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Haruo Sugiyama - One of the best experts on this subject based on the ideXlab platform.
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clinical phase ii study of WT1 peptide vaccination in pediatric leukemia patients after stem cell transplantation and analysis of immunological monitoring
Blood, 2014Co-Authors: Yoshiko Hashii, Takako Miyamura, Haruo Sugiyama, Akihiro Tsuboi, Naoki Hosen, Keiichi OzonoAbstract:Introduction Allogeneic stem cell transplantation (allo-SCT) is a radical treatment for pediatric patients with relapsed and/or chemotherapy-resistant leukemia. Because these leukemia cells seem refractory to chemotherapy and irradiation, immunotherapy are needed. WT1 is highly expressed in pediatric leukemia and essential for leukemia cell growth, leading to the hypothesis that the WT1 Protein-based immunization after allo-SCT may improve the clinical outcome of the SCT therapy. We investigated the clinical efficacy and immunological effect of immunotherapy targeting WT1 Protein for pediatric patients after allo-SCT. Method Major inclusion criteria were as follows: Patients with HLA-A*2402 and/or HLA-*A0201 aged 20 years or younger and WT1 mRNA expression in leukemic cells; donors with HLA-A*2402 and/or HLA-*A0201. The HLA A*2402 restricted 9mer modified WT1 peptide or HLA A*0201 restricted natural WT1 peptide emulsified in Montanide ISA 51 adjuvant was injected intradermally. The dose of WT1 peptide depended on patient weight. The vaccinations were scheduled to be given weekly for 12 consecutive weeks, and if no recurrence and severe adverse effect were observed, vaccination was continued. Absolute number and frequency of WT1 specific cytotoxic T lymphocytes were analyzed by WT1 tetramer assay. Results A total of 18 patients with 9 AML/MDS, 8 ALL and 1 NHL were enrolled. Seven patients were those with high risk (HR) of relapse who had received SCT at 3rd CR or on disease. The other 11 patients were at standard risk (SR) but had either relapsed after SCT or shown induction failure. 16 had HLA-A*2402 and the other 2 had HLA-A*0201. 12 of the 18 patients received WT1 peptide vaccination monthly after the first 12-time consecutive vaccinations with one-week interval and are still in complete remission for 16-72 months (median 31 mo.). One patient discontinued the vaccination because of pancreatitis due to treatment with steroid for GVHD. One year probabilities of PFS is 66.7% in all, and it is notable that 4 of 7 HR patients are maintained in long-term CR. Although, of these 12 patients with persistence of CR, seven patients had high WT1 mRNA level before vaccination in peripheral blood (PB) or bone marrow (BM), WT1 mRNA levels decreased to within normal level after vaccination. WT1-specific CTLs in PB were detected and increased after vaccination in all cases and absolute numbers of these cells were significantly increased after vaccination in all cases (p< 0.05). The fold increase of WT1-specific CTL numbers after the vaccination was significantly higher in cases which maintained CR (p< 0.05). The WT1-specific CTLs with high tetramer staining were detected during the first 12-time vaccinations in 14 patients, and of these patients, 12 patients are still in CR. In the other 4 patients, only very few WT1 specific CTLs were detected, and three of the four patients showed recurrence. Five patients (1AMKL, 4 ALL) had recurrence. There was no significant difference in the fold increase of WT1-specific CTL numbers after the vaccination between cases with immunosuppressive agents for the prevention of GVHD and those without the agents. Discussion We reported the interim result of the phase II clinical study of WT1 peptide immunotherapy after SCT for pediatric patients, in which promising clinical efficacy of the therapy was suggested. In addition, we found that an increase in WT1-specific CTLs was associated with a decrease in WT1 mRNA, suggesting occurrence of WT1-driven graft versus leukemia (GVL) effect, which is consistent with strategies to enhance leukemia antigen-specific GVL response without deterioration of GVHD. The following three mechanisms are suggested. 1) After SCT, many kinds of cytokines are produced and proliferation of T lymphocytes occur; 2) lymphodepletion after SCT allows development of T cells specific to WT1 peptide; and resultantly, 3) efficient homeostatic expansion of WT1-specific CTLs is induced. Post-SCT vaccination with WT1 peptide can be a safe and effective strategy to prevent the recurrence of the disease without deterioration of GVHD in pediatric hematological malignancy. For avoiding late side effect due to preconditioning regimen and GVHD, effective post SCT immunotherapy is a promising strategy for pediatric patient. Larger studies are warranted on application of WT1 targeted immunotherapy to prevent recurrence after pediatric SCT Disclosures No relevant conflicts of interest to declare.
