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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. [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]

Diffuse midline glioma (DMG) in children is a highly aggressive, malignant brain tumor that is fatal when relapsed. Wilms tumor 1 (WT1) is a high-priority antigen target for cancer immunotherapy. We hereby report on a pediatric patient who had DMG that regrew after chemoradiotherapy and underwent WT1 peptide vaccination. A 13-year-old Japanese boy presented with vertigo, diplopia, and right hemiplegia at the initial visit to another hospital, where he was diagnosed with DMG by magnetic resonance imaging (MRI); DMG was categorized to histological grade IV glioma. The patient underwent radiotherapy and chemotherapy with temozolomide. After three cycles of chemotherapy, MRI revealed tumor regrowth that translated into deteriorated clinical manifestations. Immunohistochemically, the H3.3K27M mutation in the biopsy specimen was confirmed and the specimen was positive for WT1 protein. The patient underwent WT1-targeting immunotherapy with the WT1-specific peptide vaccine because of having HLA-A*24:02. Consequently, his quality of life drastically improved so much as to the extent that the patient became capable of conducting nearly normal daily activities at weeks 8 to 12 of vaccination. MRI at week 8 of vaccination revealed an obvious reduction in the signal intensity of the tumor. Furthermore, betamethasone dose could be reduced successively (4, 1, and 0.5 mg/day at weeks 4, 5, and 7, respectively) without deteriorating clinical manifestations. Best response among responses assessed according to the Response Assessment in Neuro-Oncology criteria was stable disease. Overall survival was 6.5 months after vaccination onset and was 8.3 months after relapse; the latter was markedly longer than the reported median OS of 3.2 months for pediatric patients with relapsed DMG in the literature. Modified WT1 tetramer staining revealed the WT1 peptide vaccine-induced production of WT1-specific cytotoxic T cells, and the interferon-γ (IFN-γ) ELISpot assay of peripheral blood mononuclear cells disclosed the production of IFN-γ. Delayed-type hypersensitivity test became positive. Any treatment-emergent adverse events did not occur except injection site erythema. Our pediatric patient exhibited an encouraging clinical evolution as manifested by stable disease, improved clinical manifestations, steroid dose reductions, a WT1-specific immune response, and a good safety profile. Therefore, WT1-targeting immunotherapy warrants further investigation in pediatric patients with relapsed DMG. [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]

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]

Wilms' tumor gene 1 (WT1) protein is a promising tumor-associated antigen for cancer immunotherapy. We have been performing WT1 peptide vaccination with good clinical responses in over 750 patients with leukemia or solid cancers. In this study, we generated single-cell gene-expression profiles of the effector memory (EM) subset of WT1-specific cytotoxic T lymphocytes (CTLs) in peripheral blood of nine acute myeloid leukemia patients treated with WT1 peptide vaccine, in order to discriminate responders ( WT1 mRNA levels in peripheral blood decreased to undetectable levels, decreased but stayed at abnormal levels, were stable at undetectable levels, or remained unchanged from the initial abnormal levels more than 6 months after WT1 vaccination) from non-responders (leukemic blast cells and/or WT1 mRNA levels increased relative to the initial state within 6 months of WT1 vaccination) prior to WT1 vaccination. Cluster and principal component analyses performed using 83 genes did not discriminate between responders and non-responders prior to WT1 vaccination. However, these analyses revealed that EM subset of WT1-specific CTLs could be divided into two groups: the 'activated' and 'quiescent' states; in responders, EM subset of the CTLs shifted to the 'quiescent' state, whereas in non-responders, those shifted to the 'activated' state following WT1 vaccination. These results demonstrate for the first time the existence of two distinct EM states, each of which was characteristic of responders or non-responders, of WT1-specific CTLs in AML patients, and raises the possibility of using advanced gene-expression profile analysis to clearly discriminate between responders and non-responders prior to WT1 vaccination. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Wilms’ tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3’ end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms. [ABSTRACT FROM AUTHOR]

Immunotherapy using a Wilms tumor (WT1) peptide has been undergoing clinical trials for adulthood leukemia and solid cancer with promising results. In this study, the authors used WT1 peptide vaccination to treat a 6-year-old girl with metastatic alveolar rhabdomyosarcoma. She received weekly intradermal injection with HLA-A*2404-restricted, 9-mer WT1 peptide against residual bone disease. After 3 months her bone disease disappeared, concurrent with an increase in the frequency of WT1-specific cytotoxic T lymphocytes (CTLs). A high proportion of WT1-specific CTLs with effector or effector memory phenotype were detected in peripheral blood of this patient. She is currently still on continued WT1 peptide immunotherapy in a disease-free condition for 22 months. WT1 peptide-based immunotherapy should be a promising option for high-risk rhabdomyosarcoma in childhood. [ABSTRACT FROM AUTHOR]

The Wilms' tumor gene WT1 is overexpressed in various tumors, and the WT1 protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. A WT1 protein-derived 16-mer peptide, WT1332 (KRYFKLSHLQMHSRKH), which was naturally generated through processing in cells and could elicit Th1-type CD4+ helper T cell responses with an HLA-DRB1*0405-restriction has previously been identified by us. In the present study, it has been demonstrated that WT1332 can induce WT1332-specific CD4+ T cell responses with the restriction of not only HLA-DRB1*0405 but also HLA-DRB1*1501, -DRB1*1502, or -DPB1*0901. These HLA class II-restricted WT1332-specific CD4+ T cell lines produced IFN-γ but neither IL-4 nor IL-10 with WT1332 stimulation, thus showing a Th1-type cytokine profile. Furthermore, HLA-DRB1*1501 or -DRB1*1502-restricted WT1332-specific CD4+ T cell lines responded to WT1-expressing transformed cells in an HLA-DRB1-restricted manner, which is consistent with our previous finding that WT1332 is a naturally processed peptide. These results indicate that the natural peptide, WT1332, is a promiscuous WT1-specific helper epitope. WT1332 is expected to apply to cancer patients with various types of HLA class II as a WT1-specific helper peptide in combination with HLA class I-restricted WT1 peptides. [ABSTRACT FROM AUTHOR]

