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Akihiro Tsuboi - One of the best experts on this subject based on the ideXlab platform.
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Identification of two distinct populations of WT1-specific cytotoxic T lymphocytes in co-vaccination of WT1 killer and helper peptides.
Cancer immunology immunotherapy : CII, 2020Co-Authors: Fumihiro Fujiki, Hiroko Nakajima, Naoya Hashimoto, Jun Nakata, Soyoko Morimoto, Sumiyuki Nishida, Akihiro Tsuboi, Miki Inatome, Kana Hasegawa, Naoki HosenAbstract:Simultaneous induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) is required for an optimal anti-tumor immune response. WT1332, 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 WT1332 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 WT1332-specific HTL responses. Interestingly, two novel distinct populations of WT1-tetramerlow WT1-TCRlow CD5low and WT1-tetramerhigh WT1-TCRhigh CD5high 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.
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Association of WT1 IgG antibody against WT1 peptide with prolonged survival in glioblastoma multiforme patients vaccinated with WT1 peptide
International Journal of Cancer, 2016Co-Authors: Yusuke Oji, Olga A Elisseeva, Yui Murakami, Miki Iwai, Yasuyoshi Chiba, Shuichi Izumoto, Naoya Hashimoto, Akihiro Tsuboi, Naoki Kagawa, Ryo IchinohasamaAbstract: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.
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Two distinct effector memory cell populations of WT1 (Wilms’ tumor gene 1)-specific cytotoxic T lymphocytes in acute myeloid leukemia patients
Cancer Immunology Immunotherapy, 2015Co-Authors: Yoshiki Nakae, Hiroko Nakajima, Satoshi Takashima, Jun Nakata, Soyoko Morimoto, Fumihiro Fujiki, Sumiyuki Nishida, Naoki Hosen, Toshio Kamiya, Akihiro TsuboiAbstract: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.
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Recognition of a natural WT1 epitope by a modified WT1 peptide-specific T-cell receptor.
Anticancer research, 2012Co-Authors: Taichi Tamanaka, Hiroko Nakajima, Yoshihiro Oka, Fumihiro Fujiki, Sumiyuki Nishida, Akihiro Tsuboi, Naoki Hosen, Akiko Katsuhara, Yu-hung Lin, Sho TachinoAbstract:Wilms' tumor gene WT1 is highly expressed in leukemia and in various types of solid tumors and exerts an oncogenic function. Thus, WT1 protein is a most promising tumor-associated antigen. We have been successfully performing WT1 vaccination with a 9-mer modified WT1 235 peptide, which has one amino acid substitution (MY) at position 2 of 9-mer natural WT1 235 peptide (235-243 a.a.), for close to 700 HLA-A*24:02-positive patients with leukemia or solid tumors. Although vaccination of modified WT1 235 peptide induced natural WT1 235 peptide-recognizing cytotoxic T-lymphocytes (CTLs) and exerted cytotoxic activity towards leukemia and solid tumor cells that expressed the natural WT1 235 peptide (epitope) but not the vaccinated modified WT1 235 peptide (epitope), the molecular basis has remained unclear. In this study, we established a modified WT1 235 peptide-specific CTL clone, we isolated T-cell receptor (TCR) genes from it and transduced the TCR genes into CD8 + T-cells. The TCR-transduced CD8 + T-cells produced interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) in response to stimulation not only with the modified WT1 235 peptide but also with the natural WT1 235 peptide and lysed modified or natural WT1 235 peptide-pulsed target cells and endogenously WT1-expressing leukemia cells in a HLA- A*24:02-restriction manner. These results provided us, for the first time at molecular basis, with a proof-of-concept of modified WT1 235 peptide-based immunotherapy for natural WT1 235 peptide-expressing malignancies. It is evident that T-cell-mediated immunity plays a crucial role in tumor regression and eradication, and the main
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Sensitive immunohistochemical detection of WT1 protein in tumors with anti‐WT1 antibody against WT1 235 peptide
