WT1 peptide-based immunotherapy for advanced thymic epithelial malignancies.

Thymic epithelial tumors are rare malignancies, and no optimal therapeutic regimen has been defined for patients with advanced disease. Patients with advanced thymic epithelial tumors, which were resistant or intolerable to prior therapies, were eligible for this study. Patients received 9 mer-WT1-derived peptide emulsified with Montanide ISA51 adjuvant via intradermal administration once a week as a monotherapy. After the 3-month-protocol treatment, the treatment was continued mostly at intervals of 2-4 weeks until disease progression or intolerable adverse events occurred. Of the 15 patients enrolled, 11 had thymic carcinoma (TC) and 4 had invasive thymoma (IT). Median period from diagnosis to the start of treatment was 13.3 and 65.5 months for TC and IT, respectively. No patients achieved a complete or partial response. Of the 8 evaluable TC patients, 6 (75.0%) had stable disease (SD) and 2 had progressive disease (PD). Of the 4 evaluable IT patients, 3 (75.0%) had SD and 1 (25.0%) had PD. Median period of monotherapy treatment was 133 and 683 days in TC and IT patients, respectively. No severe adverse events occurred during the 3-month-protocol treatment. As adverse events in long responders, thymoma-related autoimmune complications, pure red cell aplasia and myasthenia gravis occurred in two IT patients. Cerebellar hemorrhage developed in a TC patient complicated with Von Willebrand disease. Induction of WT1-specific immune responses was observed in the majority of the patients. WT1 peptide vaccine immunotherapy may have antitumor potential against thymic malignancies.

Association of WT1 IgG antibody against WT1 peptide with prolonged survival in glioblastoma multiforme patients vaccinated with WT1 peptide.

We previously evaluated Wilms' tumor gene 1 (WT1) peptide vaccination in a large number of patients with leukemia or solid tumors and have reported that HLA-A*24:02 restricted, 9-mer WT1-235 peptide (CYTWNQMNL) vaccine induces cellular immune responses and elicits WT1-235-specific cytotoxic T lymphocytes (CTLs). However, whether this vaccine induces humoral immune responses to produce WT1 antibody remains unknown. Thus, we measured IgG antibody levels against the WT1-235 peptide (WT1-235 IgG antibody) in patients with glioblastoma multiforme (GBM) receiving the WT1 peptide vaccine. The WT1-235 IgG antibody, which was undetectable before vaccination, became detectable in 30 (50.8%) of a total of 59 patients during 3 months of WT1 peptide vaccination. The dominant WT1-235 IgG antibody subclass was Th1-type, IgG1 and IgG3 . WT1-235 IgG antibody production was significantly and positively correlated with both progression-free survival (PFS) and overall survival (OS). Importantly, the combination of WT1-235 IgG antibody production and positive delayed type-hypersensitivity (DTH) to the WT1-235 peptide was a better prognostic marker for long-term OS than either parameter alone. These results suggested that WT1-235 peptide vaccination induces not only WT1-235-specific CTLs as previously described but also WT1-235-specific humoral immune responses associated with antitumor cellular immune response. Our results indicate that the WT1 IgG antibody against the WT1 peptide may be a useful predictive marker, with better predictive performance in combination with DTH to WT1 peptide, and provide a new insight into the antitumor immune response induction in WT1 peptide vaccine-treated patients.

[Successful treatment of an overwhelming infection with granulocyte transfusion in severe aplastic anemia patient undergoing allogeneic peripheral blood stem cell transplantation].

A 19-year-old woman complaining of fever and a sore throat was diagnosed with very severe aplastic anemia (AA) by bone marrow examination at a local hospital. Despite administration of antibiotics and granulocyte-colony stimulating factor to treat the soft tissue infection in her neck, her neutrophil count showed no increase. Because emergent allogeneic stem cell transplantation (SCT) was necessary, she was referred to our hospital. On admission, computed tomography revealed right-sided severe pharyngitis and lymphadenitis causing tracheal stenosis, and emergent intubation was required the next day. Granulocyte transfusion therapy (GTX) from related donors coupled with broad-spectrum antibiotic administration controlled the otherwise overwhelming infection. The patient received allogeneic peripheral blood SCT using a reduced-intensity conditioning regimen. After allogeneic SCT, successful engraftment was obtained. She was discharged from the hospital 59 days after allogeneic SCT. She remains alive and well, as of the latest follow up. This case clearly demonstrates that GTX is useful for controlling severe infection and enables patients with severe AA to receive allogeneic SCT safely.

