WT1 Peptide Cancer Vaccine for Patients with Hematopoietic Malignancies and Solid Cancers

Wild-type Wilms' tumor gene WT1 is expressed at a high level in hematopoietic malignancies including acute leukemia, chronic myelogenous leukemia, and myelodysplastic syndromes, as well as in various kinds of solid cancers. Human cytotoxic T lymphocytes (CTLs), which could specifically lyse WT1-expressing tumor cells with HLA class I restriction, were generated in vitro. It was also demonstrated that mice immunized with the WT1 peptide rejected challenges by WT1-expressing cancer cells and survived with no signs of autoaggression to normal organs that physiologically expressed WT1. Furthermore, we and others detected IgM and IgG WT1 antibodies in patients with hematopoietic malignancies, indicating that the WT1 protein was highly immunogenic, and that immunoglobulin class-switch-inducing, WT1-specific, cellular immune responses were elicited in these patients. CD8+ WT1-specific CTLs were also detected in peripheral blood or tumor-draining lymph nodes of cancer patients. These results provided us with the rationale for elicitation of CTL responses targeting the WT1 product for cancer immunotherapy. On the basis of these findings, we performed a phase I clinical trial of a WT1 peptide cancer vaccine for the patients with malignant neoplasms. These results strongly suggested that the WT1 peptide cancer vaccine had efficacy in the clinical setting because clinical responses, including reduction of leukemic blast cells or regression of tumor masses, were observed after the WT1 vaccination in patients with hematopoietic malignancies or solid cancers. The power of a tumor-associated-antigen (TAA)-derived cancer vaccine may be enhanced in combination with stronger adjuvants, helper peptide, molecular-target-based drugs, or some chemotherapy drugs, such as gemcitabine, which has been revealed to suppress regulartory T-cell function. In contrast, reduction of WT1 peptide dose may be needed for the treatment of patients with hematological stem cell diseases, because rapid and strong destruction of malignant cell-sustained hematopoiesis before recovery of normal hematopoiesis may lead to pancytopenia in these patients

Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms.

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

Functional CTLs specific for the antigen expressed in leukemia cells were highly expanded in mice that did not develop leukemia.

<p>Analysis of BM and spleen cells from mice 4 weeks (A–C, n = 5) or 6 weeks (D–F, n = 3) after transplant with 3 × 10⁴ MLL/AF9-OVA leukemia cells. (A, D) Flow-cytometry analysis of the frequencies of GFP⁺ leukemia cells in the 7AAD^- whole BM or spleen cells and those of H-2K^b/OVA tetramer-positive cells in CD8⁺ T cells. (B, E) Flow-cytometry analysis of cytokine production by CD8⁺ BM and spleen T cells, with or without SIINFEKL peptide stimulation. (C, F) Percentages of IFN-γ- and/or TNF-α-producing cells in BM or spleen CD8⁺ T cells, with or without SIINFEKL peptide stimulation. *: p < 0.05.</p

In mice with advanced leukemia, CTLs specific for the antigen expressed in leukemia cells were also expanded, but could not suppress disease progression.

<p>(A-C) Analysis of BM and spleen cells from mice with advanced MLL/AF9-OVA leukemia (A) Flow-cytometry analysis of the frequencies of GFP⁺ leukemia cells among the whole BM or spleen cells and the frequencies of H-2K^b/OVA tetramer-positive cells among CD8⁺ T cells. (B) Flow-cytometry analysis of cytokine production by CD8⁺ T cells in BM and spleen with or without SIINFEKL peptide stimulation. (C) Percentages of IFN-γ- and/or TNF-α-producing cells among CD8⁺ BM or spleen T cells, with or without SIINFEKL peptide stimulation (n = 3). *: p < 0.05 (D) Analysis of the expression of T-cell exhaustion–associated markers in H-2K^b/OVA tetramer-positive CD8⁺ T cells. BM cells from non-leukemic mice and mice with advanced leukemia were analyzed (n = 3 for each). Representative flow-cytometry analysis and bar graphs for mean fluorescence intensities (MFI) are shown. Dotted lines represent isotype controls.*: p < 0.05, N.S.: not statistically significant. (E) Analysis of the expression of H-2K^b, GFP, and the presentation of SIINFEKL peptide in leukemia cells that developed in wild-type or <i>Rag2</i>^-/- recipients. Representative flow-cytometry analysis and bar graphs for MFI are shown. Dotted lines represent isotype controls.</p

Spontaneous regression of leukemia was observed in the presence, but not in the absence, of adaptive immunity.