Cancer science, 2010Co-Authors: Ryo Ichinohasama, Yusuke Oji, Yoshihiro Oka, Akihiro Tsuboi, Hisayuki Yokoyama, Kengo Takeuchi, Tohru Fujiwara, Kenichi Ishizawa, Osamu Taniguchi, Haruo SugiyamaAbstract:The Wilms' tumor 1 (WT1) gene is overexpressed in leukemia and various types of solid tumor, such as lung and colorectal cancer, and plays an oncogenic role in their tumorigenesis. Recent studies have demonstrated the potential of WT1-targeting cancer immunotherapy in clinical settings. As expression of WT1 protein in tumor cells is a prerequisite for WT1-targeting immunotherapy, immunohistochemical methods to detect WT1 protein with high sensitivity and specificity are required. In the present study, we developed a rabbit polyclonal antibody (WT1-R) against the 9-mer WT1 235 peptide, which is used for vaccination. The specificity of WT1-R was confirmed by immunoprecipitation, western blotting analysis, and competitive enzyme-linked immunosorbent assay. Immunocytochemistry showed the same reactivity against five cell lines (K562, Daudi, HT-180, SW480, and PC-14), whereas levels of WT1 mRNA expression determined by real-time qPCR (RT-PCR) analysis were not equivalent. Next, we examined the reactivity of WT1-R in tissue samples compared with a previously developed anti-WT1 antibody, 6F-H2. WT1-R showed greater sensitivity for detecting WT1 protein expression in samples from four different breast cancer patients than 6F-H2 antibody. The discrepancy in WT1 expression between these methods suggested that immunohistochemical detection of WT1 peptide may be advantageous for predicting the efficacy of WT1 vaccine compared to RT-PCR, and the highly sensitive WT1 antibody, WT1-R, may be useful to detect WT1 protein in tumors.
Haruo Sugiyama - One of the best experts on this subject based on the ideXlab platform.
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WT1 (Wilms' Tumor Gene 1) : biology and cancer immunotherapy
Japanese Journal of Clinical Oncology, 2010Co-Authors: Haruo SugiyamaAbstract:: Wilms' tumor gene WT1 encodes a transcription factor and plays an important role in cell growth and differentiation. The WT1 gene is highly expressed in leukemia and various types of solid tumors, whereas WT1 is a tumor marker convenient for the detection of minimal residual disease of leukemia. The WT1 gene was originally defined as a tumor suppressor gene, but we proposed that it was, on the contrary, an oncogene. Furthermore, the WT1 protein has proven to be a promising tumor-associated antigen, in which many human leukocyte antigen class I- or II-restricted WT1 epitopes have been identified. Clinical trials of WT1-targeted immunotherapy have confirmed its safety and clinical efficacy. WT1-specific cytotoxic T lymphocytes and WT1 antibodies are spontaneously induced in tumor-bearing patients, probably because of high immunogenicity of the WT1 protein. WT1-specific cytotoxic T lymphocytes make a major contribution to the graft-versus-leukemia effect after allogenic stem cell transplantation. When 75 cancer antigens including WT1 were prioritized according to several criteria such as therapeutic function and immunogenicity, WT1 was ranked as the top antigen. These findings suggest that a new era of WT1 immunotherapy is imminent.
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Sensitive immunohistochemical detection of WT1 protein in tumors with anti‐WT1 antibody against WT1 235 peptide
Cancer science, 2010Co-Authors: Ryo Ichinohasama, Yusuke Oji, Yoshihiro Oka, Akihiro Tsuboi, Hisayuki Yokoyama, Kengo Takeuchi, Tohru Fujiwara, Kenichi Ishizawa, Osamu Taniguchi, Haruo SugiyamaAbstract:The Wilms' tumor 1 (WT1) gene is overexpressed in leukemia and various types of solid tumor, such as lung and colorectal cancer, and plays an oncogenic role in their tumorigenesis. Recent studies have demonstrated the potential of WT1-targeting cancer immunotherapy in clinical settings. As expression of WT1 protein in tumor cells is a prerequisite for WT1-targeting immunotherapy, immunohistochemical methods to detect WT1 protein with high sensitivity and specificity are required. In the present study, we developed a rabbit polyclonal antibody (WT1-R) against the 9-mer WT1 235 peptide, which is used for vaccination. The specificity of WT1-R was confirmed by immunoprecipitation, western blotting analysis, and competitive enzyme-linked immunosorbent assay. Immunocytochemistry showed the same reactivity against five cell lines (K562, Daudi, HT-180, SW480, and PC-14), whereas levels of WT1 mRNA expression determined by real-time qPCR (RT-PCR) analysis were not equivalent. Next, we examined the reactivity of WT1-R in tissue samples compared with a previously developed anti-WT1 antibody, 6F-H2. WT1-R showed greater sensitivity for detecting WT1 protein expression in samples from four different breast cancer patients than 6F-H2 antibody. The discrepancy in WT1 expression between these methods suggested that immunohistochemical detection of WT1 peptide may be advantageous for predicting the efficacy of WT1 vaccine compared to RT-PCR, and the highly sensitive WT1 antibody, WT1-R, may be useful to detect WT1 protein in tumors.
