Toshiaki Shirakata

Phase II clinical trial of Wilms tumor 1 peptide vaccination for patients with recurrent glioblastoma multiforme

OBJECT The object of this study was to investigate the safety and clinical responses of immunotherapy targeting the WT1 (Wilms tumor 1) gene product in patients with recurrent glioblastoma multiforme (GBM). METHODS Twenty-one patients with WT1/HLA-A*2402-positive recurrent GBM were included in a Phase II clinical study of WT1 vaccine therapy. In all patients, the tumors were resistant to standard therapy. Patients received intra-dermal injections of an HLA-A*2402-restricted, modified 9-mer WT1 peptide every week for 12 weeks. Tumor size, which was obtained by measuring the contrast-enhanced area on magnetic resonance images, was determined every 4 weeks. The responses were analyzed according to Response Evaluation Criteria in Solid Tumors (RECIST) 12 weeks after the initial vaccination. Patients who achieved an effective response continued to be vaccinated until tumor progression occurred. Progression-free survival and overall survival after initial WT1 treatment were estimated. RESULTS The protocol was well tolerated; only local erythema occurred at the WT1 vaccine injection site. The clinical responses were as follows: partial response in 2 patients, stable disease in 10 patients, and progressive disease in 9 patients. No patient had a complete response. The overall response rate (cases with complete or partial response) was 9.5%, and the disease control rate (cases with complete or partial response as well as those in which disease was stable) was 57.1%. The median progression-free survival (PFS) period was 20.0 weeks, and the 6-month (26-week) PFS rate was 33.3%. CONCLUSIONS 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.

Antiapoptotic function of 17AA(+)WT1 (Wilms' tumor gene) isoforms on the intrinsic apoptosis pathway.

The WT1 gene is overexpressed in human primary leukemia and a wide variety of solid cancers. The WT1 gene is alternatively spliced at two sites, yielding four isoforms: 17AA(+)KTS(+), 17AA(+)KTS(−), 17AA(−)KTS(+), and 17AA(−)KTS(−). Here, we showed that 17AA(+)WT1-specific siRNA induced apoptosis in three WT1-expressing leukemia cell lines (K562, HL-60, and Kasumi-1), but not in WT1-non-expressing lymphoma cell line (Daudi). 17AA(+)WT1-specific siRNA activated caspase-3 and -9 in the intrinsic apoptosis pathway but not caspase-8 in the extrinsic one. On the other hand, 17AA(−)WT1-specific siRNA did not induce apoptosis in the three WT1-expressing cell lines. The apoptosis was associated with activation of proapoptotic Bax, which was activated upstream of the mitochondria. Constitutive expression of 17AA(+)WT1 isoforms inhibited apoptosis of K562 leukemia cells induced by apoptosis-inducing agents, etoposide and doxorubicin, through the protection of mitochondrial membrane damages, and DNA-binding zinc-finger region of 17AA(+)WT1 isoform was essential for the antiapoptotic functions. We further studied the gene(s) whose expression was altered by the expression of 17AA(+)WT1 isoforms and showed that the expression of proapoptotic Bak was decreased by the expression of 17AA(+)KTS(−)WT1 isoform. Taken together, these results indicated that 17AA(+)WT1 isoforms played antiapoptotic roles at some points upstream of the mitochondria in the intrinsic apoptosis pathway.

Wilms’ tumor gene WT1 17AA(–)/KTS(–) isoform induces morphological changes and promotes cell migration and invasion in vitro

The wild‐type Wilms’ tumor gene WT1 is overexpressed in human primary leukemia and in a wide variety of solid cancers. All of the four WT1 isoforms are expressed in primary cancers and each is considered to have a different function. However, the functions of each of the WT1 isoforms in cancer cells remain unclear. The present study demonstrated that constitutive expression of the WT1 17AA(–)/KTS(–) isoform induces morphological changes characterized by a small‐sized cell shape in TYK‐nu.CP‐r (TYK) ovarian cancer cells. In the WT1 17AA(–)/KTS(–) isoform‐transduced TYK cells, cell–substratum adhesion was suppressed, and cell migration and in vitro invasion were enhanced compared to that in mock vector‐transduced TYK cells. Constitutive expression of the WT1 17AA(–)/KTS(–) isoform also induced morphological changes in five (one gastric, one esophageal, two breast and one fibrosarcoma) of eight cancer cell lines examined. No WT1 isoforms other than the WT1 17AA(–)/KTS(–) isoform induced the phenotypic changes. A decrease in α‐actinin 1 and cofilin expression and an increase in gelsolin expression were observed in WT1 17AA(–)/KTS(–) isoform‐transduced TYK cells. In contrast, co‐expression of α‐actinin 1 and cofilin or knockdown of gelsolin expression by small interfering RNA restored WT1 17AA(–)/KTS(–) isoform‐transduced TYK cells to a phenotype that was comparable to that of the parent TYK cells. These results indicated that the WT1 17AA(–)/KTS(–) isoform exerted its oncogenic functions through modulation of cytoskeletal dynamics. The present results may provide a novel insight into the signaling pathway of the WT1 gene for its oncogenic functions. (Cancer Sci 2006; 97: 259–270)

The Wilms’ tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis

The Wilms’ tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1GFP/+) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (<1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFβR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit+lin−Sca-1+ (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1GFP/+ mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.

