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. The WT1 gene was isolated as a gene responsible for Wilms' tumor, a pediatric renal cancer, and encodes a zinc finger transcription factor, which is involved in cell proliferation and differentiation, apoptosis, and organ development (3-6). Although the WT1 gene was first categorized as a tumor suppressor gene, we have proposed that the wild-type WT1 gene functions as an oncogene rather than a tumor-suppressor gene on the basis of the following findings. The first is high expression of the wild-type WT1 gene in both leukemias and solid tumors (7-18), the second is growth inhibition of leukemic and solid tumor cells by treatment with WT1 antisense oligomers (14,19), and the third is block of differentiation, but induction of proliferation, of wild-type WT1 gene-transfected myeloid progenitor cells in response to granulocyte colony-stimulating factor (20, 21). The last two are block of thymocyte differentiation but induction of thymocyte proliferation in the transgenic mice with the lck promoter-driven WT1 gene (22), and WT1 gene expression in the majority of dimethylbenzanthracene-induced erythroblastic leukemia and a stronger tendency of the cells with high levels of WT1 to develop into leukemias (23).Expression of the wild-type WT1 gene has been found in most cases of acute myelocytic leukemia (AML), acute lymphocytic leukemia, chronic myelocytic leukemia, and myelodysplastic syndrome (MDS) at higher levels than those in normal bone marrow (BM) or peripheral blood (7-13). Furthermore, various types of solid tumors, including lung, breast, thyroid, and colorectal cancers, expressed the wild-type WT1 gene at higher levels compared to those in corresponding normal tissues (15-18). These results indicated that the wild-type WT1 gene product may be a promising target for cancer immunotherapy (24,25).We tested the potential of the WT1 gene product to serve as a target antigen for tumor-specific immunotherapy. Human WT1-specific CTLs have been found to induce lysis of endogenously WT1-expressing tumor cells in vitro, but not to cause damage to physiologically WT1-expressing normal cells (24,(26)(27)(28). We used a mouse in vivo system to demonstrate that immunization of mice with either MHC class I-restricted WT1 peptide or WT1 cDNA induced WT1-specific CTLs. We also showed that the immunized mice rejected challenges of WT1-expressing tumor cells, whereas the induced CTLs did not affect normal healthy tissues that physiologically expressed WT1 nor damaged the normal tissues (25, 29). These results indicated that the WT1 protein could be a novel tumor rejection antigen for cancer immunotherapy (24)(25)(26)(27)(28)(29)(30)(31)(32).In...

The product of the Wilms' tumor gene WT1 is a transcription factor overexpressed not only in leukemic blast cells of almost all patients with acute myeloid leukemia, acute lymphoid leukemia, and chronic myeloid leukemia, but also in various types of solid tumor cells. Thus, it is suggested that the WT1 gene plays an important role in both leukemogenesis and tumorigenesis. Here we tested the potential of WT1 to serve as a target for immunotherapy against leukemia and solid tumors. Four 9-mer WT1 peptides that contain HLA-A2.1-binding anchor motifs were synthesized. Two of them, Db126 and WH187, were determined to bind to HLA-A2.1 molecules in a binding assay using transporter associated with antigen processing-deficient T2 cells. Peripheral blood mononuclear cells from an HLA-A2.1-positive healthy donor were repeatedly sensitized in vitro with T2 cells pulsed with each of these two WT1 peptides, and CD8(+) cytotoxic T lymphocytes (CTLs) that specifically lyse WT1 peptide-pulsed T2 cells in an HLA-A2.1-restricted fashion were induced. The CTLs also exerted specific lysis against WT1-expressing, HLA-A2.1-positive leukemia cells, but not against WT1-expressing, HLA-A2.1-negative leukemia cells, or WT1-nonexpressing, HLA-A2. 1-positive B-lymphoblastoid cells. These data provide the first evidence of human CTL responses specific for the WT1 peptides, and provide a rationale for developing WT1 peptide-based adoptive T-cell therapy and vaccination against leukemia and solid tumors.

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.

Although a small uncontrolled nonrandomized trial, this study showed that WT1 vaccine therapy for patients with WT1/HLA-A*2402-positive recurrent GBM was safe and produced a clinical response. Based on these results, further clinical studies of WT1 vaccine therapy in patients with malignant glioma are warranted.

