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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. [ABSTRACT FROM AUTHOR]

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]

We have previously revealed the overexpression of Wilms' tumor gene 1 (WT1) in malignant glioma and developed WT1 peptide vaccine cancer immunotherapy. A phase II clinical trial indicated the clinical efficacy of the WT1 peptide vaccine for recurrent malignant glioma. Here, we aimed to investigate the immunological microenvironment in glioma tissues before and after WT1 peptide vaccine treatment. Paired tissue samples were obtained from 20 malignant glioma patients who had received the WT1 peptide vaccine for > 3 months and experienced tumor progression, confirmed radiographically and/or clinically, during vaccination. We discovered that the expression of WT1 and HLA class I antigens in the tumor cells significantly decreased after vaccination. Maintenance of WT1 expression, which is the target molecule of immunotherapy, in tumor cells during the vaccination period was significantly associated with a longer progression-free and overall survival. A high expression of HLA class I antigens and low CD4+/CD8+ tumor-infiltrating lymphocytes (TIL) ratio in pre-vaccination specimens, were also associated with a good prognosis. No statistically significant difference existed in the number of infiltrating CD3+ or CD8+ T cells between the pre- and post-vaccination specimens, whereas the number of infiltrating CD4+ T cells significantly decreased in the post-vaccination specimens. This study provides insight into the mechanisms of intra-tumoral immune reaction/escape during WT1 peptide vaccine treatment and suggests potential clinical strategies for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

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]