodies is now being considered.

Wilms tumor gene (WT1) protein is an attractive target for cancer immunotherapy. We aimed to investigate the feasibility of a combination therapy consisting of gemcitabine and WT1 peptide-based vaccine for patients with advanced pancreatic cancer and to make initial assessments of its clinical efficacy and immunologic response. Thirty-two HLA-A*24:02 patients with advanced pancreatic cancer were enrolled. Patients received HLA-A*24:02-restricted, modified 9-mer WT1 peptide (3 mg/body) emulsified with Montanide ISA51 adjuvant (WT1 vaccine) intradermally biweekly and gemcitabine (1000 mg/m) on days 1, 8, and 15 of a 28-day cycle. This combination therapy was well tolerated. The frequencies of grade 3-4 adverse events for this combination therapy were similar to those for gemcitabine alone. Objective response rate was 20.0% (6/30 evaluable patients). Median survival time and 1-year survival rate were 8.1 months and 29%, respectively. The association between longer survival and positive delayed-type hypersensitivity to WT1 peptide was statistically significant, and longer survivors featured a higher frequency of memory-phenotype WT1-specific cytotoxic T lymphocytes both before and after treatment. WT1 vaccine in combination with gemcitabine was well tolerated for patients with advanced pancreatic cancer. Delayed-type hypersensitivity-positivity to WT1 peptide and a higher frequency of memory-phenotype WT1-specific cytotoxic T lymphocytes could be useful prognostic markers for survival in the combination therapy with gemcitabine and WT1 vaccine. Further clinical investigation is warranted to determine the effectiveness of this combination therapy.

Object: Immunotherapy targeting the Wilms tumor 1 (WT1) gene product is a promising treatment modality for patients with malignant gliomas, and there have been reports of encouraging results. It has become clear, however, that Gd-enhanced MR imaging does not reflect prognosis, thereby necessitating a more robust imaging evaluation system for monitoring response to WT1 immunotherapy. To meet this demand, the authors performed a voxel-wise parametric response map (PRM) analysis of (11)C-methionine PET (MET-PET) in WT1 immunotherapy and compared the data with the overall survival after initiation of WT1 immunotherapy (OS(WT1))., Methods: Fourteen patients with recurrent malignant glioma were included in the study, and OS(WT1) was compared with: 1) volume and length change in the contrast area of the tumor on Gd-enhanced MR images; 2) change in maximum uptake of (11)C-methionine; and 3) a more detailed voxel-wise PRM analysis of MET-PET pre- and post-WT1 immunotherapy., Results: The PRM analysis was able to identify the following 3 areas within the tumor core: 1) area with no change in (11)C-methionine uptake pre- and posttreatment; 2) area with increased (11)C-methionine uptake posttreatment (PRM(+MET)); and 3) area with decreased (11)C-methionine uptake posttreatment. While the results of Gd-enhanced MR imaging volumetric and conventional MET-PET analysis did not correlate with OS(WT1) (p = 0.270 for Gd-enhanced MR imaging length, p = 0.960 for Gd-enhanced MR imaging volume, and p = 0.110 for MET-PET), the percentage of PRM(+MET) area showed excellent correlation (p = 0.008) with OS(WT1)., Conclusions: This study describes the limited value of Gd-enhanced MR imaging and highlights the potential of voxel-wise PRM analysis of MET-PET for monitoring treatment response in immunotherapy for malignant gliomas. Clinical trial registration no.: UMIN000002001.

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-β. 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-β was injected three times a week. Mice in control groups were treated with WT1 peptide alone, IFN-β 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-β, while it was 7, 7, and 0% for those treated with WT1 peptide alone, IFN-β 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-β enhanced expression of MHC class I molecules on the implanted tumor cells. In conclusion, our results showed that co-administration of WT1 peptide+IFN-β 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-β administration can thus be expected to enhance the clinical efficacy of WT1 immunotherapy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