The Wilms tumor gene, WT1, is overexpressed not only in leukemias and myelodysplastic syndrome (MDS) but also in various types of solid tumors, including lung and breast cancer, and the WT1 protein is a tumor antigen for these malignancies. In clinical trials of WT1 peptide-based cancer immunotherapy, patients with overt leukemia from MDS or MDS with myelofibrosis were injected intradermally with 0.3 mg of an HLA-A*2402-restricted, 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant. Only a single dose of WT1 vaccination resulted in an increase in WT1-specific cytotoxic T-lymphocytes, which was followed by a rapid reduction in leukemic blast cells. Severe leukopenia and local erythema at the injection sites of WT1 peptide were observed as adverse effects. These results have provided us with the first clinical evidence suggesting that WT1 peptide-based immunotherapy is an attractive treatment for patients with leukemias or MDS.

IntroductionWilms tumor gene, WT1, is responsible for the tumorigenesis of a childhood renal neoplasm, Wilms tumor, which is thought to arise as a result of the inactivation of both alleles of the WT1 gene. 1,2 The WT1 gene has been considered a tumor-suppressor gene on the basis of findings such as intragenic deletions or mutations in Wilms tumor, germline mutations in patients with leukemia predisposition syndromes, and WT1-mediated growth suppression of Wilms tumor cells. [3][4][5][6][7] This gene encodes a zinc finger transcription factor involved in tissue development, in cell proliferation and differentiation, and in apoptosis. 8 The WT1 gene product represses the transcription of growth factor (platelet-derived growth factor ␣ chain, 9 colony-stimulating factor-1, 10 and insulinlike growth factor-II [IGF-II] 11 ) and growth factor receptor genes (IGF-IR 12 and EGFR 13 ), and the other genes (RAR-␣, 14 c-myb, 15 c-myc, 16 bcl-2, 16 ornithine decarboxylase, 17 and N-myc 18 ), whereas it activates the transcription of some genes (retinoblastoma suppressor-associated protein 46,20 and bcl-2 21 ). Unlike tumor-suppressor genes such as Rb and p53 that are ubiquitously expressed, WT1 gene expression is restricted to a limited set of tissues, including gonads, uterus, kidney, mesothelium, and hematopoietic progenitors. [22][23][24] WT1 knock-out mice have been shown to have defects in the urogenital system and to die at embryonic day 13.5, probably because of heart failure. 25 The WT1 gene was originally defined as a tumor-suppressor gene, as mentioned earlier. However, we recently proposed that the wild-type WT1 gene performs an oncogenic rather than a tumorsuppressor function in leukemogenesis and tumorigenesis in various types of solid tumors on the basis of the following findings: (1) high expression of the wild-type WT1 gene in leukemias [26][27][28][29][30][31] and various types of solid tumors, including ovarian tumors, Leydig cell tumors, mesothelioma, gastric cancer, colon cancer, lung cancer, and breast cancer 23,[32][33][34][35][36][37][38][39][40][41][42][43] ; (2) growth inhibition of leukemic 44,45 and solid tumor cells 41 by treatment with WT1 antisense oligomers; (3) promotion of cell growth, but blocking of cell differentiation, in the myeloid progenitor cell line 32D 46 and in normal bone marrow myeloid cells 47 as a result of constitutive WT1 gene expression caused by transfection with the wild-type WT1 gene; and (4) WT1 expression detected in most 7,12-dimethylbenzanthracene-induced erythroblastic leukemias and a tendency for cells with high levels of WT1 expression to develop into leukemias. 48 Stimulation in vitro of HLA-A2.1-positive or -A24.2-positive peripheral blood mononuclear cells with 9-mer WT1 peptides containing major histocompatibility complex (MHC) class 1 binding anchor motifs elicited WT1-specific cytotoxic T lymphocytes (CTLs). [49][50][51] These CTLs specifically killed WT1-expressing tumor cells in an HLA class 1-restricted manner and inhibited colony (1) cancer-testis ant...

The Wilms' tumor gene WT1 is overexpressed in various types of solid tumors, including lung and breast cancer and WT1 protein is a tumor antigen for these malignancies. In phase I clinical trials of WT1 peptide‐based cancer immunotherapy, two patients with advanced lung cancer were intradermally injected with 0.3 mg of an HLA‐A*2402‐restricted, 9‐mer WT1 peptide emulsified with Montanide ISA51 adjuvant. Consecutive WT1 vaccination at 2‐week intervals resulted in a reduction in tumor markers such as chorio‐embryonic antigen (CEA) and sialyl Lewis (x) (SLX) and by a transient decrease in tumor size. No adverse effects except for local erythema at the injection sites of WT1 vaccine were observed. These results provided us with the first clinical evidence demonstrating that WT1 peptide‐based immunotherapy should be a promising treatment for patients with lung cancer.

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