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Immunohistochemical detection of WT1 protein in endometrial cancer.
Anticancer research, 2009Co-Authors: Satoshi Ohno, Haruo Sugiyama, Satoshi Dohi, Yumiko Ohno, Satoru Kyo, Nobutaka Suzuki, Masaki InoueAbstract:Background: The Wilms' tumor gene WTI is overexpressed in various kinds of solid tumors. However, it remains unclear whether WT1 plays a pathophysiological role in endometrial cancer. Patients and Methods: A series of 70 endometrial cancer patients who had undergone a curative resection was studied to determine the correlation between WT1 expression, clinicopathological characteristics and prognosis. Tissue specimens were evaluated for WT1 expression by immunohistochemistry. Results: The expression of WT1 was strong in 31 patients (44% ) and weak in 39 patients (56% ). WT1 overexpression was associated with advanced FIGO stage (p=0.0266), myometrial invasion (p=0.0477) and high-grade histological differentiation (p=0.0049). The expression level of WT1 was found to be a significant predictor of disease relapse in univariate analysis (p=0.0233), but not in multivariate analysis (p=0.4757). Conclusion: These results suggested that tumor-produced WT1 provided additional prognostic information in endometrial cancer patients.
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WT1 peptide vaccine for the treatment of cancer.
Current opinion in immunology, 2008Co-Authors: Yoshihiro Oka, Yusuke Oji, Akihiro Tsuboi, Ichiro Kawase, Haruo SugiyamaAbstract: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.
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The Wilms’ Tumor Gene WT1 Is Over-Expressed in Immature Leukemia Cells but Not Necessary for Leukemia Development in Mouse Leukemia Models.
Blood, 2006Co-Authors: Naoki Hosen, Haruo Sugiyama, Irving L WeissmanAbstract:The Wilms tumor gene WT1, which is over-expressed in almost all leukemia, is one of the most promising targets for immunotherapy. To clarify which cells express WT1, we generated a knock-in reporter GFP mice (WT1 GFP/+) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term hematopoietic stem cells (HSCs) and very few (
Yusuke Oji - One of the best experts on this subject based on the ideXlab platform.
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Association of WT1 IgG antibody against WT1 peptide with prolonged survival in glioblastoma multiforme patients vaccinated with WT1 peptide
International Journal of Cancer, 2016Co-Authors: Yusuke Oji, Olga A Elisseeva, Yui Murakami, Miki Iwai, Yasuyoshi Chiba, Shuichi Izumoto, Naoya Hashimoto, Akihiro Tsuboi, Naoki Kagawa, Ryo IchinohasamaAbstract: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.
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The role of WT1 gene in neuroblastoma.
Journal of pediatric surgery, 2011Co-Authors: Jingfu Wang, Yusuke Oji, Takaharu Oue, Shuichiro Uehara, Hiroaki Yamanaka, Masahiro FukuzawaAbstract:Abstract Background/Purpose The oncogenic properties of the Wilms' tumor gene ( WT1 ) have recently been reported in various malignancies. However, the role of WT1 in pediatric tumors is unclear. To elucidate the role of WT1 in the development of neuroblastoma (NB), we examined the WT1 expression in NB and the effect of WT1 suppression on NB cell proliferation. Methods We examined the expression of the WT1 protein in 20 NBs and 5 ganglioneuromas (GNs) by performing immunohistochemical analysis. We determined WT1 messenger RNA expression in 22 NBs, 5 GNs, and 4 NB cell lines by real-time reverse transcription polymerase chain reaction. We studied the effects of WT1 suppression on cell proliferation using small interfering RNA against WT1 . Results Expression of WT1 was higher in mature ganglionic cells, and in the immunohistochemical analysis, the WT1 positivity for GNs was significantly higher than that for NBs ( P WT1 messenger RNA expression did not correlate with histologic grade, clinical stage, and prognosis of the tumor. Knockdown of WT1 gene promoted the proliferation of NB69 cells ( P Conclusions The WT1 may govern cell differentiation and suppress cell proliferation in NB. The WT1 does not act as an oncogene, but it may participate in the maturation of NB.