How to treat CML patients who are resistant to inhibitors of BCR-ABL tyrosine kinase such as Imatinib is a very important and urgent issue in clinical hematology. Here, we report a case of Imatinib-treated CML in which intradermally administered WT1 peptide vaccine elicited WT1-specific immune responses and the resultant reduction in the persistent residual disease in co-administration of Imatinib. BCR-ABL mRNA levels were being maintained under the detection limit for 8 months since week 77 of vaccination. No adverse effects except local erythema at the injection sites were observed. The tetramer assay revealed that the decrease in BCR-ABL mRNA levels was associated with the increase in frequency of WT1-specific cytotoxic T lymphocytes, notably effector-memory type of that, in the patient's peripheral blood. The case presented here indicates that WT1 peptide vaccine may become a safe and cure-oriented therapy for CML patients who have residual disease regardless of the treatment with Imatinib., (© 2010 John Wiley & Sons A/S.)

Clinical studies of WT1-targeted cancer vaccine are being performed. However, WT1-specific Th response in cancer patients remains unclear. Using quantitative real-time RT-PCR, we investigated IFN-gamma and IL-10 mRNA expression from Th cells by stimulation with helper peptide WT1(332). Seventeen patients, of whom 10 had achieved stable disease and the remaining 7 had progressive disease, were weekly vaccinated with WT1 CTL epitope (modified WT1(235)) and examined for WT1(332)-specific Th response. A clear correlation between WT1(332)-specific Th response and clinical response was observed at 4 weeks post-vaccination. In patients who responded, a clear inverse correlation between IL-10-type and IFN-gamma-type WT1(332)-specific Th response was detected at pre- and 4 weeks post-vaccination, and the shift of the Th response from IL-10-type dominancy at early phase to IFN-gamma-type dominancy at late phase was observed. From this study we concluded that occurrence of WT1(332)-specific Th response could predict good clinical response of WT1 CTL epitope vaccination.

Objective: Immunotherapy targeting the Wilms' tumour 1 gene product has been proven safe and effective for treating malignant glioma in a phase II clinical study. Currently, radiation/temozolomide therapy is the standard treatment with only modest benefit. Whether combining radiation/temozolomide therapy with WT1 immunotherapy will have a negating effect on immunotherapy is still controversial because of the significant lymphocytopaenia induced by the former therapy. To address this issue, we investigated the changes in frequency and number of WT1-specific T-cells in patients with malignant gliomas., Methods: Twenty-two patients with newly diagnosed malignant glioma who received standard radiation/temozolomide therapy were recruited for the study. Blood samples were collected before treatment and on the sixth week of therapy. The frequencies and numbers of lymphocytes, CD8(+) T-cells, WT1-specific T-cells, regulatory T-cells, natural killer cells and natural killer T-cells were measured and analysed using T-tests., Results: Analysis of the frequency of T lymphocytes and its subpopulation showed an increase in regulatory T-cells, but no significant change was noted in the populations of T-cells, WT1-specific T-cells, NK cells and NKT cells. Reductions in the total numbers of T-cells, WT1-specific T-cells, NK cells and NKT cells were mainly a consequence of the decrease in the total lymphocyte count., Conclusions: Radiation/temozolomide therapy did not significantly affect the frequency of WT1-specific T-cells, suggesting that the combination with WT1 immunotherapy may be possible, although further assessment in the clinical setting is warranted.

Background: The object of this study was to investigate the safety and clinical response of immunotherapy targeting the WT1 (Wilms' tumor 1) gene product in patients with gynecological cancer., Patients and Methods: Twelve patients with WT1/human leukocyte antigen (HLA)-A*2402-positive gynecological cancer were included in a Phase II clinical trial of WT1 vaccine therapy. In all the patients, the tumors were resistant to standard therapy. The patients received intradermal injections of a HLA-A*2402-restricted, modified 9-mer WT1 peptide every week for 12 weeks. Tumor size, which was measured by computed tomography (CT), was determined every 4 weeks. The responses were analyzed according to Response Evaluation Criteria in Solid Tumors (RECIST)., Results: The protocol was well tolerated; only local erythema occurred at the WT1 vaccine injection site. The clinical responses were as follows: stable disease (SD) in 3 patients and progressive disease (PD) in 9 patients. No patients had a complete (CR) or partial response (PR). The disease control rate was 25.0%., Conclusion: Although a small, uncontrolled, nonrandomized trial, this study showed that WT1 vaccine therapy for patients with gynecological cancer was safe and produced a clinical response.