Irreversible neurological defects in the lower extremities after haploidentical stem cell transplantation: possible association with nelarabine.

Severe peripheral neuropathy and myelopathy are rare complications after stem cell transplantation (SCT). In our institution, seven patients of precursor T lymphoblastic leukemia/lymphoma without the central nervous involvement who had been treated by nelarabine to control their diseases received SCT from HLA-haploidentical familial donor (HLA-haploidentical SCT) with the conditioning regimen including high-dose cytarabine (HDAC). Three of evaluable six patients developed irreversible paresthesia and muscle weakness in both lower extremities after neutrophil engraftment. The results of nerve conduction studies and short latency somatosensory evoked potentials suggested axonal neuropathy of both lower extremities in all three patients and myelopathy in two patients. Negative findings of PET-CT, and analyses of repeated cerebrospinal fluid samples and the bone marrow also indicated that tumor involvement was improbable. In all three patients, the symptoms worsened or persisted despite administration of corticosteroid and intravenous immunoglobulin. The high frequency of the neurological symptoms in our patients previously treated by nelarabine strongly suggested the association of the nelarabine use. Furthermore, the HLA-haploidentical SCT setting and the use of a potentially neurotoxic agent, HDAC might augment the neurotoxicity of nelarabine. It may be desirable that HLA-haploidentical SCT candidates avoid receiving nelarabine.

[Alleviation of carotid sinus syncope and removal of cardiac pacing after regression of cervical malignant lymphoma].

A 68-year-old man developed a rapidly-growing right cervical tumor, a biopsy of which allowed for the diagnosis of diffuse large B-cell lymphoma, not otherwise specified. Magnetic resonance imaging demonstrated a right cervical mass lesion of 80 mm in diameter that extended from the medial region of the parotid gland to the posterior region of the neck. While undergoing a chest X-ray in an upright position, he lost consciousness and briefly fell. A transient loss of consciousness recurred while changing his position on the bed, and an electrocardiogram at that time revealed sinus arrest of a seven second duration. This syncope was considered to be a carotid sinus syncope (CSS) induced by the compression of the carotid sinus by his cervical bulky lymphoma. Temporary cardiac pacing was immediately started and rituximab was administered. Three days later, CHOP therapy was started. As his cervical tumor rapidly shrank, the frequency of sensed sinus arrests decreased to zero per day by day 9 of CHOP therapy, resulting into the removal of the pacemaker. In certain cases with CSS due to cervical lymphoma, cardiac pacing, if needed at the onset, is considered to become removable early after chemotherapy in association with tumor shrinkage.

CD48 as a novel molecular target for antibody therapy in multiple myeloma.

Monoclonal antibody (mAb) drugs are desirable for the improvement of multiple myeloma (MM) treatment. In this study, we found for the first time that CD48 was highly expressed on MM plasma cells. In 22 out of 24 MM patients, CD48 was expressed on more than 90% of MM plasma cells at significantly higher levels than it was on normal lymphocytes and monocytes. CD48 was only weakly expressed on some CD34(+) haematopoietic stem/progenitor cells, and not expressed on erythrocytes or platelets. We next examined whether CD48 could serve as a target antigen for mAb therapy against MM. A newly generated in-house anti-CD48 mAb induced mild antibody-dependent cell-mediated cytotoxicity and marked complement-dependent cytotoxicity against not only MM cell lines but also primary MM plasma cells in vitro. Administration of the anti-CD48 mAb significantly inhibited tumour growth in severe combined immunodeficient mice inoculated subcutaneously with MM cells. Furthermore, anti-CD48 mAb treatment inhibited growth of MM cells transplanted directly into murine bone marrow. Finally and importantly, we demonstrated that the anti-CD48 mAb did not damage normal CD34(+) haematopoietic stem/progenitor cells. These results suggest that the anti-CD48 mAb has the potential to become an effective therapeutic mAb against MM.