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Sensitive immunohistochemical detection of WT1 protein in tumors with anti‐WT1 antibody against WT1 235 peptide
Cancer science, 2010Co-Authors: Ryo Ichinohasama, Yusuke Oji, Yoshihiro Oka, Akihiro Tsuboi, Hisayuki Yokoyama, Kengo Takeuchi, Tohru Fujiwara, Kenichi Ishizawa, Osamu Taniguchi, Haruo SugiyamaAbstract:The Wilms' tumor 1 (WT1) gene is overexpressed in leukemia and various types of solid tumor, such as lung and colorectal cancer, and plays an oncogenic role in their tumorigenesis. Recent studies have demonstrated the potential of WT1-targeting cancer immunotherapy in clinical settings. As expression of WT1 protein in tumor cells is a prerequisite for WT1-targeting immunotherapy, immunohistochemical methods to detect WT1 protein with high sensitivity and specificity are required. In the present study, we developed a rabbit polyclonal antibody (WT1-R) against the 9-mer WT1 235 peptide, which is used for vaccination. The specificity of WT1-R was confirmed by immunoprecipitation, western blotting analysis, and competitive enzyme-linked immunosorbent assay. Immunocytochemistry showed the same reactivity against five cell lines (K562, Daudi, HT-180, SW480, and PC-14), whereas levels of WT1 mRNA expression determined by real-time qPCR (RT-PCR) analysis were not equivalent. Next, we examined the reactivity of WT1-R in tissue samples compared with a previously developed anti-WT1 antibody, 6F-H2. WT1-R showed greater sensitivity for detecting WT1 protein expression in samples from four different breast cancer patients than 6F-H2 antibody. The discrepancy in WT1 expression between these methods suggested that immunohistochemical detection of WT1 peptide may be advantageous for predicting the efficacy of WT1 vaccine compared to RT-PCR, and the highly sensitive WT1 antibody, WT1-R, may be useful to detect WT1 protein in tumors.
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WT1 igg antibody for early detection of nonsmall cell lung cancer and as its prognostic factor
International Journal of Cancer, 2009Co-Authors: Yusuke Oji, Shin-ichi Nakatsuka, Yayoi Kitamura, Eriko Kamino, Aiko Kitano, Noriyoshi Sawabata, Masayoshi Inoue, Masahide Mori, Nao Sakaguchi, Kaori MiyazakiAbstract:There are urgent needs to develop methods for early detection of nonsmall cell lung cancer (NSCLC) because of its increasing incidence and poor prognosis. Here, we analyzed the production of IgG antibody (WT1 Ab) against WT1 (Wilms' tumor gene) protein that was overexpressed in the majority of NSCLC. Enzyme-linked immuno-sorbent assay showed that WT1 Ab was produced in all of 91 NSCLC patients and 70 healthy individuals and that WT1 Ab titers were significantly higher in NSCLC patients compared with healthy individuals. When the cut-off level of WT1 Ab titers were fixed at mean + 3SD of those in healthy individuals, 26.4% of NSCLC patients had WT1 Ab titers over the cut-off level, and positive rates of WT1 Ab at each clinical stage were 25.0, 30.8 and 38.4% in stage I, II and III NSCLC, respectively. When WT1 Ab was combined with CEA or CYFRA for detection of NSCLC, positive detection rates increased from 25.0 to 34.1 and 31.8%, respectively, in stage I and from 38.4 to 69.2 and 46.1%, respectively, in stage III, but not changed in stage II. Western blot analysis showed that dominant subclass of WT1 Ab was Th1-type IgG2. Interestingly, elevation of WT1 Ab titers was significantly associated with longer disease-free survival in patients with stages I-III NSCLC. These results showed that WT1 Ab could be a useful marker for early detection of NSCLC and its prognostic prediction. These results also suggested that WT1-specific immune responses played an important role in anti-cancer immunity in NSCLC.
