Hiroko Nakajima

Wilms tumor gene peptide-based immunotherapy for patients with overt leukemia from myelodysplastic syndrome (MDS) or MDS with myelofibrosis.

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.

CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19+ clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19+ B cells. We found that in vitro MM colony-forming cells were enriched in CD138−CD19−CD38++ plasma cells, while CD19+ B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138−CD19−CD38++ plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19+ B cells was detected. In 4 out of 9 cases, CD138+ plasma cells propagated MM, although more slowly than CD138− cells. Finally, we transplanted CD19+ B cells from 13 MM patients into NOD/SCID IL2Rγc−/− mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138− and CD138+ cells have the potential to propagate MM clones in vivo in the absence of CD19+ B cells.

Identification and characterization of a WT1 (Wilms Tumor Gene) protein-derived HLA-DRB1*0405-restricted 16-mer helper peptide that promotes the induction and activation of WT1-specific cytotoxic T lymphocytes.

Effective tumor vaccine may be required to induce both cytotoxic T lymphocyte (CTL) and CD4+ helper T-cell responses against tumor-associated antigens. CD4+ helper T cells that recognize HLA class II-restricted epitopes play a central role in the initiation and maintenance of antitumor immune responses. The Wilms tumor gene WT1 is overexpressed in both leukemias and solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for cancer immunotherapy. In this study, we identified a WT1 protein-derived 16-mer peptide, WT1332 (KRYFKLSHLQMHSRKH), which was restricted with HLA-DRB1*0405, one of the most common HLA class II types in Japanese, as a helper epitope that could elicit WT1-specific CD4+ T-cell responses. We established a WT1332-specific CD4+ helper T-cell clone (E04.1), which could respond to both HLA-DRB1*0405-positive, WT1-expressing transformed hematopoietic cells and autologous dendritic cells pulsed with apoptosis-induced WT1-expressing cells, indicating that the WT1332 was a naturally processed helper epitope. Stimulation of peripheral blood mononuclear cells with both the CTL epitope (WT1235) and the helper epitope (WT1332) in the presence of WT1332-specific TH1-type CD4+ T cell clone strikingly enhanced the induction and the functional activity of WT1235-specific CTLs compared with that of peripheral blood mononuclear cells with the WT1235 alone. These results indicated that a helper epitope, WT1332 should be useful for improvement of the efficacy of CTL epitope-based cancer vaccine targeting WT1 in the clinical setting.

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.

Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease.

Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease

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 vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone

A Wilms’ tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer. WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models. We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a “prophylactic” model). However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a “therapeutic” setting. In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a “therapeutic” model) compared with WT1 peptide vaccination alone. An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells. These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.

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.

Maintenance of complete remission after allogeneic stem cell transplantation in leukemia patients treated with Wilms tumor 1 peptide vaccine