Wilms Tumor gene WT1 peptide-based immunotherapy induced a minimal response in a patient with advanced therapy-resistant multiple myeloma

The product of the Wilms tumor gene, WT1, is a universal tumor antigen. We performed WT1 peptide-based immunotherapy for a patient with multiple myeloma (MM). This patient was a 57-year-old woman with chemotherapy-resistant MM (Bence Jones к type). The patient received weekly intradermal injections of an HLA-A*2402-restricted 9-mer WT1 peptide emulsified with Montanide ISA 51 adjuvant for 12 weeks and achieved a minimal response according to European Group for Blood and Marrow Transplantation criteria without experiencing systemic adverse effects. The proportion of myeloma cells in the bone marrow (BM) decreased from 85% to 25%, and the amount of M protein in the urine decreased from 3.6 to 0.6 g/day after WT1 vaccination. Furthermore, a bone scintigram showed an improvement after the vaccination. As for immunologic parameters, the frequency of WT1 tetramer-positive cells among CD8+ T-cells, which was higher than in healthy donors, temporarily decreased at weeks 4 and 8 but increased at week 12, whereas the frequency of WT1 peptide-responding CD107a/b+ cells among WT1 tetramer-positive T-cells increased from 27.0% to 38.6% after the vaccination. After WT1 vaccination, the frequency of CXCR4+ cells among WT1 tetramer-positive T-cells increased in the BM, where stromal cells expressed the ligand for CXCR4, stromal-derived factor 1 (SDF-1), but decreased in the peripheral blood (PB), implying that WT1-specific cytotoxic T-lymphocytes had migrated from the PB to the BM, a tumor site.

Clinical and Immunologic Responses to Very Low-Dose Vaccination with WT1 Peptide (5 µg/Body) in a Patient with Chronic Myelomonocytic Leukemia

The wild-type Wilms tumor gene, WT1, is overexpressed in myelodysplastic syndrome (MDS) as well as acute myeloid leukemia. In a phase I clinical trial of biweekly vaccination with HLA-A*2402-restricted WT1 peptide for these malignancies, 2 patients with MDS developed severe leukocytopenia in association with a reduction in leukemic blast cells and levels of WT1 messenger RNA (mRNA) after only a single vaccination with 0.3 mg of WT1 peptide. These results indicated that the WT1-specific cytotoxic T-lymphocytes (CTLs) elicited by WT1 vaccination eradicated the WT1-expressing transformed stem or progenitor cells and that MDS patients with little normal hematopoiesis required a new strategy of WT1 vaccination to avoid severe leukocytopenia. We describe the first trial for a 57-year-old male patient with chronic myelomonocytic leukemia who was vaccinated biweekly with a small quantity (5 µg/body) of WT1 peptide. After the start of vaccination, the leukocyte and monocyte counts (13,780/µL and 1930/µL, respectively) gradually decreased to within the normal range in association with a reduction in the WT1 mRNA level. Simultaneously, the percentage of WT1-specific CTLs as measured by the HLA-WT1 tetramer assay increased. This case demonstrates for the first time that vaccination with as little as 5 µg of WT1 peptide can induce WT1-specific immune responses and resultant clinical responses.

WT1 peptide vaccine induces reduction in minimal residual disease in an Imatinib-treated CML patient.

How to treat CML patients who are resistant to inhibitors of BCR‐ABL tyrosine kinase such as Imatinib is a very important and urgent issue in clinical hematology. Here, we report a case of Imatinib‐treated CML in which intradermally administered WT1 peptide vaccine elicited WT1‐specific immune responses and the resultant reduction in the persistent residual disease in co‐administration of Imatinib. BCR‐ABL mRNA levels were being maintained under the detection limit for 8 months since week 77 of vaccination. No adverse effects except local erythema at the injection sites were observed. The tetramer assay revealed that the decrease in BCR‐ABL mRNA levels was associated with the increase in frequency of WT1‐specific cytotoxic T lymphocytes, notably effector‐memory type of that, in the patient’s peripheral blood. The case presented here indicates that WT1 peptide vaccine may become a safe and cure‐oriented therapy for CML patients who have residual disease regardless of the treatment with Imatinib.

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

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 naïve (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.

Biased usage of BV gene families of T‐cell receptors of WT1 (Wilms’ tumor gene)‐specific CD8+ T cells in patients with myeloid malignancies

(Cancer Sci 2010; 101: 594–600)

Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34 + CD38 − cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells

Primitive hematopoietic progenitor cells such as severe combined immunodeficiency- repopulating cells and long-term culture-initiating cells are enriched in CD34+CD38− cells derived from various stem cell sources. In this study, to elucidate the features of such primitive cells at the molecular level, we tried to isolate genes that were preferentially expressed in umbilical cord blood (CB)-derived CD34+CD38− cells by subtractive hybridization. The gene for VPAC1 receptor, a receptor for the neuropeptide vasoactive intestinal peptide (VIP), was thereby isolated and it was shown that this gene was expressed in both CD34+CD38− and CD34+CD38+ CB cells and that the expression levels were higher in CD34+CD38− CB cells. Next, we assessed the effects of VIP on the proliferation of CD34+ CB cells using in vitro culture systems. In serum-free single-cell suspension culture, VIP enhanced clonal growth of CD34+ CB cells in synergy with FLT3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO). In serum-free clonogenic assays, VIP promoted myeloid (colony-forming unit-granulocyte/macrophage (CFU-GM)) and mixed (CFU-Mix) colony formations. Furthermore, in Dexter-type long-term cultures, VIP increased colony-forming cells at week 5 of culture. These results suggest that VIP functions as a growth-promoting factor of CB-derived hematopoetic progenitor cells.