Pretreatment levels of serum soluble interleukin-2 receptor are useful in selecting the treatment regimen for newly diagnosed advanced-stage follicular lymphoma with low tumor burden.

High pre-treatment serum soluble interleukin-2 receptor (sIL-2R) levels are associated with poor overall survival (OS) of patients with newly diagnosed follicular lymphoma (FL). We evaluated the usefulness of pre-treatment sIL-2R levels in selecting a treatment regimen for advanced-stage FL with low tumor burden (FL-LTB). This retrospective, multicenter observational study enrolled consecutive patients who received a rituximab-containing regimen for newly diagnosed advanced stage FL-LTB (grade 1-3a) between 2008 and 2018. We applied a previously reported cut-off value of 1800 IU/mL for sIL-2R. A total of 211 patients were eligible for the analysis. Among patients with high sIL-2R (47 patients, 22.3%), the OS rates for patients treated by rituximab monotherapy (R-mono) (11 patients) were significantly lower than those treated by rituximab-combination chemotherapy (R-chemo) (36 patients): 5-year OS rates were 66.7% and 94.4%, respectively (P = 0.007). Among patients with low sIL-2R (164 patients, 77.7%), OS rates were comparably good between the R-mono group (34 patients) and the R-chemo group (130 patients): 5-year OS rates were 100% and 98.3%, respectively (P = 0.38). Our results suggest that R-chemo may yield better OS than R-mono for patients with newly diagnosed advanced-stage FL-LTB and high pre-treatment serum sIL-2R levels.

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

WT1 (Wilms' tumor gene 1) protein is a potent pan-tumor-associated antigen (TAA) and WT1-specific cytotoxic T lymphocytes (WT1 tetramer(+) CD8(+) T cells) are spontaneously induced in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We conducted a single-cell level comparative analysis of T-cell receptor beta-chain variable region (TCR-BV) gene families of a total of 1242 spontaneously induced WT1 tetramer(+) CD8(+) T cells in HLA-A*2402(+) patients with AML or MDS and those in healthy donors (HDs). This is the first report of direct usage analysis of TCR-BV gene families of individual TAA-specific CD8(+) T cells at single-cell level. Usage analysis using single-cell RT-PCR of TCR-BV gene families of individual FACS-sorted WT1 tetramer(+) CD8(+) T cells showed for the first time (i) that BVs 5, 6, 20, and 27 were commonly biased in both HDs and patients; (ii) that BV4 was commonly biased in HDs and MDS patients; (iii) that BV19 was commonly biased in the patients; and (iv) that BVs 7 and 28, BVs 9 and 15, and BVs 12 and 29 were specifically biased in HDs, AML, and MDS patients, respectively. However, statistical analysis of similarity among HD, AML, and MDS of individual usage frequencies of 24 kinds of TCR-BV gene families indicated that the usage frequencies of TCR-BV gene families in AML and MDS patients reflect those in HDs. These findings represent a novel insight for a better understanding of WT1-specific immune response.

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.

WT1 peptide vaccination in combination with imatinib therapy for a patient with CML in the chronic phase.

Although tyrosine kinase inhibitors is effective for dramatically reducing CML cells, it might be difficult to eradicate completely the CML stem cells. We aimed to clarify the safety and effects of WT1 peptide vaccination in combination with imatinib therapy for a CML patient. A 51 year-old male with CML in CP, who showed a resistance against imatinib therapy for 2.5 years, began to be treated with 9 mer modified-type WT1 peptides in combination with standard dose of imatinib. Although every 2-week-administration of WT1 peptides for 22 weeks did not show definite effects on the quantification of bcr-abl transcripts, by changing the administration from every 2 weeks to 4 weeks bcr-abl transcripts decreased remarkably. After 11 months of every 4-week-administration of the peptides and 12 months post cessation of the peptides bcr-abl transcripts achieved to the level below detection by RQ/RT-PCR (complete molecular response). WT1/MHC tetramer(+)CD8(+) CTLs, which appeared after the second administration of WT1 peptides and remained more than 15 in number among 10(6) CD8(+) T cells throughout the administration of WT1 peptides, are still present in the blood on 14th month post cessation of the peptides. An in vitro study as to the cytotoxicity of lymphocytes induced by mixed lymphocyte peptide culture demonstrated that cultured lymphocytes possessed cytotoxicity against WT1 expressing leukemia cells and the cytotoxicity was WT1-specific and MHC class I restricted. The present study showed that WT1 peptide vaccination in combination with TKI is feasible and effective in the therapy for imatinib-resistant CML.

