WT1-specific Cytotoxic T Lymphocytes Research Articles

A leukemic plasmacytoid dendritic cell line (PMDC05) as potent antigen presenting cells for anti-tumor cellular immunothrerapy (95.8)

Abstract We established a leukemic plasmacytoid dendritic cell (pDC) line (PMDC05) with potent antigen presenting capacity from leukemia cells of a patient with pDC acute leukemia. PMDC05 was positive for CD4, CD56, CD33, HLA-DR, CD123 (IL-3Rα) and CD86 in the absence of lineage markers. mRNA of TLR1, TLR2, TLR4, TLR7 and TLR9 were clearly expressed and among these TLRs, TLR7 was prominent. By culturing PMDC05 with IL-3, CpG-B or LPS, the expression of CD1a, CD80, CD83, CD86 or HLA-DR was remarkably enhanced. Production of IFN-α and IL-12p70 was enhanced by the stimulation with CpG-A and LPS, respectively. PMDC05, which was stimulated with 0.1 μg/mL LPS and loaded with 10 μg/mL WT1 peptides (HLA-A*2402-restricted, modified-type 9-mer peptide; CYTWNQMNL) for 24 hours, was irradiated (60 Gy) and co-cultured with allogeneic CD8+ T cells purified from PB-MNCs of HLA-A*2402+ healthy donor (PMDC05:CD8+ Tcells = 1:10). Induction of WT1-specific CTLs was evaluated by flow cytometry analysis using HLA-A*2402 WT1 tetramer every week. Although Daudi pulsed with WT1 peptides could not induce WT1 tetramer+CD8+ T cells during the co-culture up to 7 weeks, PMDC05 began to induce WT1 tetramer+CD8+ T cells at week 4 and the percentage of WT1 tetramer+ T cells in CD8+ T cells rose to more than 75% at week 7. These data suggested that PMDC05 could be efficiently used for generating CTLs specific for antigens of tumors and could be applicable for cellular immunotherapy for tumors.

Effectiveness of WT1 Peptide Vaccination Combined with Imatinib in a Patient with CML in Chronic Phase.

Abstract 4253Cytotoxic T lymphocytes (CTLs) are presumed to kill the relevant antigen-expressing leukemia cells including leukemic stem cells, which display intrinsic resistance against tyrosine kinase inhibitors such as imatinib in CML patients. In order to clarify the safety and effectiveness of WT1 peptide vaccination for the patients with CML, we started WT1 peptide vaccination in combination with imatinib therapy for a CML patient who could not acquire a major molecular response by the administration of imatinib. In addition, we tried to evaluate the kinetics of WT1-specific CTLs in peripheral blood (PB) during WT1 peptide vaccination. A 51 years-old male with CML in chronic phase had been treated with 400 mg imatinib for 4 years. BCR-ABL transcripts decreased transiently to less than 700 copies in 1 μg RNA (3-log reduction: 300 copies in 1 μg RNA extracted from PB cells; median in our laboratory) but gradually increased to more than 1,000 copies thereafter. Since the patient was HLA-A*2402+ and an informed consent was obtained, HLA-A*2402-restricted 9mer WT1 peptides (CYTWNQMNL; a.a. 235-243), which had been identified to possess an anti-tumor immunogenicity, were administered subcutaneously at the dose of 1 mg/day every 2 weeks in combination with 400 mg imatinib for first 5 months and thereafter every 4 weeks for 12 months. No adverse effects due to WT1 peptide vaccination was observed except for skin induration and redness at the sites of WT1 peptide injection. Although BCR-ABL transcripts increased up to more than 2,000 copies after transient decrease to less than 1,000 copies by the administration of WT1 peptides every 2 weeks, the transcripts have decreased to less than 500 copies by the administration of WT1 peptides every 4 weeks. The appearance of WT1-specific CTLs in PB was confirmed by evaluating the frequency of MHC/WT1 tetramer+CD8+ T cells by using MLPC (mixed lymphocyte peptide culture). MLPC was performed by culturing 1-3 ×105 PB mononuclear cells (MNCs) in 200 l of 5% autologous serum-containing medium with 10 μg WT1 peptides in 96 well plates. Frequency of MHC/WT1 tetramer+ cells in PB-CD8+ cells was calculated by the following formula. Number of wells containing a lump of tetramer+CD8+ cells / [(Number of PB-MNCs seeded in a well of MLPC) x (total number of wells for MLPC) x (ratio of PB-CD8+ cells in PB-MNCs)]. Although WT1-specific CTLs were not detected in PB before WT1 peptide vaccination, the CTLs appeared after the second vaccination and maintained at the level of nearly 15/106 CD8+ cells thereafter. The MHC/WT1 tetramer+ cells showed cytotoxicity against only leukemia cells expressing WT1 and HLA-A*2402. The cytotoxicity was blocked clearly by adding unlabeled cold target cells in cold inhibition test and blocked also by antibodies against MHC class I but not by antibodies against MHC class II. In vitro study demonstrated that the stimulation with WT1 peptides made WT1-specific CTLs, which were generated by MLPC, less reactive to MHC/WT1 tetramers and the unreactivity lasted at least for 2 weeks. The present study showed that WT1 peptide vaccination for an imatinib-pretreated CML patient is feasible and effective, which is due to the generation of WT1-specific CTLs with cytotoxicity against WT1-expressing leukemia cells. In addition, since the CTLs are suggested to get tolerant to WT1 peptides presented by leukemia cells by the exposure with WT1 peptides, the interval of peptide administration has to be considered for the clinical efficacy of peptide vaccination. The present findings including in vivo and in vitro studies revealed that the administration of the peptides every 4 weeks is superior to every 2 weeks. Disclosures:No relevant conflicts of interest to declare.