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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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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 Immunotherapy Targeting Wilms’ Tumor Gene WT1 Product
Journal of Immunology, 2000Co-Authors: Keiko Udaka, Olga A Elisseeva, Katsuyuki Aozasa, Akihiro Tsuboi, Tadamitsu Kishimoto, Hiroyasu Ogawa, Haruo SugiyamaAbstract:The Wilms’ tumor gene WT1 is expressed at high levels not only in acute myelocytic and lymphocytic leukemia and in chronic myelocytic leukemia but also in various types of solid tumors including lung cancers. To determine whether the WT1 Protein can serve as a target Ag for tumor-specific immunity, three 9-mer WT1 peptides (Db126, Db221, and Db235), which contain H-2Db-binding anchor motifs and have a comparatively higher binding affinity for H-2Db molecules, were tested in mice (C57BL/6, H-2Db) for in vivo induction of CTLs directed against these WT1 peptides. Only one peptide, Db126, with the highest binding affinity for H-2Db molecules induced vigorous CTL responses. The CTLs specifically lysed not only Db126-pulsed target cells dependently upon Db126 concentrations but also WT1-expressing tumor cells in an H-2Db-restricted manner. The sensitizing activity to the Db126-specific CTLs was recovered from the cell extract of WT1-expressing tumor cells targeted by the CTLs in the same retention time as that needed for the synthetic Db126 peptide in RP-HPLC, indicating that the Db126-specific CTLs recognize the Db126 peptide to kill WT1-expressing target cells. Furthermore, mice immunized with the Db126 peptide rejected challenges by WT1-expressing tumor cells and survived for a long time with no signs of autoaggression by the CTLs. Thus, the WT1 Protein was identified as a novel tumor Ag. Immunotherapy targeting the WT1 Protein should find clinical application for various types of human cancers.
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Growth inhibition of human leukemic cells by WT1 (Wilms tumor gene) antisense oligodeoxynucleotides: implications for the involvement of WT1 in leukemogenesis
Blood, 1996Co-Authors: Tamotsu Yamagami, Haruo Sugiyama, Hiroyasu Ogawa, Kazushi Inoue, Toyoshi Tatekawa, Moritoshi Hirata, Tetsuhiro Kudoh, Tetsu Akiyama, Akira Murakami, Taira MaekawaAbstract:We have previously reported expression of WT1 in acute leukemia. To elucidate its biological significance, we examined the effect of the suppression of the WT1 expression by WT1 antisense oligomers on the growth of the leukemic cells expressing WT1. When 20 different WT1 antisense (AS) oligomers covering from the 5' cap sites of the WT1 gene to the 3' end were examined for the inhibitory effect on the growth of K562 cells expressing WT1, four WT1 AS oligomers inhibited the cell growth, whereas WT1 sense and random sequence oligomers had no effect on the cell growth of K562. Moreover, WT1 AS oligomers significantly inhibited the growth of the clonogenic cells of fresh leukemic cells in six of 14 patients with acute myeloid leukemia, in one of two patients with chronic myelogenous leukemia (CML) chronic phase, and in one of one patient with CML blastic crisis. However, these oligomers did not inhibit normal colony-forming unit-granulocyte-macrophage. Western blot analysis clearly demonstrated the significant reduction in the WT1 Protein levels in the K562 and fresh leukemic cells that were treated with the WT1 AS oligomers, confirming that the inhibitory effect of the WT1 AS oligomers on the cell growth operates via the reduction in the WT1 Protein levels. These results show that WT1 plays an important role in leukemogenesis.
Nicholas D. Hastie - One of the best experts on this subject based on the ideXlab platform.
