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.