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WT1 peptide vaccine for the treatment of cancer.
Current opinion in immunology, 2008Co-Authors: Yoshihiro Oka, Yusuke Oji, Akihiro Tsuboi, Ichiro Kawase, Haruo SugiyamaAbstract: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.
Yoshihiro Oka - One of the best experts on this subject based on the ideXlab platform.
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Recognition of a natural WT1 epitope by a modified WT1 peptide-specific T-cell receptor.
Anticancer research, 2012Co-Authors: Taichi Tamanaka, Hiroko Nakajima, Yoshihiro Oka, Fumihiro Fujiki, Sumiyuki Nishida, Akihiro Tsuboi, Naoki Hosen, Akiko Katsuhara, Yu-hung Lin, Sho TachinoAbstract:Wilms' tumor gene WT1 is highly expressed in leukemia and in various types of solid tumors and exerts an oncogenic function. Thus, WT1 protein is a most promising tumor-associated antigen. We have been successfully performing WT1 vaccination with a 9-mer modified WT1 235 peptide, which has one amino acid substitution (MY) at position 2 of 9-mer natural WT1 235 peptide (235-243 a.a.), for close to 700 HLA-A*24:02-positive patients with leukemia or solid tumors. Although vaccination of modified WT1 235 peptide induced natural WT1 235 peptide-recognizing cytotoxic T-lymphocytes (CTLs) and exerted cytotoxic activity towards leukemia and solid tumor cells that expressed the natural WT1 235 peptide (epitope) but not the vaccinated modified WT1 235 peptide (epitope), the molecular basis has remained unclear. In this study, we established a modified WT1 235 peptide-specific CTL clone, we isolated T-cell receptor (TCR) genes from it and transduced the TCR genes into CD8 + T-cells. The TCR-transduced CD8 + T-cells produced interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) in response to stimulation not only with the modified WT1 235 peptide but also with the natural WT1 235 peptide and lysed modified or natural WT1 235 peptide-pulsed target cells and endogenously WT1-expressing leukemia cells in a HLA- A*24:02-restriction manner. These results provided us, for the first time at molecular basis, with a proof-of-concept of modified WT1 235 peptide-based immunotherapy for natural WT1 235 peptide-expressing malignancies. It is evident that T-cell-mediated immunity plays a crucial role in tumor regression and eradication, and the main
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Sensitive immunohistochemical detection of WT1 protein in tumors with anti‐WT1 antibody against WT1 235 peptide
Cancer science, 2010Co-Authors: Ryo Ichinohasama, Yusuke Oji, Yoshihiro Oka, Akihiro Tsuboi, Hisayuki Yokoyama, Kengo Takeuchi, Tohru Fujiwara, Kenichi Ishizawa, Osamu Taniguchi, Haruo SugiyamaAbstract:The Wilms' tumor 1 (WT1) gene is overexpressed in leukemia and various types of solid tumor, such as lung and colorectal cancer, and plays an oncogenic role in their tumorigenesis. Recent studies have demonstrated the potential of WT1-targeting cancer immunotherapy in clinical settings. As expression of WT1 protein in tumor cells is a prerequisite for WT1-targeting immunotherapy, immunohistochemical methods to detect WT1 protein with high sensitivity and specificity are required. In the present study, we developed a rabbit polyclonal antibody (WT1-R) against the 9-mer WT1 235 peptide, which is used for vaccination. The specificity of WT1-R was confirmed by immunoprecipitation, western blotting analysis, and competitive enzyme-linked immunosorbent assay. Immunocytochemistry showed the same reactivity against five cell lines (K562, Daudi, HT-180, SW480, and PC-14), whereas levels of WT1 mRNA expression determined by real-time qPCR (RT-PCR) analysis were not equivalent. Next, we examined the reactivity of WT1-R in tissue samples compared with a previously developed anti-WT1 antibody, 6F-H2. WT1-R showed greater sensitivity for detecting WT1 protein expression in samples from four different breast cancer patients than 6F-H2 antibody. The discrepancy in WT1 expression between these methods suggested that immunohistochemical detection of WT1 peptide may be advantageous for predicting the efficacy of WT1 vaccine compared to RT-PCR, and the highly sensitive WT1 antibody, WT1-R, may be useful to detect WT1 protein in tumors.