WT1 Peptide Vaccine as a Paradigm for “Cancer Antigen-Derived Peptide”-Based Immunotherapy for Malignancies: Successful Induction of Anti-Cancer Effect by Vaccination with a Single Kind of WT1 Peptide

Wilms' tumor gene (WT1) possesses oncogenic functions and is expressed in various kinds of malignancies, which suggests that the gene's product, the WT1 protein, should be one of the most promising cancer antigens. In fact, the WT1 protein was shown to be highly immunogenic in cancer patients. WT1 peptides that could induce WT1-specific CTLs (WT1 CTL peptides) were identified, and vaccination of cancer patients with these WT1 CTL peptides induced immunological responses, which were assessed by ex vivo immuno-monitoring, such as the tetramer assay, and in vivo immuno-monitoring, such as the peptide-specific delayed type hypersensitivity reaction. The induced immunological responses then led to clinical responses such as solid tumor shrinkage, a decrease in leukemia cells, and reduction of M-protein (multiple myeloma). Long-term stabilization of disease with good quality of life, which might be characteristic of cancer vaccine therapy, was also reported. It is noteworthy that injection with a "single" kind of WT1 peptide elicited an immunological response strong enough to induce a clinical response, indicating that the WT1 peptide vaccine has therapeutic potential. The number of reports of the successful treatment of cancer patients (not only adult but also childhood malignancies) with WT1 vaccination is increasing. Strategies for further improvement in the efficacy of therapy, including combined use of chemotherapy drugs, molecular-target-based drugs, or WT1 helper peptides, are being proposed. WT1 peptide vaccination in an "adjuvant setting" should be considered a promising treatment to protect against progression or relapse of malignancies in cases with minimal residual disease.

WT1 (WILMS TUMOR 1) PEPTIDE IMMUNOTHERAPY FOR CHILDHOOD RHABDOMYOSARCOMA: A Case Report

Immunotherapy using a Wilms tumor (WT1) peptide has been undergoing clinical trials for adulthood leukemia and solid cancer with promising results. In this study, the authors used WT1 peptide vaccination to treat a 6-year-old girl with metastatic alveolar rhabdomyosarcoma. She received weekly intradermal injection with HLA-A*2404-restricted, 9-mer WT1 peptide against residual bone disease. After 3 months her bone disease disappeared, concurrent with an increase in the frequency of WT1-specific cytotoxic T lymphocytes (CTLs). A high proportion of WT1-specific CTLs with effector or effector memory phenotype were detected in peripheral blood of this patient. She is currently still on continued WT1 peptide immunotherapy in a disease-free condition for 22 months. WT1 peptide-based immunotherapy should be a promising option for high-risk rhabdomyosarcoma in childhood.