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the wilms tumour Protein WT1 shuttles between nucleus and cytoplasm and is present in functional polysomes
Human Molecular Genetics, 2003Co-Authors: Martina Niksic, Joan Slight, Jeremy R Sanford, Javier F Caceres, Nicholas D. HastieAbstract:Mutations of the Wilms' tumour-1 (WT1) gene in humans can lead to childhood kidney cancer, life-threatening glomerular nephropathy and gonadal dysgenesis. The WT1 Protein is normally expressed in the developing genitourinary tract, heart, spleen and adrenal glands and is crucial for their development, however it's function at the molecular level is yet to be fully understood. The Protein is predominantly nuclear and there is evidence that the two different isoforms of WT1 (-KTS and +KTS) are involved in two different steps of gene expression control: transcription and RNA processing. In this study we report a novel property of WT1, namely that it shuttles between the nucleus and cytoplasm. Moreover, western blot analysis showed that between 10 and 50% of total cellular WT1 can be detected in the cytoplasm depending on the cell type. A significant proportion of cytoplasmic WT1 is in association with ribonucleoProtein particles (RNPs), which strengthens the idea of its involvement in RNA metabolism. Furthermore, we report that WT1 is associated with actively translating polysomes, extending even further the potential roles of WT1 and opening the possibility that it is involved in the regulation of translation. Interestingly, despite the functional differences between two of the WT1 isoforms (+/-KTS) within the nucleus, both isoforms share the shuttling property and are found in translating polysomes.
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faithful expression of a tagged fugu WT1 Protein from a genomic transgene in zebrafish
Nucleic Acids Research, 2003Co-Authors: Colin G Miles, Lesley Rankin, Shirley I Smith, Martina Niksic, Greg Elgar, Nicholas D. HastieAbstract:The teleost ®sh are widely used as model organisms in vertebrate biology. The compact genome of the puffer®sh, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identi®cation of conserved regulatory elements, whilst the zebra®sh is particularly suited to genetic and developmental studies. We demonstrate that a puffer®sh WT1 transgene can be expressed and spliced appropriately in transgenic zebra®sh, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebra®sh with the same construct, we show that Fugu RNA is processed correctly in zebra®sh but not in mice. Furthermore, we show for the ®rst time that a Fugu genomic construct can produce Protein in transgenic zebra®sh: a full-length Fugu WT1 transgene with a C-terminal b-galactosidase fusion is spliced and translated correctly in zebra®sh, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebra®sh:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.
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faithful expression of a tagged fugu WT1 Protein from a genomic transgene in zebrafish efficient splicing of pufferfish genes in zebrafish but not mice
Nucleic Acids Research, 2003Co-Authors: Colin G Miles, Lesley Rankin, Shirley I Smith, Martina Niksic, Greg Elgar, Nicholas D. HastieAbstract:The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce Protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal β-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.
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presence of WT1 the wilm s tumor suppressor gene product in nuclear poly a ribonucleoProtein
Journal of Biological Chemistry, 1999Co-Authors: Michael Ladomery, Joan Slight, Sharon Mc Ghee, Nicholas D. HastieAbstract:The tumor suppressor gene WT1 encodes a zinc finger Protein, which consists of four C-terminal C2-H2 zinc fingers of the Kruppel type, and at the N terminus a Q/P-rich trans-regulatory domain, both characteristic of transcription factors. However, recent findings suggest that WT1 may also be involved in a post-transcriptional process. Specifically, WT1 isoforms containing the alternatively spliced exon 9 (+lysine-threonine-serine (KTS)) preferentially associate with nuclear speckles and co-immunoprecipitate splicing antigens (Larsson, S. H., Charlieu, J.-P., Miyagawa, K., Engelkamp, D., Rassoulzadegan, M., Ross, A., Cuzin, F., van Heyningen, V., and Hastie, N. D. (1995) Cell 81, 391–401); furthermore, WT1 has been shown to interact with the ubiquitous splicing factor U2AF65 (Davies, R. C., Calvo, C., Larsson, S. H., Lamond, A. I., and Hastie, N. D. (1998) Genes Dev. 12, 3217–3225) and binds to RNA in vitro(Caricasole, A., Duarte, A., Larsson, S. H., Hastie, N. D., Little, M., Holmes, G., Todorov, I., and Ward, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7562–7566; Bardeesy, N., and Pelletier, J. (1998) Nucleic Acids Res. 26, 1784–1792). To extend these findings, we have fractionated nuclear extracts to see if particles containing WT1 have the properties of ribonucleoProtein (RNP). In summary, WT1 is enriched by oligo(dT) chromatography, as are U2AF65, the U5 small nuclear RNP-associated Protein p116 and hnRNP A1. Gel filtration and sedimentation profiles suggest that WT1 is present in RNase-sensitive particles, >2 MDa in size, peaking at ∼60 S, and ∼1.27 g/cm3 on Nycodenz. Similar results were obtained from two cell lines expressing WT1, fetal kidneys (day E17), and transiently transfected cells, suggesting that the presence of WT1 Protein in nuclear poly(A)+ RNP is a general aspect of WT1 function.
