WT1 Peptide Vaccination Research Articles

Clinical Phase II Study of WT1 Peptide Vaccination in Pediatric Leukemia Patients after Stem Cell Transplantation and Analysis of Immunological Monitoring

IntroductionAllogeneic stem cell transplantation (allo-SCT) is a radical treatment for pediatric patients with relapsed and/or chemotherapy-resistant leukemia. Because these leukemia cells seem refractory to chemotherapy and irradiation, immunotherapy are needed. WT1 is highly expressed in pediatric leukemia and essential for leukemia cell growth, leading to the hypothesis that the WT1 protein-based immunization after allo-SCT may improve the clinical outcome of the SCT therapy. We investigated the clinical efficacy and immunological effect of immunotherapy targeting WT1 protein for pediatric patients after allo-SCT. MethodMajor inclusion criteria were as follows: Patients with HLA-A*2402 and/or HLA-*A0201 aged 20 years or younger and WT1 mRNA expression in leukemic cells; donors with HLA-A*2402 and/or HLA-*A0201. The HLA A*2402 restricted 9mer modified WT1 peptide or HLA A*0201 restricted natural WT1 peptide emulsified in Montanide ISA 51 adjuvant was injected intradermally. The dose of WT1 peptide depended on patient weight. The vaccinations were scheduled to be given weekly for 12 consecutive weeks, and if no recurrence and severe adverse effect were observed, vaccination was continued. Absolute number and frequency of WT1 specific cytotoxic T lymphocytes were analyzed by WT1 tetramer assay.ResultsA total of 18 patients with 9 AML/MDS, 8 ALL and 1 NHL were enrolled. Seven patients were those with high risk (HR) of relapse who had received SCT at 3rd CR or on disease. The other 11 patients were at standard risk (SR) but had either relapsed after SCT or shown induction failure. 16 had HLA-A*2402 and the other 2 had HLA-A*0201. 12 of the 18 patients received WT1 peptide vaccination monthly after the first 12-time consecutive vaccinations with one-week interval and are still in complete remission for 16-72 months (median 31 mo.). One patient discontinued the vaccination because of pancreatitis due to treatment with steroid for GVHD. One year probabilities of PFS is 66.7% in all, and it is notable that 4 of 7 HR patients are maintained in long-term CR. Although, of these 12 patients with persistence of CR, seven patients had high WT1 mRNA level before vaccination in peripheral blood (PB) or bone marrow (BM), WT1 mRNA levels decreased to within normal level after vaccination. WT1-specific CTLs in PB were detected and increased after vaccination in all cases and absolute numbers of these cells were significantly increased after vaccination in all cases (p< 0.05). The fold increase of WT1-specific CTL numbers after the vaccination was significantly higher in cases which maintained CR (p< 0.05). The WT1-specific CTLs with high tetramer staining were detected during the first 12-time vaccinations in 14 patients, and of these patients, 12 patients are still in CR. In the other 4 patients, only very few WT1 specific CTLs were detected, and three of the four patients showed recurrence. Five patients (1AMKL, 4 ALL) had recurrence. There was no significant difference in the fold increase of WT1-specific CTL numbers after the vaccination between cases with immunosuppressive agents for the prevention of GVHD and those without the agents.DiscussionWe reported the interim result of the phase II clinical study of WT1 peptide immunotherapy after SCT for pediatric patients, in which promising clinical efficacy of the therapy was suggested. In addition, we found that an increase in WT1-specific CTLs was associated with a decrease in WT1 mRNA, suggesting occurrence of WT1-driven graft versus leukemia (GVL) effect, which is consistent with strategies to enhance leukemia antigen-specific GVL response without deterioration of GVHD. The following three mechanisms are suggested. 1) After SCT, many kinds of cytokines are produced and proliferation of T lymphocytes occur; 2) lymphodepletion after SCT allows development of T cells specific to WT1 peptide; and resultantly, 3) efficient homeostatic expansion of WT1-specific CTLs is induced. Post-SCT vaccination with WT1 peptide can be a safe and effective strategy to prevent the recurrence of the disease without deterioration of GVHD in pediatric hematological malignancy. For avoiding late side effect due to preconditioning regimen and GVHD, effective post SCT immunotherapy is a promising strategy for pediatric patient. Larger studies are warranted on application of WT1 targeted immunotherapy to prevent recurrence after pediatric SCT DisclosuresNo relevant conflicts of interest to declare.