Wilms' tumor gene (WT1) possesses oncogenic functions and is expressed in various kinds of malignancies, which suggests that the gene's product, the WT1 protein, should be one of the most promising cancer antigens. In fact, the WT1 protein was shown to be highly immunogenic in cancer patients. WT1 peptides that could induce WT1-specific CTLs (WT1 CTL peptides) were identified, and vaccination of cancer patients with these WT1 CTL peptides induced immunological responses, which were assessed by ex vivo immuno-monitoring, such as the tetramer assay, and in vivo immuno-monitoring, such as the peptide-specific delayed type hypersensitivity reaction. The induced immunological responses then led to clinical responses such as solid tumor shrinkage, a decrease in leukemia cells, and reduction of M-protein (multiple myeloma). Long-term stabilization of disease with good quality of life, which might be characteristic of cancer vaccine therapy, was also reported. It is noteworthy that injection with a "single" kind of WT1 peptide elicited an immunological response strong enough to induce a clinical response, indicating that the WT1 peptide vaccine has therapeutic potential. The number of reports of the successful treatment of cancer patients (not only adult but also childhood malignancies) with WT1 vaccination is increasing. Strategies for further improvement in the efficacy of therapy, including combined use of chemotherapy drugs, molecular-target-based drugs, or WT1 helper peptides, are being proposed. WT1 peptide vaccination in an "adjuvant setting" should be considered a promising treatment to protect against progression or relapse of malignancies in cases with minimal residual disease.

In this paper, we review the current status of immunotherapy targeting Wilms' tumor 1 (WT1) peptide in malignant brain tumors as well as in other hematological and solid malignancies. WT1 is expressed in various kinds of malignancies, and is involved in oncogenesis. The titers of antibodies against WT1 and the frequency of WT1-specific cytotoxic T lymphocytes (CTLs) were higher in cancer patients than in healthy donors, indicating that WT1 protein has immunogenic function. These findings provided us with a rationale for developing cancer immunotherapy that targets the WT1 peptide. Clinical trials of the WT1 peptide vaccination were initiated, and definite immunological and clinical responses were observed. The disease control rate of 57.1% was obtained especially in the case of recurrent glioblastomas, with a median progression-free survival period of 20.0 weeks and progression-free survival rate at 6 months of 33.3%. The trial showed that WT1 vaccination for malignant gliomas, which is generally believed to be an intractable disease, was safe and elicited a favorable clinical response. Further clinical studies of WT1 vaccination in patients with malignant gliomas as well as other cancers are warranted. An enhancement of the efficacy of WT1 vaccination can be expected with a combined treatment using the WT1-specific helper peptide or anti-cancer chemotherapeutic agents. Administration of WT1 vaccination along with other therapeutic modalities during initial treatment or in the case showing minimal residual disease may prolong the survival time of the cancer patients.

Object: The object of this study was to investigate the safety and clinical responses of immunotherapy targeting the WT1 (Wilms tumor 1) gene product in patients with recurrent glioblastoma multiforme (GBM)., Methods: Twenty-one patients with WT1/HLA-A*2402-positive recurrent GBM were included in a Phase II clinical study of WT1 vaccine therapy. In all patients, the tumors were resistant to standard therapy. Patients received intra-dermal injections of an HLA-A*2402-restricted, modified 9-mer WT1 peptide every week for 12 weeks. Tumor size, which was obtained by measuring the contrast-enhanced area on magnetic resonance images, was determined every 4 weeks. The responses were analyzed according to Response Evaluation Criteria in Solid Tumors (RECIST) 12 weeks after the initial vaccination. Patients who achieved an effective response continued to be vaccinated until tumor progression occurred. Progression-free survival and overall survival after initial WT1 treatment were estimated., Results: The protocol was well tolerated; only local erythema occurred at the WT1 vaccine injection site. The clinical responses were as follows: partial response in 2 patients, stable disease in 10 patients, and progressive disease in 9 patients. No patient had a complete response. The overall response rate (cases with complete or partial response) was 9.5%, and the disease control rate (cases with complete or partial response as well as those in which disease was stable) was 57.1%. The median progression-free survival (PFS) period was 20.0 weeks, and the 6-month (26-week) PFS rate was 33.3%., Conclusions: Although a small uncontrolled nonrandomized trial, this study showed that WT1 vaccine therapy for patients with WT1/HLA-A*2402-positive recurrent GBM was safe and produced a clinical response. Based on these results, further clinical studies of WT1 vaccine therapy in patients with malignant glioma are warranted.