The prognosis of patients after allogeneic hematopoietic stem cell transplantation (HSCT) is still not satisfactory because, while treatment-related mortalities have decreased, relapse after HSCT remains a major concern. The effectiveness of allogeneic HSCT for hematological malignancies is the result of immunologic rejection of recipient leukemia cells by donor T cells, known as the graft-versus-leukemia (GVL) effect.1 It is thus obviously important to be able to exploit the GVL effect while minimizing graft-versus-host disease (GVHD). A targeted anti-leukemic immunotherapy, such as use of a leukemia vaccine,2 is a promising strategy to boost the GVL effect. Wilms tumor1 (WT1) protein is one of the best targets for leukemia vaccines. Overexpression of the wild-type WT1 gene has been detected in all types of human leukemia.3, 4, 5 We performed a phase I clinical study of immunotherapy targeting the WT1 protein in patients with leukemia, and were able to show that WT1 vaccination was safe and could induce WT1-specific cytotoxic T lymphocyte (CTL).6 Furthermore, reduction of minimal residual disease and long-lasting complete remission (CR) was observed in some leukemia patients who were given the WT1 vaccine.7 This report presents the results of phase I clinical study of WT1 vaccination for HLA-A*2402-positivie post-HSCT patients who were at high risk of relapse (HSCT in non-CR and 2nd HSCT for post-transplant relapse) or had already relapsed. The HLA-A*2402-restricted modified 9-mer WT1 peptide (amino acids 235–243 CYTWNQMNL)8 was emulsified with Montanide ISA51 adjuvant. Patients were intradermally injected with 1.0 mg (three patients: UPNs 1, 4 and 6) or 3.0 mg (other six patients) of WT1 peptide four times weekly. When no adverse effects and no obvious disease progression were observed after the fourth injection, further WT1 vaccinations at 2-week intervals were administered. Nine patients (five with acute myeloid leukemia (AML), one each with acute lymphoblastic leukemia, chronic myelomonocytic leukemia, multiple myeloma and T-cell lymphoblastic lymphoma) were enrolled in this study (Supplementary Tables 1 and 2). Local inflammatory response was observed at the vaccine injection sites of all patients. One patient (UPN5) suffered mild hypoxia (PaO2 65 mm Hg at room air) and restrictive pulmonary dysfunction (FEV1.0 40%) 65 days after the start of WT1 vaccination (day 199 after HSCT; Figure 1a). He was diagnosed with bronchioleitis obliterans (BO), which was a symptom of chronic GVHD. The patient recovered soon after administration of inhaled steroids. While early and sudden discontinuation of prednisolone and tacrolimus (day 103 after HSCT) were considered to be the reason for development of BO, the possibility of an association between BO and WT1 vaccination cannot be entirely ruled out. In other eight patients, no severe toxicities related to WT1 vaccine were observed (Table1). Figure 1 Clinical course of patients who attained CR after the start of WT1 peptide vaccination. (a) Clinical course of UPN5 who achieved CR after administration of WT1 vaccine but stopped vaccination because of the development of bronchioleitis obliterans. ( ... Table 1 Patient outcomes Three AML patients (UPN1–3), who had undergone HSCT in non-CR, started WT1 vaccine in CR (Supplementary Tables 1 and 2). They started WT1 vaccination on post-HSCT days 141, 76 and 93 and have remained in CR for 1038, 973 and 662 days, respectively (as of 8 April 2013; Table1), suggesting the potential of WT1 vaccination as a maintenance therapy after HSCT. Six patients started WT1 vaccination in non-CR and two of them became CR after WT1 vaccination. One B-ALL patient (UPN4) with MLL-AF4 underwent bone marrow transplantation from an HLA-matched unrelated donor during the first CR. On post-HSCT day 111, MLL-AF4 and WT1 mRNA in peripheral blood (PB) had increased to 16 000 and 15 000 copies/μg RNA, indicating that the disease had relapsed. Tacrolimus and prednisolone doses were tapered off to induce GVL effects. The expression levels of MLL-AF4 and WT1 mRNA in PB had decreased to 2700 and 190 copies/μg RNA by day 132, and WT1 vaccination was started on day 133. MLL-AF4 mRNA had become undetectable by day 146, and had never appeared until post-HSCT day 1312 (day 1179 after the start of WT1 vaccination as of 8 April 2013; Figure 1b). Skin tumors appeared in UPN5 (AML-M5) on post-HSCT day 103 and was diagnosed by biopsy as leukemia relapse. Tacrolimus was discontinued on day103, and WT1 vaccination was started on day 130. Cutaneous tumors had regressed 2 weeks after the start of WT1 vaccination, but vaccination was terminated after the second injection because of the development of BO as described earlier (Figure 1a). This patient has been remained in CR until post-HSCT day 972 (day 842 after the start of WT1 vaccination at 8 April 2013). While the exact contribution of the vaccination effect to the disease remission in addition to the GVL effect was unclear, the fact that both of these two patients still have remained in CR until now is encouraging to continue this trial. In the following phase II trials, the enumeration of WT1-specific CTLs should be performed more frequently after the start of vaccination to clarify the relationship between the effect of WT1 peptide vaccination and leukemia regression. WT1 (a natural 9-mer WT1 peptide) HLA-A*2402 tetramer assays could be performed with peripheral blood mononuclear cell in seven of the nine patients to determine whether WT1235 peptide-specific CD8+ T cells had increased after WT1 vaccination. The gates for WT1 tetramer+ cells were drawn as <0.1% of CD8+ T cells were included in the tetramer-positive gate in multiple healthy individuals (Supplementary Figure 1A). WT1235 tetramer+ cells increased after the start of vaccination in three (UPNs1, 2 and 4) of the four patients who have remained in CR (Figure 1b and Supplementary Figure 1B). In the cases with progressive disease, continuous increase in the frequencies of WT1235 tetramer+ cells was not observed (Supplementary Figure 1B). Our results suggest that WT1 vaccination should be started when the leukemia burden is minimal. The timing of the start of WT1 vaccination may be also important. For the cases with good outcomes, WT1 vaccination was started 76–140 days after transplantation (UPNs1–5), and at later times (days 299–1815) for PD cases (UPNs 6–9). A lymphopenic environment a few months after transplantation may be favorable for rapid and extensive expansion of tumor antigen-specific CTLs. In summary, this report suggests that WT1 vaccine can be safely administrated for post-HSCT patients with hematological malignancies and has potential as a maintenance therapy. Clinical benefit of WT1 vaccination for post-HSCT patients will be evaluated in the subsequent phase II trials.

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.