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WT1 peptide vaccine for the treatment of cancer.
Current opinion in immunology, 2008Co-Authors: Yoshihiro Oka, Yusuke Oji, Akihiro Tsuboi, Ichiro Kawase, Haruo SugiyamaAbstract: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.
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Cancer antigen WT1-targeting treatment for the malignancies
Nihon Rinsho Men'eki Gakkai kaishi = Japanese journal of clinical immunology, 2008Co-Authors: Yoshihiro Oka, Ichiro KawaseAbstract:Wilm's tumor gene WT1, which has an oncogenic function, is expressed in various kinds of hematological malignancies and solid cancers. WT1 antibodies at higher titers and WT1-specific cytotoxic T lymphocytes (CTLs) at higher frequencies were detected in cancer patients than in healthy donors, indicating that WT1 protein was immunogenic. Furthermore, WT1-specific immune responses are considered to be involved with Graft versus Leukemia effect in the context of hematopoietic stem cell transplantation. 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-driven immunological responses and clinical responses, including reduction of leukemic cells, reduction of M-protein amount in myeloma, and shrinkage of solid cancer, were observed. Further enhancement of efficacy of WT1 peptide vaccine can be expected by co-administration of WT1-specific helper peptide or anti-cancer chemotherapy agent. WT1 peptide vaccination in the setting of MRD (minimal residual disease) may prolong "progression-free survival time", or decrease "relapse rate".
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WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone
Cancer immunology immunotherapy : CII, 2004Co-Authors: Hiroko Nakajima, Manabu Kawakami, Yoshihiro Oka, Kazuhiro Ikegame, Kotomi Kawasaki, Fumihiro Fujiki, Akihiro Tsuboi, Akiko Nakano, Yoshihiko Hoshida, Tomoki MasudaAbstract: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-Guerin 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.
Andreas Schedl - One of the best experts on this subject based on the ideXlab platform.
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the wilms tumour suppressor WT1 is involved in endothelial cell proliferation and migration expression in tumour vessels in vivo
Oncogene, 2008Co-Authors: Kay-dietrich Wagner, Andreas Schedl, Nicole Wagner, J F MichielsAbstract:Vascularization is an important step in tumour growth. Although a variety of molecules, for example, VEGF, ETS-1 or nestin have been implicated in tumour angiogenesis, the molecular mechanisms of vessel formation are not fully characterized. We showed that the Wilms' tumour suppressor WT1 activates nestin during development. Here we tested whether WT1 might also be involved in tumour angiogenesis. Endothelial WT1 expression was detected in 95% of 113 tumours of different origin. To analyse the function of WT1 in endothelial cells, we used an RNAi approach in vitro and showed that inhibition of WT1 reduces cell proliferation, migration and endothelial tube formation. On a molecular level, WT1 silencing diminished expression of the ETS-1 transcription factor. WT1 and ETS-1 shared an overlapping expression in tumour endothelia. The ETS-1 promoter was stimulated approximately 10-fold by transient co-transfection of a WT1 expression construct and WT1 bound to the ETS-1 promoter in chromatin immunoprecipitation and electrophoretic mobility shift assays. Deletion of the identified WT1-binding site abolished stimulation of the ETS-1 promoter by WT1. These findings suggest that transcriptional activation of ETS-1 by the Wilms' tumour suppressor WT1 is a crucial step in tumour vascularization via regulation of endothelial cell proliferation and migration.
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The complex life of WT1.