The wild-type Wilms’ tumor gene WT1 is overexpressed in human primary leukemia and in a wide variety of solid cancers. All of the four WT1 isoforms are expressed in primary cancers and each is considered to have a different function. However, the functions of each of the WT1 isoforms in cancer cells remain unclear. The present study demonstrated that constitutive expression of the WT1 17AA(–)/KTS(–) isoform induces morphological changes characterized by a small-sized cell shape in TYK-nu.CP-r (TYK) ovarian cancer cells. In the WT1 17AA(–)/KTS(–) isoform-transduced TYK cells, cell–substratum adhesion was suppressed, and cell migration and in vitro invasion were enhanced compared to that in mock vector-transduced TYK cells. Constitutive expression of the WT1 17AA(–)/KTS(–) isoform also induced morphological changes in five (one gastric, one esophageal, two breast and one fibrosarcoma) of eight cancer cell lines examined. No WT1 isoforms other than the WT1 17AA(–)/KTS(–) isoform induced the phenotypic changes. A decrease in α-actinin 1 and cofilin expression and an increase in gelsolin expression were observed in WT1 17AA(–)/KTS(–) isoform-transduced TYK cells. In contrast, co-expression of α-actinin 1 and cofilin or knockdown of gelsolin expression by small interfering RNA restored WT1 17AA(–)/KTS(–) isoform-transduced TYK cells to a phenotype that was comparable to that of the parent TYK cells. These results indicated that the WT1 17AA(–)/KTS(–) isoform exerted its oncogenic functions through modulation of cytoskeletal dynamics. The present results may provide a novel insight into the signaling pathway of the WT1 gene for its oncogenic functions. ( Cancer Sci 2006; 97: 259–270) [ABSTRACT FROM AUTHOR]

The Wilms' tumor gene WT1 is overexpressed in most types of leukemias and various kinds of solid tumors, including lung and breast cancer, and participates in leukemogenesis and tumorigenesis. WT1 protein has been reported to be a promising tumor antigen in mouse and human. In the present study, a single amino-acid substitution, M→Y, was introduced into the first anchor motif at position 2 of the natural immunogenic HLA-A*2402-restricted 9-mer WT1 peptide (CMTWNQMNL; a.a. 235–243). This substitution increased the binding affinity of the 9-mer WT1 peptide to HLA-A*2402 molecules from 1.82×10–5 to 6.40×10–7 M. As expected from the increased binding affinity, the modified 9-mer WT1 peptide (CYTWNQMNL) elicited WT1-specific cytotoxic T lymphocytes (CTL) more effectively than the natural 9-mer WT1 peptide from peripheral blood mononuclear cells (PBMC) of HLA-A*2402-positive healthy volunteers. CTL induced by the modified 9-mer WT1 peptide killed the natural 9-mer WT1 peptide-pulsed CIR-A*2402 cells, primary leukemia cells with endogenous WT1 expression and lung cancer cell lines in a WT1-specific HLA-A*2402-restricted manner. These results showed that this modified 9-mer WT1 peptide was more immunogenic for the induction of WT1-specific CTL than the natural 9-mer WT1 peptide, and that CTL induced by the modified 9-mer WT1 peptide could effectively recognize and kill tumor cells with endogenous WT1 expression. Therefore, cancer immunotherapy using this modified 9-mer WT1 peptide should provide efficacious treatment for HLA–A*2402-positive patients with leukemias and solid tumors. [ABSTRACT FROM AUTHOR]

Summary. The Wilms' tumour gene, WT1, is expressed at high levels in leukaemia cells and plays an important role in leukaemogenesis. WT1 is also expressed in human normal CD34+ bone marrow (BM) cells at about 100 times lower levels than in leukaemia cells. To identify and characterize WT1-expressing cells in CD34+ BM cells, they were sorted into single cells and analysed for WT1 expression using two kinds of single-cell reverse transcriptase polymerase chain reaction (RT–PCR) methods. Using the semiquantitative single-cell polyA-PCR + sequence-specific (SS)-PCR method, WT1 expression was detected in four (1·3%) out of 319 CD34+ BM single cells. To confirm the above results, a single-cell nested sequence-specific (NSS)-RT–PCR method that was less quantitative but more sensitive than the polyA-PCR + SS-PCR method was also performed, and WT1 expression was detected in 15 (1·1%) out of 1315 CD34+ BM single cells. In total, WT1 expression was found in 19 (1·2%) out of 1634 CD34+ BM single cells. No significant differences in the frequencies of WT1-expressing cells were found between CD34+ CD38– and CD34+ CD38+ BM single cells. Furthermore, WT1-expressing CD34+ BM single cells expressed WT1 at levels similar to those in K562 leukaemia single cells. Analysis of lineage-specific and cell cycle gene expression in WT1-expressing CD34+ BM single cells showed that the WT1 gene could be expressed in both uncommitted, dormant CD34+ CD38– and lineage-committed, proliferating CD34+ CD38+ BM cells. Our results could indicate that these WT1-expressing CD34+ BM cells were normal counterparts of leukaemia cells. [ABSTRACT FROM AUTHOR]