Commissioning of total body irradiation using plastic bead bags.

The goal of total body irradiation (TBI) is to deliver a dose to the whole body with uniformity within ±10%. The purpose of this study was to establish the technique of TBI using plastic bead bags. A lifting TBI bed, Model ORP-TBI-MN, was used. The space between the patient's body and the acrylic walls of the bed was filled with polyacetal bead bags. Patients were irradiated by a 10 MV photon beam with a source to mid-plane distance of 400 cm. The monitor unit (MU) was calculated by dose-per-MU, tissue-phantom-ratio and a spoiler factor measured in solid water using an ionization chamber. The phantom-scatter correction factor, off-center ratio and the effective density of the beads were also measured. Diode detectors were used for in vivo dosimetry (IVD). The effective density of the beads was 0.90 ± 0.09. The point doses calculated in an I'mRT phantom with and without heterogeneity material showed good agreement, with measurements within 3%. An end-to-end test was performed using a RANDO phantom. The mean ± SD (range) of the differences between the calculated and IVD-measured mid-plane doses was 1.1 ± 4.8% (-5.9 to 5.0%). The differences between the IVD-measured doses and the doses calculated with Acuros XB of the Eclipse treatment planning system (TPS) were within 5%. For two patients treated with this method, the differences between the calculated and IVD-measured doses were within ±6% when excluding the chest region. We have established the technique of TBI using plastic bead bags. The TPS may be useful to roughly estimate patient dose.

Pretreatment serum soluble interleukin-2 receptor level predicts survival in patients with newly diagnosed follicular lymphoma.

This retrospective, multicenter observational study investigated the prognostic value of pretreatment serum soluble interleukin-2 receptor (sIL-2R) level for outcomes of newly diagnosed follicular lymphoma (FL) grade 1-3a who required treatment at diagnosis. A total of 628 patients were recorded, and 502 of these were eligible for analysis. Patients were divided into four quartiles, based on their serum sIL-2R levels as follows: Q1 (sIL-2R < 520 IU/mL), Q2 (520 ≤ sIL-2R < 1030 IU/mL), Q3 (1030 ≤ sIL-2R < 2530 IU/mL) and Q4 (sIL-2R ≥ 2530 IU/mL). Using a multivariable Cox proportional-hazards model, we showed the adjusted probability of overall survival (OS) decreased with increasing serum sIL-2R levels (p for trend = .007). Similar trends were observed for disease-specific survival (DSS) and progression-free survival (PFS). In conclusion, pretreatment serum sIL-2R levels significantly and dose-dependently associate with worse outcomes (OS, DSS and PFS) of patients with newly diagnosed FL.

Recovery from established graft-vs-host disease achieved by bone marrow transplantation from a third-party allogeneic donor.

OBJECTIVE: We investigated whether established graft-vs-host disease (GVHD) could be successfully treated by a second allogeneic bone marrow transplantation (BMT) through elimination of first donor-derived lymphocytes responsible for GVHD. MATERIALS AND METHODS: In a murine GVHD model of BDF1 (H-2(b/d))-->B6C3F1(H-2(b/k)), GVHD mice underwent a second BMT using a graft (1 x 10(7) bone marrow and 3 x 10(7) spleen cells) from a major histocompatibility complex (MHC) antigen haploidentically mismatched (to host and also to first donor) mouse strain, B6B10F1(H-2(b/s)), following low-dose total body irradiation (TBI) 2 to 3 weeks after the first BMT. RESULTS: Results demonstrated that severe GVHD could be successfully and stably treated by a second allogeneic BMT. For successful treatment of GVHD, rapid achievement of full second-donor T-cell chimerism was required. Furthermore, we showed that mice with GVHD could easily accept MHC haploidentically mismatched second-donor hematopoietic cells even after minimal conditioning (2-4 Gy TBI) because they were in a profoundly immunosuppressed state, and that the mice were relatively resistant to new development of GVHD by second-donor grafts. Furthermore, the timing of the second BMT, the intensity of conditioning treatment (GVHD mice are very sensitive), and donor selection were also found to be important for obtaining successful outcomes. Increased regulatory T cells and reduction of interferon-gamma levels may be involved in tolerance induction. CONCLUSIONS: We demonstrated that established GVHD in a murine GVHD model could be successfully treated by a second BMT from a third-party allogeneic donor.