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dna binding capacity of the WT1 Protein is abolished by denys drash syndrome WT1 point mutations
Human Molecular Genetics, 1995Co-Authors: Melissa H. Little, Veronica Van Heyningen, Nicholas D. Hastie, Gregory Holmes, Wendy A. Bickmore, Brandon J. WainwrightAbstract:Constitutional point mutations in the zinc finger (ZF) region of the Wilms' tumour suppressor gene 1 (WT1) lead to Denys-Drash syndrome (DDS). Patients with this syndrome display renal failure, Wilms' tumour (WT) and pseudohermaphroditism. DDS WT1 mutations fall into three major categories: (a) missense mutations altering amino acids which directly interact with the DNA target; (b) substitution of amino acids involved in zinc complexing; and (c) nonsense mutations leading to the removal of at least two zinc fingers. We have expressed the WT1 zinc fingers as glutathione-S-transferase fusion Proteins, with the lysine-threonine-serine (KTS) alternate splice between ZF3 and ZF4 either present or absent. WT1 fusion constructs with all three classes of DDS mutation were also created. Wild-type and mutant fusion Proteins were assayed for their DNA-binding affinity using four previously identified WT1 DNA targets: an EGR1 consensus site; murine insulin-like growth factor 2 promoter 2 (IGF2P2); a (TCC)n motif from the PDGFA-chain promoter; and +P5, a genomic fragment isolated by its affinity for WT1 + KTS. WT1-KTS bound all four targets, but WT1 + KTS only bound +P5. All three classes of DDS mutation investigated, with or without KTS, abolished binding to all four targets. This provides evidence that DDS mutations act either as dominant-negative antimorphs, or elicit their effect through disturbed isoform dosage balance.
Renier J Brentjens - One of the best experts on this subject based on the ideXlab platform.
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phase 2 trial of a multivalent WT1 peptide vaccine galinpepimut s in acute myeloid leukemia
Blood Advances, 2018Co-Authors: Tao Dao, P Maslak, Yvette Bernal, Suzanne Chanel, Rong Zhang, Mark G Frattini, Todd L Rosenblat, Joseph G Jurcic, Renier J BrentjensAbstract:A National Cancer Institute consensus study on prioritization of cancer antigens ranked the Wilms tumor 1 (WT1) Protein as the top immunotherapy target in cancer. We previously reported a pilot study of a multivalent WT1 peptide vaccine (galinpepimut-S) in acute myeloid leukemia (AML) patients. We have now conducted a phase 2 study investigating this vaccine in adults with AML in first complete remission (CR1). Patients received 6 vaccinations administered over 10 weeks with the potential to receive 6 additional monthly doses if they remained in CR1. Immune responses (IRs) were evaluated after the 6th and 12th vaccinations by CD4+ T-cell proliferation, CD8+ T-cell interferon-γ secretion (enzyme-linked immunospot), or the CD8-relevant WT1 peptide major histocompatibility complex tetramer assay (HLA-A*02 patients only). Twenty-two patients (7 males; median age, 64 years) were treated. Fourteen patients (64%) completed ≥6 vaccinations, and 9 (41%) received all 12 vaccine doses. Fifteen patients (68%) relapsed, and 10 (46%) died. The vaccine was well tolerated, with the most common toxicities being grade 1/2 injection site reactions (46%), fatigue (32%), and skin induration (32%). Median disease-free survival from CR1 was 16.9 months, whereas the overall survival from diagnosis has not yet been reached but is estimated to be ≥67.6 months. Nine of 14 tested patients (64%) had an IR in ≥1 assay (CD4 or CD8). These results indicated that the WT1 vaccine was well tolerated, stimulated a specific IR, and was associated with survival in excess of 5 years in this cohort of patients. This trial was registered at www.clinicaltrials.gov as #NCT01266083.