Long term efficacy of WT1 peptide vaccine in patients with WT1 overexpressing hematopoeitic and solid malignancies

We have performed two separate phase II peptide vaccine trials in patients with AML/MDS and in patients with WT1 overexpressing solid tumors with identical vaccine schedule. Here we concentrate on patients remaining on vaccination for at least one year in order to analyze characteristics for long-term vaccination success. Factors considered were disease type, intensity of prior chemotherapy, tumor load and toxicity. Patients with AML and high-risk MDS (n=25) as well as with ovarian (n=7), thyroid (n=2), breast (n=1), gastric (n=1), and larynx cancer (n=1), astrocytoma (n=1) and mesothelioma (n=4) received 0.2mg HLA-A*0201-restricted WT1.126-134 peptide admixed with 1 mg KLH (day 3), and 62.5µg GMCSF (day 1-4) every 2 weeks x 4, followed by either biweekly or monthly for 10 months and at increased intervals of up to 3-monthly thereafter. Standard criteria were used for response assessment. Patients remained on study in absence of limiting toxicity and disease progression. Toxicity to treatment was mild, and no patient went off protocol for adverse events or consent withdrawal. Eleven patients remained on study for more than 1 year with disease control, including 3/19 patients with active AML, 4/9 patients with AML in CR, 2/7 patients with ovarian cancer and 2/4 patients with mesothelioma. Of these 11 patients 4 remained on study for more than 3 years: A 70 year old man with sAML from MDS, with 5% marrow blasts at baseline and severe anemia after 3 cycles of induction and consolidation chemotherapy, who was 58 months on study. A previously untreated woman with AML with PD and 40% of marrow blasts at baseline, who is 78+ months in SD on study, and a woman with AML in CR after induction and consolidation therapy, who achieved a second CR after initial relapse with 30% marrow blasts during vaccination and 48+ months on study. In addition a man with mesothelioma and PR after initial pleurectomy, who is after 50+ months in SD still being vaccinated. Overall, no correlation between prior treatment and duration on vaccine was apparent, as 2/4 patients in long-term disease control had previously aggressive chemotherapy. Of the solid tumor patients, chemotherapy-pretreated ovarian cancer and mesothelioma appear to be sensitive to WT1 vaccine, although other histologies cannot be excluded due to low patient numbers. These results further underline the potential of WT1 targeting immunotherapies in cancer patients and detailed immunological and molecular analyses might be able to impact on improved vaccine design and to characterize a certain subgroup of patients with specific benefit from such therapies.

WS4–4 - WT1 Peptide Vaccine for Non-Small Lung Cancer After Operation

WS4–4 - WT1 Peptide Vaccine for Non-Small Lung Cancer After Operation

A vaccine for leukemia: one step closer? Cure-oriented WT1 peptide vaccination therapy is being developed

Leukemia antigen-targeting vaccination therapy is a naturally occurring demand & its establishment is now desired For the treatment of hematological malignancies, including leukemia, striking advances in chemotherapy, targeted therapy such as tyrosine kinase inhibitors, and hematopoietic stem cell transplantation (HSCT), have been made. However, unfortunately, a substantial proportion of patients relapse after treatment. In this context, a novel treatment is desired and one of the more attractive candidates is a vaccine therapy. HSCT is the most intensive treatment for leukemia. The HSCT confers a powerful T-cell-mediated graft-versus-leukemia effect to eradicate leukemic cells and, therefore, is a direct evidence for the effectiveness of T-cell-mediated immune reaction in leukemia treatment. The undisputed effect of graft-versus-leukemia provided us with an idea that autologous T lymphocytes, as well as HSCT donor-derived T cells, might eradicate leukemic cells if the autologous T cells could recognize leukemia antigens and be activated strongly enough.