Comparative Analysis of CD8+ t Cell Priming Efficiencies against a Panel of Leukemia-Associated HLA-A2 Restricted Epitopes Identifies PRAME as a Possible Vaccination Target in AML

Background Therapeutic vaccination with dendritic cells (DCs) is regarded as an option for the treatment of minimal residual disease in acute myeloid leukemia (AML). In this respect, defined epitope sequences derived from Leukemia-Associated Antigens (LAA) are an attractive source of antigen. WT1, PRAME, hTERT, and survivin are all LAA that are expressed in various hematological malignancies and that share a potential for the induction of cellular immune responses. Expression rates in AML of each of these LAA are high, ranging from 60 to 90%. To evaluate the feasibility of using these LAA in DC based vaccine formulations in AML, we investigated the relative Cytotoxic T Lymphocyte (CTL) priming efficiencies in response to a panel of LAA-derived HLA-A2 binding epitopes.Methods CD8+ cells were isolated from peripheral blood of healthy donors (HD) and stimulated with peptide-loaded DCs. DCs were derived from the human AML cell line MUTZ-3 (MUTZ3-DCs).1 The peptides used were WT1 (126–134), PRAME (100–108), hTERT (540–548), and survivin (96–104). Irradiated CD8− cells from the same donor were also added. CD8+ cells were plated at one million per well in 8 to 30 wells. Weekly CD8+ cells were restimulated with peptide-loaded MUTZ3-DCs. HLA-tetramer positivity (Tm+) for WT1, PRAME, hTERT or survivin epitopes was weekly screened by flow cytometry. Wells were scored positive if more than 0.02% Tm+, viable CTLs were present for at least 2 weeks. Functionality of Tm+ cells was studied by analysis of IFN-gamma production after stimulation with the JY cell line loaded with relevant LAA peptides or with an irrelevant control peptide (BCR-ABL).Results Percentages of the induced peptide-specific CTLs were low as shown in the table below. Priming efficiencies were highest for PRAME. Furthermore, PRAME and WT1 specific CTLs produced IFN-gamma in an epitope specific fashion, confirming functionality of the primed T cells.Number of HD that were Tm+ after co-cultureNumber of wells that were Tm+ after co-cultureMedian % Tm+ cells per positive well (range)Number of restimulations at maximum of Tm+PRAME3/428/600.10% (0.02–1.07)1–5WT12/54/1040.07% (0.03–0.09)1–4hTERT1/21/240.25%5Survivin1/12/180.17% (0.12–0.21)8Conclusions On the whole, the detected LAA-specific CD8+ T cell frequencies were low. Most consistently successful priming results were obtained for PRAME, indicating that this peptide may be an attractive candidate for inclusion in AML-specific DC-based vaccine regimens.

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 kappa 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 microg/body) of WT1 peptide. After the start of vaccination, the leukocyte and monocyte counts (13,780/microL and 1930/microL, 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 microg of WT1 peptide can induce WT1-specific immune responses and resultant clinical responses.

Dendritic Cell Immunization Induces Nonprotective WT1-specific CTL Responses in Mouse

In the present article we describe the immunogenicity in the mouse of 2 epitopes from the tumor-associated antigen Wilms tumor 1 antigen (WT1). The newly described K-restricted pWT330 epitope stimulates high-avidity allo-major histocompatibility complex restricted cytotoxic T lymphocyte (CTL) capable of killing WT1-expressing tumor cell lines. The epitope pWT126 has been previously described as a D-restricted CTL epitope. Both epitopes are weakly immunogenic as immunization with incomplete Freund adjuvant induced poor CTL responses. In contrast, when coated onto dendritic cells (DCs) both peptides readily induced CTL responses. However, these peptide-specific CTL were of low avidity and unable to recognize WT1-expressing tumor cells in vitro and to protect against tumor challenge in vivo. In contrast, vaccination with DCs coated with peptides derived from the nonself antigen ovalbumin (OVA) induced CTL that recognized OVA-expressing tumor cells and protected against tumor growth in vivo. These data show that although DC vaccination readily stimulated CTL against WT1 peptides, these CTL did not display antitumor activity in vitro and in vivo. This suggests that tolerance to the 2 WT1 epitopes interferes with the generation of protective CTL immunity in mice.