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393 engineering armored t cell receptor mimic tcrm chimeric antigen receptor car t cells specific for the intracellular Protein wilms tumor 1 WT1 for treatment of hematologic and solid malignancies
Molecular Therapy, 2016Co-Authors: Sarwish Rafiq, Tao Dao, David A. Scheinberg, Terence J Purdon, Chen Liu, Renier J BrentjensAbstract:Adoptive therapy with chimeric antigen receptor (CAR) T cells specific for CD19 is clinically successful in a limited set of leukemias and most CARs studied are targeted against external antigens. Wilms Tumor Antigen 1 (WT1) Protein (WT1) is an intracellular antigen overexpressed in many cancers, including leukemias and solid malignancies and is thus an appealing, broadly applicable target. We have engineered the first armored T cell receptor-mimic (TCRm) CAR against WT1. Derived from the ESK1 antibody, the second generation CAR, WT1-28z, is reactive with the RMFPNAPYL peptide of WT1 that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1-28z was further modified to secrete human IL-12 cytokine, thus creating the armored CAR WT1-28z/IL-12. T cells expressing WT1-28z or WT1-28z/IL-12 are cytotoxic against a range of both hematological and solid tumors. Importantly, both WT1-directed T cells are specific for the WT1-HLA-A*02:01 complex and are not reactive against cells that do not express both HLA-A*02:01 and WT1. In established SCID/Beige mouse models of either acute leukemia or ovarian cancer, one dose of WT1-28z T cells prolongs survival of mice over untreated or irrelevant antigen-specific CAR T cell treated mice. Furthermore, one dose of the armored WT1-28z/IL12 CAR T cells further significantly prolongs survival of mice in both models over WT1-28z CAR T cell treated mice, with a subset of mice whose disease was eradicated. The armored TCRm CAR T cells against WT1 are effective in eradicating disease in both hematologic and solid tumors and may hold great clinical potential to expand on the success of CAR T cell therapy.
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engineered t cell receptor mimic antibody tcrm chimeric antigen receptor car t cells against the intracellular Protein wilms tumor 1 WT1 for treatment of hematologic and solid cancers
Blood, 2014Co-Authors: Sarwish Rafiq, Tao Dao, David A. Scheinberg, Cheng Liu, Renier J BrentjensAbstract:Adoptive transfer therapy of T cells expressing chimeric antigen receptors (CAR) against tumor-associated antigens has been shown to be clinically successful in a limited set of leukemia. However, novel antigen targets for both hematological and solid malignancies are required. Most CARs described thus far are targeted against external antigens on particular cell types. We have designed and engineered the first CAR T cell against a human intracellular Protein, WT1. WT1 is overexpressed in many cancers, including acute and chronic leukemias and numerous solid tumors. Our TCRm CAR, derived from the ESK1 TCRm mAb, termed WT1 28z, is reactive with the RMFPNAPYL peptide of the WT1 Protein that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1 28z expressing T cells have high expression of the CAR on their surface. They are cytotoxic in standard 51 Cr assays against a range of cancer cell lines, including the megakaryoblastic cell line SET2, the acute myeloid leukemia (AML) cell line AML14, the multiple myeloma cell line KARPAS, and the ovarian cancer line, OVCAR3, as compared to CAR T cells against an irrelevant antigen. The WT1 28z CAR T cells are also cytotoxic against primary AML bone marrow blasts in this assay. When co-cultured with these primary cells or cancer cell lines, the WT1 28z CAR T cells have enhanced production of proinflammatory cytokines such as IFN-g, IL-2, and GM-CSF, as compared to irrelevant CAR T cells. Importantly, WT1 28z T cells are specific for the WT1-HLA-A*02:01 complex. The cells do not show cytotoxicity against cell lines or primary cells that are not both HLA-A*02:01- positive and WT1 positive. WT1 28z T cells are currently being tested alongside irrelevant antigen CAR T cells in AML and ovarian cancer murine models in vivo to assess efficacy, with the ultimate goal of translating this novel approach into the clinical setting for both hematological and solid cancers. The data provide the proof-of-concept that CAR T cells also may be directed at intracellular antigens. Disclosures Dao: Novartis: Patents & Royalties. Liu: Eureka: Employment, Inventor Other. Scheinberg: Novartis: Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.
Yoshihiro Oka - One of the best experts on this subject based on the ideXlab platform.