Wilms' tumor gene WT1 is expressed in various kinds of cancers. Human WT1-specific cytotoxic T lymphocytes (CTLs) were generated, and mice immunized with WT1 peptide rejected challenges by WT1-expressing cancer cells without auto-aggression to normal organs. Furthermore, WT1 antibodies and WT1-specific CTLs were detected in cancer patients, indicating that WT1 protein was immunogenic. These findings provided us with the rationale for cancer immunotherapy targeting WT1. Clinical trials of WT1 peptide vaccination for cancer patients were started, and WT1 vaccination-related immunological responses and clinical responses, including reduction of leukemic cells, reduction of M-protein amount in myeloma, and shrinkage of solid cancer, were observed. Valuable information about immune responses against tumor antigens can be obtained by the analysis of samples from the vaccinated patients, which should lead to further improvement of cancer vaccine.

The product of the Wilms tumor gene, WT1, is a universal tumor antigen. We performed WT1 peptide-based immunotherapy for a patient with multiple myeloma (MM). This patient was a 57-year-old woman with chemotherapy-resistant MM (Bence Jones kappa type). The patient received weekly intradermal injections of an HLA-A*2402-restricted 9-mer WT1 peptide emulsified with Montanide ISA 51 adjuvant for 12 weeks and achieved a minimal response according to European Group for Blood and Marrow Transplantation criteria without experiencing systemic adverse effects. The proportion of myeloma cells in the bone marrow (BM) decreased from 85% to 25%, and the amount of M protein in the urine decreased from 3.6 to 0.6 g/day after WT1 vaccination. Furthermore, a bone scintigram showed an improvement after the vaccination. As for immunologic parameters, the frequency of WT1 tetramer-positive cells among CD8+ T-cells, which was higher than in healthy donors, temporarily decreased at weeks 4 and 8 but increased at week 12, whereas the frequency of WT1 peptide-responding CD107a/b+ cells among WT1 tetramer-positive T-cells increased from 27.0% to 38.6% after the vaccination. After WT1 vaccination, the frequency of CXCR4+ cells among WT1 tetramer-positive T-cells increased in the BM, where stromal cells expressed the ligand for CXCR4, stromal-derived factor 1 (SDF-1), but decreased in the peripheral blood (PB), implying that WT1-specific cytotoxic T-lymphocytes had migrated from the PB to the BM, a tumor site.

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.

Wild-type Wilms' tumor gene WT1 is expressed at a high level in hematopoietic malignancies including acute leukemia, chronic myelogenous leukemia, and myelodysplastic syndromes, as well as in various kinds of solid cancers. Human cytotoxic T lymphocytes (CTLs), which could specifically lyse WT1-expressing tumor cells with HLA class I restriction, were generated in vitro. It was also demonstrated that mice immunized with the WT1 peptide rejected challenges by WT1-expressing cancer cells and survived with no signs of autoaggression to normal organs that physiologically expressed WT1. Furthermore, we and others detected IgM and IgG WT1 antibodies in patients with hematopoietic malignancies, indicating that the WT1 protein was highly immunogenic, and that immunoglobulin class-switch-inducing, WT1-specific, cellular immune responses were elicited in these patients. CD8+ WT1-specific CTLs were also detected in peripheral blood or tumor-draining lymph nodes of cancer patients. These results provided us with the rationale for elicitation of CTL responses targeting the WT1 product for cancer immunotherapy. On the basis of these findings, we performed a phase I clinical trial of a WT1 peptide cancer vaccine for the patients with malignant neoplasms. These results strongly suggested that the WT1 peptide cancer vaccine had efficacy in the clinical setting because clinical responses, including reduction of leukemic blast cells or regression of tumor masses, were observed after the WT1 vaccination in patients with hematopoietic malignancies or solid cancers. The power of a tumor-associated-antigen (TAA)-derived cancer vaccine may be enhanced in combination with stronger adjuvants, helper peptide, molecular-target-based drugs, or some chemotherapy drugs, such as gemcitabine, which has been revealed to suppress regulatory T-cell function. In contrast, reduction of WT1 peptide dose may be needed for the treatment of patients with hematological stem cell diseases, because rapid and strong destruction of malignant cell-sustained hematopoiesis before recovery of normal hematopoiesis may lead to pancytopenia in these patients.