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.

Development of WT1 Peptide Cancer Vaccine Against Hematopoietic Malignancies and Solid Cancers

Wild-type Wilms' tumor gene WT1 is highly expressed not only in hematopoietic malignancies, including leukemia and myelodysplastic syndromes (MDS), but also in various kinds of solid tumors. Human cytotoxic T lymphocytes (CTLs) which could specifically lyse WT1-expressing tumor cells with HLA class I restriction were generated in vitro. We have also demonstrated that mice immunized with the WT1 peptide or WT1 cDNA rejected challenges by WT1-expressing tumor cells and survived with no signs of auto-aggression to normal organs which physiologically expressed WT1 in prophylactic and therapeutic models. Furthermore, we and others detected IgM and IgG WT1 antibodies in the patients with hematopoietic malignancies, indicating that WT1 protein was highly immunogenic, and that immunoglobulin class-switch-inducing WT1-specific cellular immune responses were elicited in the 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 the findings mentioned above, we performed a phase I clinical trial of WT1 peptide cancer vaccine for the patients with malignant neoplasms. These results strongly suggested that 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 TAA-derived cancer vaccine may be enhanced by combination with stronger adjuvants, helper peptide, or conventional treatments such as molecular-target-based drugs.

Monitoring of WT1‐specific cytotoxic T lymphocytes after allogeneic hematopoietic stem cell transplantation

Donor-derived cytotoxic T lymphocytes (CTL) that respond to tumor antigens emerge after hematopoietic stem cell transplantation (HSCT), particularly in association with the status of immune recovery. To analyze the frequency of CTL against PR1, PRAME and WT1 after HSCT, a tetramer-based analysis was performed in 97 samples taken from 35 patients (9 AML, 11 MDS, 2 CML, 4 ALL, 7 lymphoma and 2 renal cell carcinoma [RCC]) with the HLA-A02 phenotype. Regarding PR1, only 1 sample showed the presence of tetramer-positive cells (0.04%/lymphocyte). Similarly, in PRAME, only 10 of 97 samples were sporadically positive with low titers. For WT1, positive results were detected in 39 of 97 samples and 7 (2 CML, 1 ALL, 2 lymphoma and 2 RCC) patients clearly showed positive results more than once. On the basis of these results, we performed serial analyses of WT1-specific CTL during the clinical course in 2 patients with RCC, who underwent HSCT with a reduced-intensity regimen, to examine the precise correlation between the kinetics of CTL, the occurrence of GVHD and the observed clinical response. A higher positive rate for WT1-specific CTL and a correlation with the clinical response suggest that WT1 may be a useful antigen for a wider monitoring application.

Phenotypical Analysis of WT1-Specific CTLs in WT1 Vaccine-treated Patients: An Attempt to Find Immunological Parameters That Correlate With Clinical Response

Phenotypical Analysis of WT1-Specific CTLs in WT1 Vaccine-treated Patients: An Attempt to Find Immunological Parameters That Correlate With Clinical Response

Generation of Antigen Specific Cytotoxic T Lymphocytes (CTL) by Dendritic Cells (DCs) Transfected with In Vitro Transcribed (IVT) SART-1 and WT-1 mRNA.