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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, Hisayuki Yokoyama, Kengo Takeuchi, Tohru Fujiwara, Kenichi Ishizawa, Osamu Taniguchi, Yoshihiro OkaAbstract: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. (Cancer Sci 2010; 101: 1089–1092)
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high frequencies of less differentiated and more proliferative WT1 specific cd8 t cells in bone marrow in tumor bearing patients an important role of bone marrow as a secondary lymphoid organ
Cancer Science, 2010Co-Authors: Ayako Murao, Hiroko Nakajima, Olga A Elisseeva, Yoshihiro Oka, Akihiro Tsuboi, Naoki Hosen, Fumihiro Fujiki, Yukie Tanakaharada, Sumiyuki Nishida, Toshiaki ShirakataAbstract:In tumor-bearing patients, tumor-associated antigen (TAA)-specific CTLs are spontaneously induced as a result of immune response to TAAs and play an important role in anti-tumor immunity. Wilms’ tumor gene 1 (WT1) is overexpressed in various types of tumor and WT1 Protein is a promising pan-TAA because of its high immunogenicity. In this study, to clarify the immune response to the WT1 antigen, WT1-specific CD8+ T cells that were spontaneously induced in patients with solid tumor were comparatively analyzed in both bone marrow (BM) and peripheral blood (PB). WT1-specific CD8+ T cells more frequently existed in BM than in PB, whereas frequencies of naive (CCR7+ CD45RA+), central memory (CCR7+ CD45RA−), effector-memory (CCR7− CD45RA−), and effector (CCR7− CD45RA+) subsets were not significantly different between BM and PB. However, analysis of these subsets for the expression of CD57 and CD28, which were associated with differentiation, revealed that effector-memory and effector subsets of the WT1-specific CD8+ T cells in BM had less differentiated phenotypes and more proliferative potential than those in PB. Furthermore, CD107a/b functional assay for WT1 peptide-specific cytotoxic potential and carboxyfluorescein diacetate succinimidyl ester dilution assay for WT1 peptide-specific proliferation also showed that WT1-specific CD8+ T cells in BM were less cytotoxic and more proliferative in response to WT1 peptide than those in PB. These results implied that BM played an important role as a secondary lymphoid organ in tumor-bearing patients. Preferential residence of WT1-specific CD8+ T cells in BM could be, at least in part, explained by higher expression of chemokine receptor CCR5, whose ligand was expressed on BM fibroblasts on the WT1-specific CD8+ T cells in BM, compared to those in PB. These results should provide us with an insight into WT1-specific immune response in tumor-bearing patients and give us an idea of enhancement of clinical response in WT1 Protein-targeted immunotherapy. (Cancer Sci 2010; 101: 848–854)
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a WT1 Protein derived naturally processed 16 mer peptide WT1332 is a promiscuous helper peptide for induction of WT1 specific th1 type cd4 t cells
Microbiology and Immunology, 2008Co-Authors: Fumihiro Fujiki, Hiroko Nakajima, Olga A Elisseeva, Yoshihiro Oka, Mai Kawakatsu, Yukie Harada, Naoko Tatsumi, Eriko Kamino, Toshiaki ShirakataAbstract:The Wilms’ tumor geneWT1 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 HLADRB1 ∗ 1501, -DRB1 ∗ 1502, or -DPB1 ∗ 0901. These HLA class II-restricted WT1332-specific CD4 + Tc ell 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 + Tc ell 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.
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identification of a WT1 Protein derived peptide WT1187 as a hla a 0206 restricted WT1 specific ctl epitope
Microbiology and Immunology, 2008Co-Authors: Yoshihiro Oka, Hiroko Nakajima, Akihiro Tsuboi, Fumihiro Fujiki, Mai Kawakatsu, Yukie Harada, Soyoko Morimoto, Tomoki Masuda, Yoko Fukuda, Takamasa KatagiriAbstract:The Wilms' tumor gene WT1 is overexpressed in various kinds of hematopoietic malignancies as well as solid cancers, and this Protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. WT1-specific CTL epitopes with a restriction of HLA-A 2402 or HLA-A 0201 have been already identified. In the present study it has been demonstrated that a 9-mer WT1-derived WT1(187) peptide, which had already been shown to elicit a WT1-specific CTL response with a restriction of HLA-A 0201, can also elicit a CTL response with a restriction of HLA-A 0206. In all three different HLA-A 0206(+) healthy donors examined, WT1(187) peptide-specific CTL could be generated from peripheral blood mononuclear cells, and the CTL showed cytotoxic activity that depended on dual expression of WT1 and HLA-A 0206 molecules. The present study describes the first identification of a HLA-A 0206-restricted, WT1-specific CTL epitope. The present results should help to broaden the application of WT1 peptide-based immunotherapy from only HLA-A 0201-positive to HLA-A 0206-positive cancer patients as well.
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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.