Tumor-specific immunotherapy with a Wilms' tumor 1 (WT1) peptide has been on clinical trial for leukemia, myelodysplastic syndrome, breast and lung cancers and is producing promising results. In this study, we treated three patients with renal cell carcinoma with an anchor modified, HLA-A*2402 binding WT1 peptide which was emulsified in Freund's incomplete adjuvant. In two patients tumor growth was suppressed and clinical response was evaluated as stable disease by the RECIST criteria after 3 months of weekly immunizations. Notably, development of new metastases has stopped in these patients for a prolonged period. No deleterious side effects were observed. Peptide-specific T cells were expanded in PBMCs of the patients and a substantial fraction of them bore the surface phenotype consistent with a CD8+ cytotoxic effector population. Although established tumors did not regress further, considering the component of the vaccine, i.e. peptide alone, the stabilization effect suggested the potential of WT1 peptide to develop into a more effective vaccine. To our knowledge, this is the first report of WT1 immunotherapy for renal cell carcinoma. Hopefully, the results will stimulate more extensive clinical studies.

Objective: We conducted a phase I study to investigate the safety of a weekly WT1 tumor vaccine therapy in patients with solid tumors that had been refractory to all other anti-cancer therapies., Methods: Skin-test-negative patients were intradermally injected weekly for 12 weeks with 3.0 mg of an HLA-A*2402-restricted modified 9-mer WT1 peptide emulsified in Montanide ISA51 adjuvant. We estimated the Bayesian posterior probability of the occurrence of grade 3 or 4 toxicity when receiving the weekly WT1 vaccination. This analysis provided the basis for making a decision to terminate the phase I study and switch to phase II. Moreover, we performed an exploratory assessment of the anti-tumor effects of WT1 treatment., Results: Ten patients received 114 vaccinations with WT1 on a weekly schedule. No grade 3 or 4 toxicities were observed. Based on the Bayesian approach, it was highly likely that the probability of grade 3 or 4 toxicity was below 20% (the posterior probability = 0.914). Fifteen grade 2 and two grade 1 toxicities were observed; all of these incidents, however, were determined by the Independent Data and Safety Monitoring Committee to be unrelated to the WT1 treatment. One patient exhibited a partial response; five additional patients had stable disease while receiving weekly WT1 treatment., Conclusion: This paper confirms that the potential toxicities of the treatment schedule of weekly WT1 vaccination are acceptable and suggested a potential anti-tumor effect. Consequently, we validated the decision to continue to the phase II trial.

The Wilms' tumor gene WT1 is overexpressed in leukemias and various types of solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for immunotherapy against these malignancies. Here, we report the outcome of a phase I clinical study of WT1 peptide-based immunotherapy for patients with breast or lung cancer, myelodysplastic syndrome, or acute myeloid leukemia. Patients were intradermally injected with an HLA-A*2402-restricted, natural, or modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant at 0.3, 1.0, or 3.0 mg per body at 2-week intervals, with toxicity and clinical and immunological responses as the principal endpoints. Twenty-six patients received one or more WT1 vaccinations, and 18 of the 26 patients completed WT1 vaccination protocol with three or more injections of WT1 peptides. Toxicity consisted only of local erythema at the WT1 vaccine injection sites in patients with breast or lung cancer or acute myeloid leukemia with adequate normal hematopoiesis, whereas severe leukocytopenia occurred in patients with myelodysplastic syndrome with abnormal hematopoiesis derived from WT1-expressing, transformed hematopoietic stem cells. Twelve of the 20 patients for whom the efficacy of WT1 vaccination could be assessed showed clinical responses such as reduction in leukemic blast cells or tumor sizes and/or tumor markers. A clear correlation was observed between an increase in the frequencies of WT1-specific cytotoxic T lymphocytes after WT1 vaccination and clinical responses. It was therefore demonstrated that WT1 vaccination could induce WT1-specific cytotoxic T lymphocytes and result in cancer regression without damage to normal tissues.