Transfection with tumor antigen RNA is one of the promising tools not only because of a possible sufficient amplification of tumor antigen RNA but also because of the absence of antigen peptides-associated MHC restriction. Several succeeded experiments about generation of CTLs using DCs transfeced in vitro transcribed (IVT) cancer specific antigen mRNA such as PSA, CEA, hTERT and MUC-1 have been reported in these a few years. In addition, recent reports about the simultaneous presentation of peptides in both MHC class I and class II molecules on DCs after mRNA electroporation show another superiority of mRNA transfection into DCs. In this presentation, we demonstrate successful generation of tumor antigen specific CTLs using with DCs transfected with IVT mRNA such as SART-1 and WT-1 by electroporation. This is the first report about the generation of SART-1 and WT-1 specific CTLs by using mRNA transfected DCs. [Methods] HLA-A24 positive human PB CD14+ cell-derived DCs were transfected with IVT mRNA (SART-1and WT-1) by electroporation. MRNA transfected DCs were co-cultured with autologous lymphocytes. The bulk co-cultures were re-stimulated several times with same DCs. CD8+ cells were separated and CTL activity was evaluated by 51chromium release assay. To determine whether the induced CTL cells could recognize the target cells in an HLA class I restricted manner, anti-HLA class I monoclonal antibodies were utilized to block the cytotoxicity of effectors. [Results] Electroporation of mRNA showed no effect on the surface phenotypes and antigen presenting ability of DCs. In addition to the demonstration of efficient transfection of M1 mRNA into DCs by using RT-PCR, which eliminated the amplification of transfected mRNA by the treatment with RNase before RNA extraction from the transfected cells, we identified the definite expression of WT-1 protein in the cytoplasm of DCs by using immunoblotting. CTL assay indicated that 1) DCs transfected with mRNA stimulated the generation of antigen-specific CTLs which are capable of lysing autologous DCs transfected with the same mRNA. 2) CTLs also demonstrated cytotoxic ability against cell lines such as KE-4 presenting SART-1 peptides on HLA-A24, MEGO1 presenting WT-1 peptides on MHC class I, and HLA-A24 cDNA transfected T2 which were used as target cells after co- incubation with 9 mer SART-1 peptides with strong affinity to HLA-A24. 3) Each cytotoxicities were markedly blocked after co-incubation of target cells with anti-MHC class I antibody and not inhibited with anti-MHC class II antibody. [Conclusion] Our results showed that IVT mRNA-transfected DCs which is constructed non-virally have a highly efficient ability to stimulate specific T-cell immunity against tumor. Unlike peptide- or tumor cells extract-pulsed DCs based vaccines, anti-tumor immunotherapy using the DCs transfected with antigen mRNA could be extended to a wide range of patients who have previously been excluded from clinical trials for the reason of the un-identification of tumor specific antigens, for the reason of the impossibility of obtaining sufficient tumor specimens, or for the reason of MHC restriction of the tumor specific antigens.

Generation of Tumor-Specific CD8+ Cytotoxic T Lymphocytes and CD4+ Th1 Helper T Cells by Transfer of T-Cell Receptor Genes Isolated from a WT1-Specific CD8+ Cytotoxic T-Lymphocyte Clone.