Akihiro Tsuboi - One of the best experts on this subject based on the ideXlab platform.
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clinical phase ii study of WT1 peptide vaccination in pediatric leukemia patients after stem cell transplantation and analysis of immunological monitoring
Blood, 2014Co-Authors: Yoshiko Hashii, Takako Miyamura, Haruo Sugiyama, Akihiro Tsuboi, Naoki Hosen, Keiichi OzonoAbstract:Introduction Allogeneic stem cell transplantation (allo-SCT) is a radical treatment for pediatric patients with relapsed and/or chemotherapy-resistant leukemia. Because these leukemia cells seem refractory to chemotherapy and irradiation, immunotherapy are needed. WT1 is highly expressed in pediatric leukemia and essential for leukemia cell growth, leading to the hypothesis that the WT1 Protein-based immunization after allo-SCT may improve the clinical outcome of the SCT therapy. We investigated the clinical efficacy and immunological effect of immunotherapy targeting WT1 Protein for pediatric patients after allo-SCT. Method Major inclusion criteria were as follows: Patients with HLA-A*2402 and/or HLA-*A0201 aged 20 years or younger and WT1 mRNA expression in leukemic cells; donors with HLA-A*2402 and/or HLA-*A0201. The HLA A*2402 restricted 9mer modified WT1 peptide or HLA A*0201 restricted natural WT1 peptide emulsified in Montanide ISA 51 adjuvant was injected intradermally. The dose of WT1 peptide depended on patient weight. The vaccinations were scheduled to be given weekly for 12 consecutive weeks, and if no recurrence and severe adverse effect were observed, vaccination was continued. Absolute number and frequency of WT1 specific cytotoxic T lymphocytes were analyzed by WT1 tetramer assay. Results A total of 18 patients with 9 AML/MDS, 8 ALL and 1 NHL were enrolled. Seven patients were those with high risk (HR) of relapse who had received SCT at 3rd CR or on disease. The other 11 patients were at standard risk (SR) but had either relapsed after SCT or shown induction failure. 16 had HLA-A*2402 and the other 2 had HLA-A*0201. 12 of the 18 patients received WT1 peptide vaccination monthly after the first 12-time consecutive vaccinations with one-week interval and are still in complete remission for 16-72 months (median 31 mo.). One patient discontinued the vaccination because of pancreatitis due to treatment with steroid for GVHD. One year probabilities of PFS is 66.7% in all, and it is notable that 4 of 7 HR patients are maintained in long-term CR. Although, of these 12 patients with persistence of CR, seven patients had high WT1 mRNA level before vaccination in peripheral blood (PB) or bone marrow (BM), WT1 mRNA levels decreased to within normal level after vaccination. WT1-specific CTLs in PB were detected and increased after vaccination in all cases and absolute numbers of these cells were significantly increased after vaccination in all cases (p< 0.05). The fold increase of WT1-specific CTL numbers after the vaccination was significantly higher in cases which maintained CR (p< 0.05). The WT1-specific CTLs with high tetramer staining were detected during the first 12-time vaccinations in 14 patients, and of these patients, 12 patients are still in CR. In the other 4 patients, only very few WT1 specific CTLs were detected, and three of the four patients showed recurrence. Five patients (1AMKL, 4 ALL) had recurrence. There was no significant difference in the fold increase of WT1-specific CTL numbers after the vaccination between cases with immunosuppressive agents for the prevention of GVHD and those without the agents. Discussion We reported the interim result of the phase II clinical study of WT1 peptide immunotherapy after SCT for pediatric patients, in which promising clinical efficacy of the therapy was suggested. In addition, we found that an increase in WT1-specific CTLs was associated with a decrease in WT1 mRNA, suggesting occurrence of WT1-driven graft versus leukemia (GVL) effect, which is consistent with strategies to enhance leukemia antigen-specific GVL response without deterioration of GVHD. The following three mechanisms are suggested. 1) After SCT, many kinds of cytokines are produced and proliferation of T lymphocytes occur; 2) lymphodepletion after SCT allows development of T cells specific to WT1 peptide; and resultantly, 3) efficient homeostatic expansion of WT1-specific CTLs is induced. Post-SCT vaccination with WT1 peptide can be a safe and effective strategy to prevent the recurrence of the disease without deterioration of GVHD in pediatric hematological malignancy. For avoiding late side effect due to preconditioning regimen and GVHD, effective post SCT immunotherapy is a promising strategy for pediatric patient. Larger studies are warranted on application of WT1 targeted immunotherapy to prevent recurrence after pediatric SCT Disclosures No relevant conflicts of interest to declare.