A Wilms' tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer. WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models. We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a "prophylactic" model). However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a "therapeutic" setting. In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a "therapeutic" model) compared with WT1 peptide vaccination alone. An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells. These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings., (Copyright 2004 Springer-Verlag)

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.

Simple Summary: Therapeutic options for rare cancers are frequently limited and less effective than for common cancers. Therefore, novel therapeutic strategies to treat rare cancers are urgently required. Our clinical study on rare cancers showed that the biweekly WT1 Trio peptide vaccine comprising two WT1-cytotoxic T lymphocyte (CTL)-peptides and one WT1-helper T lymphocyte-peptide induced more robust immune responses targeting WT1 than the weekly WT1-CTL peptide (WT1-235) vaccine. In addition, the safety of WT1 Trio was confirmed without severe treatment-related adverse events, except grade 3 myasthenia gravis-like symptoms observed in a patient with thymic cancer (TC). Fifteen (33.3%) of the 45 patients with recurrent or advanced rare cancers, including malignant glioma, soft-tissue sarcoma, TC, and malignant pleural mesothelioma, achieved stable disease after 3 months of protocol treatment. Therefore, since WT1 is widely overexpressed in rare cancers, WT1-targeted immunotherapy may be a therapeutic strategy for rare cancers. No standard treatment has been established for most rare cancers. Here, we report a clinical trial of a biweekly WT1 tri-peptide-based vaccine for recurrent or advanced rare cancers. Due to the insufficient number of patients available for a traditional clinical trial, the trial was designed for rare cancers expressing shared target molecule WT1. The recruitment criteria included WT1-expressing tumors as well as HLA-A*24:02 or 02:01. The primary endpoints were immunoglobulin G (IgG) antibody (Ab) production against the WT1-235 cytotoxic T lymphocyte (CTL) epitope and delayed-type hypersensitivity (DTH) skin reactions to targeted WT1 CTL epitopes. The secondary endpoints were safety and clinical efficacy. Forty-five patients received WT1 Trio, and 25 (55.6%) completed the 3-month protocol treatment. WT1-235 IgG Ab was positive in 88.0% of patients treated with WT1 Trio at 3 months, significantly higher than 62.5% of the weekly WT1-235 CTL peptide vaccine. The DTH positivity rate in WT1 Trio was 62.9%, which was not significantly different from 60.7% in the WT1-235 CTL peptide vaccine. The WT1 Trio safety was confirmed without severe treatment-related adverse events, except grade 3 myasthenia gravis-like symptoms observed in a patient with thymic cancer. Fifteen (33.3%) patients achieved stable disease after 3 months of treatment. In conclusion, the biweekly WT1 Trio vaccine containing the WT1-332 helper T lymphocyte peptide induced more robust immune responses targeting WT1 than the weekly WT1-235 CTL peptide vaccine. Therefore, WT1-targeted immunotherapy may be a potential therapeutic strategy for rare cancers. [ABSTRACT FROM AUTHOR]

Cancer vaccine immunotherapy is a therapy that induces cellular immune responses against a target molecule to elicit clinical anti-tumor effects. These cellular immune responses against the target molecule are monitored to evaluate whether the antigen-specific cellular immune responses are induced and maintained during the vaccination period. Enzyme-linked immunospot (ELISPOT) assay is widely performed to analyze not only the frequency of immune cells, but also their effector functions as determined by their cytokine production/secretion. The present study aimed to develop a reader-free ELISPOT assay using a handy membrane-punching device termed ELI 8. With the assistance of particle analysis by ImageJ software, the results of spot counting were reproducible with high inter-assay and inter-examiner concordance. Immune cells that produce and secrete Th1 cytokines without antigen-peptide stimulation of peripheral blood mononuclear cells (PBMCs) were detected, and their frequencies in patients with cancer were significantly higher compared with those in healthy individuals. These frequencies varied between individuals, as well as between time points during the course of cancer vaccine immunotherapy in each patient. Due to the variability in spontaneous cytokine production/secretion by PBMCs, an antigen-specific immune response (IR) index is proposed, which is a ratio of the number of spot-forming cells (SFCs) subjected to antigen-stimulation to that of SFCs with spontaneous cytokine secretion without antigen-stimulation. This index may be used as a marker for antigen-specific cellular immune responses in patients treated with cancer immunotherapy. The IR index successfully detected the induction of Wilms' tumor 1-specific cellular immune responses in patients with cancer treated with cancer vaccine immunotherapy. [ABSTRACT FROM AUTHOR]