Background: Cytotoxic T lymphocytes (CTLs) and T-helper type 1 (Th1) cells undoubtedly play a crucial role in the eradication of tumors in vivo. However, the production of Th1 cytokines such as IL-2 and IFN-γ is markedly suppressed in the majority of tumor-bearing hosts. Such defects in Th1-mediated immunity in cancer patients have made it difficult to induce tumor-specific CTLs that promote tumor rejection. Adoptive transfer of tumor-specific CTLs and Th1 cells can overcome the difficulty to induce tumor-specific immune response in cancer patients; however, the generation and expansion of tumor-specific CTLs and Th1 cells in vitro are not easy. In the present study, to overcome this problem, we isolated TCR-α and -β chain genes from a WT1-specific CD8+ CTL clone, which had been shown to exert strong cytotoxicity against hematopoietic malignancies and solid tumors in an HLA-A24-restricted manner, and transduced them into nonspecifically activated human CD8+ and CD4+ T cells. Consequently, both CD8+ and CD4+ T cells appeared to acquire WT1-specific function in an HLA-A24-restricted manner.Methods: A WT1 peptide (CMTWNQMNL)-specific CD8+ CTL clone, TAK-1, was established as reported previously (Blood 95:286, 2000). TAK-1 exerts cytotoxicity against variety of tumor cells including leukemia, myeloma, and lung cancer cells but not against normal cells in an HLA-A24-restricted manner. cDNAs encoding TCR-α and -β chain genes were amplified from cDNA of TAK-1 by RT-PCR. TCR-α and -β chain cDNAs were inserted into the plasmid vector. Preparation of lentiviral vectors for transduction of TCR-α and -β chain cDNAs was performed as described previously (Cancer Res 64:1490, 2004). Peripheral blood CD4+ and CD8+ T cells isolated from healthy individuals were cultured with anti-CD3 mAb and retronectin and then infected twice with lentivirus vectors. The infected cells were expanded by culture in the presence of IL-2, IL-12, IFN-γ and anti-IL-4 mAb. Cytotoxicity of CTLs against WT1-peptide-loaded cells and various human tumor cells was examined by a standard 51Cr-release assay. Recognition of tumor cells by Th1 cells was examined by measuring IFN-γ production by ELISA.Results: CD4+ T-cell line (CD4-TCR) and CD8+ T cell line (CD8-TCR) expressing TCR-α and -β chains of TAK-1 were established. Both CD4-TCR and CD8-TCR cells exerted cytotoxicity against WT1 peptide-loaded HLA-A24-positive but not -negative cells. CD8-TCR cells appeared to be cytotoxic against human tumor cells including leukemia, myeloma, and lung cancer cells in an HLA-A24-restricted manner, but did not show any cytotoxicity against HLA-A24-positive normal cells. CD4-TCR cells produced IFN-γ in response to stimulation with HLA-A24-positive but not -negative leukemia cells.Conclusion: The present data demonstrate the functional reconstitution of CD4+ as well as CD8+ T cells by transfer of the αβ TCR complex of a WT1-specific CD8+ CTL clone. Since WT1 is a universal tumor-associated antigen, transfer of TCR genes of WT1-specific CTLs into CD4+ and CD8+ T cells would be useful for Th1-based immunotherapy of various malignancies.

Myeloma Cells Are Highly Sensitive to the Granule Exocytosis Pathway Mediated by WT1-Specific Cytotoxic T Lymphocytes

Because WT1 is a universal tumor antigen, we examined the sensitivity of myeloma cells to WT1-specific cytotoxic T lymphocyte (CTL)-mediated cytotoxicity. WT1 expression in hematologic malignant cells was examined by quantitative reverse transcription-polymerase chain reaction. The cytotoxicity of a WT1-specific CTL clone against hematologic malignant cells, including myeloma cells, was examined by standard chromium-51 release assays. The extent of membrane damage induced by purified perforin was examined. Induction of WT1-specific CTLs from the patients with multiple myeloma (MM) was attempted, and we examined their function against myeloma cells. The expression levels of WT1 mRNA in myeloma and lymphoma cells were significantly lower than that in acute leukemia cells. Although the WT1 expression levels in myeloma and lymphoma cells were almost same, only myeloma cells were lysed efficiently by WT1-specific CTLs in a HLA-restricted manner. The amounts of interferon-gamma produced by WT1-specific CTLs in response to stimulation with myeloma cells and with lymphoma cells were almost the same, suggesting that WT1 protein is processed and expressed in the context of HLA class I molecules similarly on both myeloma and lymphoma cells. The extent of membrane damage induced by purified perforin appeared to be significantly higher in myeloma cells than in lymphoma cells. WT1-specific CTLs appeared to be present in patients with MM. The present study has shown that susceptibility of membranes to perforin is an important factor determining the sensitivity of target cells to CTL-mediated cytotoxicity and that WT1 is an ideal target antigen for cellular immunotherapy of MM.

Human cytotoxic T lymphocytes specific for Wilms' tumor antigen-1 inhibit engraftment of leukemia-initiating stem cells in non-obese diabetic-severe combined immunodeficient recipients.