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high frequencies of less differentiated and more proliferative WT1 specific cd8 t cells in bone marrow in tumor bearing patients an important role of bone marrow as a secondary lymphoid organ
Cancer Science, 2010Co-Authors: Ayako Murao, Hiroko Nakajima, Olga A Elisseeva, Yoshihiro Oka, Akihiro Tsuboi, Naoki Hosen, Fumihiro Fujiki, Yukie Tanakaharada, Sumiyuki Nishida, Toshiaki ShirakataAbstract:In tumor-bearing patients, tumor-associated antigen (TAA)-specific CTLs are spontaneously induced as a result of immune response to TAAs and play an important role in anti-tumor immunity. Wilms’ tumor gene 1 (WT1) is overexpressed in various types of tumor and WT1 Protein is a promising pan-TAA because of its high immunogenicity. In this study, to clarify the immune response to the WT1 antigen, WT1-specific CD8+ T cells that were spontaneously induced in patients with solid tumor were comparatively analyzed in both bone marrow (BM) and peripheral blood (PB). WT1-specific CD8+ T cells more frequently existed in BM than in PB, whereas frequencies of naive (CCR7+ CD45RA+), central memory (CCR7+ CD45RA−), effector-memory (CCR7− CD45RA−), and effector (CCR7− CD45RA+) subsets were not significantly different between BM and PB. However, analysis of these subsets for the expression of CD57 and CD28, which were associated with differentiation, revealed that effector-memory and effector subsets of the WT1-specific CD8+ T cells in BM had less differentiated phenotypes and more proliferative potential than those in PB. Furthermore, CD107a/b functional assay for WT1 peptide-specific cytotoxic potential and carboxyfluorescein diacetate succinimidyl ester dilution assay for WT1 peptide-specific proliferation also showed that WT1-specific CD8+ T cells in BM were less cytotoxic and more proliferative in response to WT1 peptide than those in PB. These results implied that BM played an important role as a secondary lymphoid organ in tumor-bearing patients. Preferential residence of WT1-specific CD8+ T cells in BM could be, at least in part, explained by higher expression of chemokine receptor CCR5, whose ligand was expressed on BM fibroblasts on the WT1-specific CD8+ T cells in BM, compared to those in PB. These results should provide us with an insight into WT1-specific immune response in tumor-bearing patients and give us an idea of enhancement of clinical response in WT1 Protein-targeted immunotherapy. (Cancer Sci 2010; 101: 848–854)
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identification of a WT1 Protein derived peptide WT1187 as a hla a 0206 restricted WT1 specific ctl epitope
Microbiology and Immunology, 2008Co-Authors: Yoshihiro Oka, Hiroko Nakajima, Akihiro Tsuboi, Fumihiro Fujiki, Mai Kawakatsu, Yukie Harada, Soyoko Morimoto, Tomoki Masuda, Yoko Fukuda, Takamasa KatagiriAbstract:The Wilms' tumor gene WT1 is overexpressed in various kinds of hematopoietic malignancies as well as solid cancers, and this Protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. WT1-specific CTL epitopes with a restriction of HLA-A 2402 or HLA-A 0201 have been already identified. In the present study it has been demonstrated that a 9-mer WT1-derived WT1(187) peptide, which had already been shown to elicit a WT1-specific CTL response with a restriction of HLA-A 0201, can also elicit a CTL response with a restriction of HLA-A 0206. In all three different HLA-A 0206(+) healthy donors examined, WT1(187) peptide-specific CTL could be generated from peripheral blood mononuclear cells, and the CTL showed cytotoxic activity that depended on dual expression of WT1 and HLA-A 0206 molecules. The present study describes the first identification of a HLA-A 0206-restricted, WT1-specific CTL epitope. The present results should help to broaden the application of WT1 peptide-based immunotherapy from only HLA-A 0201-positive to HLA-A 0206-positive cancer patients as well.
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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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Induction of WT1 (Wilms' tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression.
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Yoshihiro Oka, Taiichi Kyo, Hiroko Nakajima, Olga A Elisseeva, Manabu Kawakami, Yusuke Oji, Akihiro Tsuboi, Tadashi Osaki, Tetsuya Taguchi, Kazuhiro IkegameAbstract: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.