Journal of cell science, 2003Co-Authors: Kay-dietrich Wagner, Nicole Wagner, Andreas SchedlAbstract:The Wilms' tumour gene, WT1, encodes a zinc-finger transcription factor that is inactivated in a subset of Wilms' tumours. Mutation analysis in human patients and genetic experiments in mice have revealed that WT1 has a role much wider than just tumour suppression. Alternative splicing, RNA editing, and the use of alternative translation initiation sites generate a multitude of isoforms, which seem to have overlapping but also distinct functions during embryonic development and the maintenance of organ function. Recently, mouse strains lacking the WT1(-KTS) or WT1(+KTS) splice variants of exon 9 were generated. More severe defects of kidneys and gonads are found in mice lacking the WT1(-KTS) variant. Animals lacking the WT1(+KTS) variant show disturbed podocyte function and male-to-female sex reversal. Alternative splicing of exon 5, however, might not modify WT1 function dramatically. Recently, it was also described that reduction of WT1 levels in the kidney results in glomerulosclerosis and upregulation of WT1 in the heart might contribute to neovascularization after infarction.
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The Wilms' tumor gene WT1 is required for normal development of the retina.
The EMBO Journal, 2002Co-Authors: Kay-dietrich Wagner, Gunnar Schley, Dagmar Wilhelm, Andreas Schedl, Christoph Englert, Valérie Vidal, Nicole Wagner, Holger ScholzAbstract:The Wilms’ tumor gene WT1 is known for its important functions during genitourinary and mesothelial formation. Here we show that WT1 is necessary for neuronal development in the vertebrate retina. Mouse embryos with targeted disruption of WT1 exhibit remarkably thinner retinas than age-matched wild-type animals. A large fraction of retinal ganglion cells is lost by apoptosis, and the growth of optic nerve fibers is severely disturbed. Strikingly, expression of the class IV POU-domain transcription factor Pou4f2 (formerly Brn-3b), which is critical for the survival of most retinal ganglion cells, is lost in WT1–/– retinas. Forced expression of WT1 in cultured cells causes an up-regulation of Pou4f2 mRNA. Moreover, the WT1(–KTS) splice variant can activate a reporter construct carrying 5′-regulatory sequences of the human POU4F2. The lack of Pou4f2 and the ocular defects in WT1–/– embryos are rescued by transgenic expression of a 280 kb yeast artificial chromosome carrying the human WT1 gene. Taken together, our findings demonstrate a continuous requirement for WT1 in normal retina formation with a critical role in Pou4f2-dependent ganglion cell differentiation.
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YAC transgenic analysis reveals Wilms' tumour 1 gene activity in the proliferating coelomic epithelium, developing diaphragm and limb
Mechanisms of Development, 1998Co-Authors: Adrian Moore, Andreas Schedl, L Mcinnes, J Hecksher-sorensen, M. Doyle, Nicholas D. HastieAbstract:Abstract Wilms' Tumour I gene (WT1) is required for the correct development of the urogenital system. To examine its regulation and expression, we created several transgenic mouse lines containing a β-galactosidase reporter driven by the human WT1 promoter. A 5 kb promoter weakly recapitulated a subset of the endogenous WT1 expression pattern. In contrast, 470 and 280 kb YAC transgenes reproduced the correct pattern with high activity and highlighted new expression sites. WT1 is expressed in the septum transversum revealing how its mutation causes diaphragmatic defects. WT1 expression in the limb demarcates a zone between chondrogenic and apoptotic domains. Finally, WT1 is expressed in mesenchymal cells derived from the coelomic epithelium. Based upon these and further data we discuss a WT1 role in epithelial↔mesenchymal transitions.
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YAC transgenic analysis reveals Wilms' tumour 1 gene activity in the proliferating coelomic epithelium, developing diaphragm and limb.
Mechanisms of development, 1998Co-Authors: A.w. Moore, Andreas Schedl, L Mcinnes, J Hecksher-sorensen, M. Doyle, Nicholas D. HastieAbstract:Wilms' Tumour 1 gene (WT1) is required for the correct development of the urogenital system. To examine its regulation and expression, we created several transgenic mouse lines containing a beta-galactosidase reporter driven by the human WT1 promoter. A 5 kb promoter weakly recapitulated a subset of the endogenous WT1 expression pattern. In contrast, 470 and 280 kb YAC transgenes reproduced the correct pattern with high activity and highlighted new expression sites. WT1 is expressed in the septum transversum revealing how its mutation causes diaphragmatic defects. WT1 expression in the limb demarcates a zone between chondrogenic and apoptotic domains. Finally, WT1 is expressed in mesenchymal cells derived from the coelomic epithelium. Based upon these and further data we discuss a WT1 role in epithelialmesenchymal transitions.