Leukemia is a disease characterized by the malignant transformation of hematopoietic stem cells. Previous studies have shown that the Wilms' tumor antigen-1 (WT1) transcription factor is expressed at elevated levels in hematopoietic stem cells of leukemia patients compared with normal stem cells. In the past, we have generated cytotoxic T lymphocytes (CTL) specific for WT1, and we have shown that they killed WT1-expressing leukemia cell lines and inhibited the in vitro colony-forming activity of leukemia cells of patients. We used a xenotransplantation model to address whether WT1-specific CTL can selectively inhibit engraftment of malignant but not normal stem cells. CD34+ hematopoietic cells isolated from individuals with chronic myeloid leukemia or normal hematopoiesis were treated with WT1-specific CTL and injected into immunodeficient non-obese diabetic-severe combined immunodeficient mice. After 5 to 8 weeks, engraftment of leukemic or normal human cells was analyzed using immunohistology, flow cytometry, and polymerase chain reaction amplification of human sequences. The data showed that exposure of chronic myeloid leukemia CD34+ cells to WT1-specifc CTL completely prevented the development of leukemia in the recipient mice, whereas CTL treatment did not inhibit engraftment of normal CD34+ stem cells. The experiments indicate that WT1-specific CTL can discriminate between stem cells that give raise to leukemia and normal hematopoiesis in the xenogenic transplantation model. This supports the use of CTL with this specificity for treatment of leukemia patients undergoing stem-cell transplantation.

Enhanced induction of human WT1-specific cytotoxic T lymphocytes with a 9-mer WT1 peptide modified at HLA-A*2402-binding residues.

The Wilms' tumor gene WT1 is overexpressed in most types of leukemias and various kinds of solid tumors, including lung and breast cancer, and participates in leukemogenesis and tumorigenesis. WT1 protein has been reported to be a promising tumor antigen in mouse and human. In the present study, a single amino-acid substitution, M-->Y, was introduced into the first anchor motif at position 2 of the natural immunogenic HLA-A*2402-restricted 9-mer WT1 peptide (CMTWNQMNL; a.a. 235-243). This substitution increased the binding affinity of the 9-mer WT1 peptide to HLA-A*2402 molecules from 1.82 x 10(-5) to 6.40 x 10(-7) M. As expected from the increased binding affinity, the modified 9-mer WT1 peptide (CYTWNQMNL) elicited WT1-specific cytotoxic T lymphocytes (CTL) more effectively than the natural 9-mer WT1 peptide from peripheral blood mononuclear cells (PBMC) of HLA-A*2402-positive healthy volunteers. CTL induced by the modified 9-mer WT1 peptide killed the natural 9-mer WT1 peptide-pulsed CIR-A*2402 cells, primary leukemia cells with endogenous WT1 expression and lung cancer cell lines in a WT1-specific HLA-A*2402-restricted manner. These results showed that this modified 9-mer WT1 peptide was more immunogenic for the induction of WT1-specific CTL than the natural 9-mer WT1 peptide, and that CTL induced by the modified 9-mer WT1 peptide could effectively recognize and kill tumor cells with endogenous WT1 expression. Therefore, cancer immunotherapy using this modified 9-mer WT1 peptide should provide efficacious treatment for HLA-A*2402-positive patients with leukemias and solid tumors.

Cancer immunotherapy targeting WT1 protein.

The Wilms tumor gene WT1 is expressed in leukemias and various kinds of solid tumors, including lung and breast cancer, and exerts an oncogenic function in these malignancies, suggesting that WT1 protein is a novel, overexpressed tumor antigen. The WT1 protein, in fact, is an attractive tumor rejection antigen in animal models. Stimulation in vitro of peripheral blood mononuclear cells with HLA-A*2402--and HLA-A*0201--restricted 9-mer WT1 peptides elicits WT1-specific cytotoxic T-lymphocytes (CTLs), and the CTLs kill endogenously WT1-expressing leukemia or solid tumor cells. Furthermore, WT1 immunoglobulin M (IgM) and IgG antibodies are detected in patients with hematopoietic malignancies such as acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndromes, indicating that WT1 protein overexpressed by leukemia cells is indeed immunogenic. Taken together, these results demonstrate that WT1 protein is a promising tumor antigen for cancer immunotherapy against leukemias and various kinds of solid tumors, including lung and breast cancer.