Unmanipulated HLA 2-3 antigen-mismatched (haploidentical) bone marrow transplantation using only pharmacological GVHD prophylaxis.

OBJECTIVE: The incidence of severe graft-vs-host disease (GVHD) in unmanipulated human leukocyte antigen (HLA) 2-3 antigen-mismatched bone marrow transplantation (BMT) using cyclosporine and methotrexate as GVHD prophylaxis is 80% to 90%. We investigated whether pharmacological GVHD prophylaxis consisting of four drugs, including a steroid, effectively suppressed GVHD in this transplantation setting. MATERIALS AND METHODS: Thirty patients who had hematologic malignancies at an advanced stage or with poor prognosis underwent allogeneic BMT using a myeloablative preconditioning regimen consisting of cyclophosphamide (60 mg/kg x 2), total body irradiation (8-10 Gy), and fludarabine (30 mg/m(2) x 4) with or without cytosine arabinoside (2 g/m(2) x 4), and GVHD prophylaxis consisting of a combination of tacrolimus, methotrexate, mycophenolate mofetil, and methylprednisone (2 mg/kg). Early therapeutic intervention for GVH reaction or grade I GVHD was performed, and steroid was slowly tapered. RESULTS: All patients achieved donor-type engraftment. Neutrophil (>0.5 x 10(9)/L) and platelet (>20 x 10(9)/L) engraftment was achieved on day 13 and on day 30, respectively. Seventeen patients (56.7%) had no GVHD. Eleven patients (36.7%) developed grade II-III acute GVHD. Seven patients (23.3%) died of transplant-related toxicity, including fungal or viral infections and thrombotic microangiopathy, and four patients died of disease progression. Estimated relapse rate at 3 years was only 20.9%. The probability of survival at 3 years was 49.9%. CONCLUSIONS: These data suggest that, in unmanipulated HLA-haploidentical allogeneic BMT, this GVHD prophylactic regimen, which includes methylprednisolone 2 mg/kg, and early therapeutic intervention for GVH reaction suppress the incidence of severe GVHD to an acceptable level, while preserving the graft-vs-leukemia effect.

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.

A case of monoclonal gammopathy of undetermined significance with abnormal low levels of plasma glycated albumin by M protein.

BACKGROUND: It is known that an immunoglobulin abnormality affects various clinical laboratory measurements and leads to abnormal values. We experienced a case of monoclonal gammopathy of undetermined significance (MGUS) showing a falsely low plasma glycated albumin (GA) level. CASE REPORT: The patient was a 75-y-old male who visited our hospital for thrombocytosis identified during a medical checkup. Based on further examinations, he was diagnosed with MGUS (IgM-κ type). Laboratory examinations revealed that the plasma GA level was significantly low at -1.3% but the serum GA level was reasonable at 15.5%. We investigated the cause of the falsely low plasma GA level. RESULTS: The patient's plasma became turbid after mixing with the first reagent for GA measurement. The plasma GA level was increased by dilution of the plasma. The plasma GA level was falsely decreased only at the time of measurement on a sample collected using a blood-collecting tube with heparin sodium. The GA level was decreased by adding heparin sodium to the patient's serum, whereas the GA level was increased by neutralization of the patient's plasma with protamine sulfate. The GA level was increased after adding polyethylene glycol to the patient's plasma. Serum GA levels in healthy controls were decreased by adding purified M protein from the patient's serum. CONCLUSIONS: We report a patient with MGUS whose plasma GA concentration was falsely decreased by M protein when blood was drawn in a heparin sodium-containing tube.

Clinical and immunologic responses to very low-dose vaccination with WT1 peptide (5 microg/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.

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.

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 regulatory 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.