The Wilms' tumor gene WT1 is highly expressed in various malignancies and may be a common target antigen for cancer immunotherapy. In our group, peptide-based cancer vaccines targeting WT1 CTL epitopes were developed as an immunotherapy for these malignancies. In the present study, WT1 epitope-specific immune responses were analyzed in 31 patients with advanced sarcoma with human leukocyte antigen-A*24:02- and WT1-expressing tumors who received the WT1-235 peptide vaccine as monotherapy. The serum levels of IgG and IgM antibodies against the target epitope WT1-235 and the non-target epitopes WT1-332 and WT1-271 were measured using ELISA. IgM antibodies against WT1-235, WT1-332 and WT1-271 were detected in three (9.6%), four (12.9%) and 20 patients (64.5%), respectively, prior to vaccine administration, indicating immune recognition of the WT1 antigen prior to administering the vaccine. Of 15 patients who had completed the 3-month treatment protocol, WT1-235 IgG was positive in five (33.3%) patients. An enzyme-linked immunospot assay revealed that WT1-235 epitope-specific IL-10 production/secretion in peripheral blood mononuclear cells declined in the first month of vaccine administration in all three patients with positivity for WT1-235 IgM at the start of the vaccine. Furthermore, positivity for both WT1-235 and WT1-271 IgM antibodies at the start of treatment was associated with unfavorable tumor control at 3 months after vaccine administration. These results suggested that WT1 epitope-specific IgG and IgM antibodies may be utilized as immune-monitoring markers for WT1 peptide cancer vaccine immunotherapy. The trials were entered in the University hospital Medical Information Network (UMIN) Clinical Trials Registry (https://www.umin.ac.jp/ctr ; no. UMIN000002001 on May 24, 2009 and no. UMIN000015997 on December 20, 2014). [ABSTRACT FROM AUTHOR]

Objective: The aim of the present study was to analyze the long-term survival effects of WT1 peptide vaccine, in addition to its anti-tumor effects and toxicity. Methods: A phase II clinical trial was conducted during the period of 2004-2010 at Osaka University Hospital, Osaka, Japan. The patients who had gynecologic malignancies progressing against previous treatments received WT1 peptide vaccine intradermally at 1-week intervals for 12 weeks. The vaccination was allowed to further continue, unless the patient's condition became significantly worse due to the disease progression. Results: Forty out of 42 patients, who met all the inclusion criteria, underwent WT1 peptide vaccine. Among these 40 patients, stable disease was observed in 16 cases (40 %). Skin toxicity of a grade 1, 2 and 3 occurred in 25 cases (63 %), 9 cases (23 %) and a single case (3 %), respectively, and liver toxicity of grade 1 in a single case (3 %). The overall survival period was significantly longer in cases positive for the WT1 peptide-specific delayed-type hypersensitivity (DTH) reaction after the vaccination, compared to those negative for the DTH reaction ( p = 0.023). Multivariate Cox proportional hazards analysis demonstrated that the adjusted hazard ratio for the negative DTH reaction was 2.73 (95 % CI 1.04-7.19, p = 0.043). Conclusion: WT1 peptide vaccine may be a potential treatment, with limited toxicity, for gynecologic malignancies that have become resistant to conventional therapies. Larger scale of clinical studies is required to establish the efficacy of the WT1 peptide vaccine for gynecologic malignancies. [ABSTRACT FROM AUTHOR]