<p>(A) Flow-cytometry analyses of BM cells of non-irradiated wild-type recipients 7 days after transplantation with different numbers (3 × 10³, 3 × 10⁴, or 3 × 10⁵) of MLL/AF9-OVA leukemia cells. (B) FACS analysis of BM from non-irradiated wild-type or <i>Rag2</i>^-/- mice transplanted with 3 × 10⁴ MLL/AF9-OVA leukemia cells. Mice were analyzed 3 weeks after transplant. (C) Kaplan–Meier curves for overall survival of non-irradiated wild-type (n = 7) or Rag2^-/- (n = 3) recipients transplanted with 3 × 10⁴ MLL/AF9-OVA leukemia cells. (D) Percentages of GFP⁺ leukemia cells in BM after transplantation into non-irradiated wild-type mice were examined every week. Each dot and line corresponds to a recipient mouse. The results of four mice in which leukemia spontaneously regressed (Exp. 3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144594#pone.0144594.t001" target="_blank">Table 1</a>) are shown. (E) FACS analysis of BM from non-irradiated wild-type or <i>Rag2</i>^<i>-/-</i> mice transplanted with 3 × 10⁴ of MLL/AF9 leukemia cells (OVA-). Mice were analyzed 3 weeks after transplant.</p

Apoptotic function of an Ex4a(+)WT1 isoform.

<p>(<b>A</b>) The role of Ex4a(+)WT1 in apoptosis. Ex4a(+) or Mock vector was transfected into WT1-expressing HT-1080 cells. Frequencies (%) of Annexin V-positive apoptotic cells and cells with loss of MMP were determined by flowcytometry after 24 h. Left, Frequencies (%) of Annexin V-positive apoptotic cells are shown. Right, Frequencies (%) of cells with mitochondrial membrane potential (MMP) loss are shown. Results are means and S.D. of three independent experiments. *, p<0.05. (<b>B</b>) Expression of Ex4a(+) and major WT1 isoforms during apoptosis. K562 cells were treated with the indicated concentrations of Dox for 12 h and analyzed for Annexin-V positive apoptotic cells and expression of Ex4a(+) and major WT1 isoforms by flowcytometry and RT-PCR, respectively. Upper, Frequencies (%) of Annexin V-positive apoptotic cells. Lower, RT-PCR using Ex4-F and Ex6-R primer pair that amplifies both Ex4a(+) and major WT1 isoforms. GAPDH is used as an internal control. Results are representative of three independent experiments. (<b>C</b>) Change of Ex4a(+)WT1 and major WT1 isoforms during apoptosis. K562 cells were treated with the indicated concentrations of Dox for 12 h and expression of Ex4a(+)WT1 and total WT1 isoforms including both Ex4a(+) and major WT1 isoforms were determined by quantitative real-time RT-PCR using Ex4a-F and Ex6-R primer pair and Ex6-F and Ex7-R primer pair, respectively. Actin is used as an internal control for normalization. Expression levels of Ex4a(+)WT1 and total WT1 in Dox-untreated cells are defined as 1.0. (<b>D</b>) Suppression of Ex4a(+)WT1 inhibits Dox-induced apoptosis. K562 cells were transfected with one μg of either of two WT1 Ex4a-specific siRNAs (si-4a-1 and si-4a-2) or a control siRNA (si-control) together with 2.0 μg of Ex4a(+)WT1 vector or 2.0 μg of empty vector (Mock), cultured for 24 h, treated with 4.0 μM Dox for 12 h, and then analyzed for Annexin-V positive apoptotic cells by flowcytometry. Frequencies (%) of Annexin V-positive apoptotic cells are shown. Results are mean and S.D. of three independent experiments. *, p<0.05.</p