WT1 peptide immunotherapy for gynecologic malignancies resistant to conventional therapies: a phase II trial

The aim of the present study was to analyze the long-term survival effects of WT1 peptide vaccine, in addition to its anti-tumor effects and toxicity. A phase II clinical trial was conducted during the period of 2004-2010 at Osaka University Hospital, Osaka, Japan. The patients who had gynecologic malignancies progressing against previous treatments received WT1 peptide vaccine intradermally at 1-week intervals for 12 weeks. The vaccination was allowed to further continue, unless the patient's condition became significantly worse due to the disease progression. Forty out of 42 patients, who met all the inclusion criteria, underwent WT1 peptide vaccine. Among these 40 patients, stable disease was observed in 16 cases (40 %). Skin toxicity of a grade 1, 2 and 3 occurred in 25 cases (63 %), 9 cases (23 %) and a single case (3 %), respectively, and liver toxicity of grade 1 in a single case (3 %). The overall survival period was significantly longer in cases positive for the WT1 peptide-specific delayed-type hypersensitivity (DTH) reaction after the vaccination, compared to those negative for the DTH reaction (p = 0.023). Multivariate Cox proportional hazards analysis demonstrated that the adjusted hazard ratio for the negative DTH reaction was 2.73 (95 % CI 1.04-7.19, p = 0.043). WT1 peptide vaccine may be a potential treatment, with limited toxicity, for gynecologic malignancies that have become resistant to conventional therapies. Larger scale of clinical studies is required to establish the efficacy of the WT1 peptide vaccine for gynecologic malignancies.

Phase II Trial of WT1 Analog Peptide Vaccine in Patients with Acute Myeloid Leukemia (AML) in Complete Remission (CR)

Abstract 3624WT1 is a transcription factor which has been implicated in leukemogenesis and has been used as a marker of minimal residual disease (MRD). We previously demonstrated the feasibility of vaccinating AML patients in CR with a multivalent WT1 peptide vaccine and inducing immune responses. In an effort to further explore the safety and efficacy of this approach, we are conducting a Phase II study in which the vaccine is administered to AML patients in first CR and who completed all planned postremission chemotherapy. Eligible patients had WT1 transcript detectable by RT-PCR. The vaccine consisted of 4 native and derived WT1 peptides administered with the immune adjuvants Montanide and GM-CSF. Patients received 6 vaccinations over 10 weeks. Early toxicity was assessed at weeks 2 and 4. Immune responses were evaluated at week 12 by CD4+ T cell proliferation, CD3+ T cell interferon-g interferon release (ELISPOT) and WT1 peptide tetramer staining. Patients who were clinically stable and without disease recurrence could continue with up to 6 more vaccinations administered approximately every month. To date, 12 patients have been accrued to the study (6-M, 6-F; median age – 66 years, range 26–73 years). Cytogenetic subtypes varied among the study patients (Favorable-3, Intermediate-5, Unfavorable-4). The median time to vaccination after achieving CR was 7.5 months (range: 3–22 months). One patient was removed early because of relapse prior to receiving the first vaccination. Four patients have received at least 6 vaccines and 2 others have completed 12 vaccinations. Eight patients are alive without evidence of disease. One of these patients has an HLA-A02 subtype and was found to have developed T cells reactive with WT1-A (native peptide) HLA tetramers following 6 vaccinations which persisted (at a lower level) until after the 12th vaccination. Three patients relapsed during vaccination (after 1, 5 and 11 vaccines) and 2 of the 3 have died. Two of the relapsed patients who had sample available for immunologic evaluation, did not develop a CD4+ response to any of the peptides tested. Two other patients discontinued vaccination because of toxicity (hypersensitivity/pain with GM-CSF administration). Both remain in CR. No episodes of anaphylaxis or generalized urticaria were observed. Neither median disease free survival nor overall survival has been reached in this small cohort of patients. These preliminary findings demonstrate that the WT1 peptide vaccine is relatively well tolerated and has immunologic activity. Trial accrual is ongoing and further follow-up is required before any beneficial effect on outcome can be determined. Disclosures:Scheinberg:Formula Pharma: WT1 Vaccine inventor, Patent held by MSKCC and Licensed to Formula Pharma, WT1 Vaccine inventor, Patent held by MSKCC and Licensed to Formula Pharma Patents & Royalties.

Achieving Selective Graft-Versus-Leukemia without Graft-Versus-Host Effects by WT1 Peptide Vaccination of Donors and Stringent Engineering of the Allo-Graft

Abstract 4108A major goal for allogeneic hematopoietic cell transplantation (HCT) is to engineer grafts to contain only cell types required to rescue hematopoiesis, recover anti-infectious immune function, and provide curative graft-versus leukemia (GVL) effects. Such grafts will avoid the life-threatening complication of graft-versus-host disease (GVHD). Before manipulative approaches can be translated into clinical practice, the dynamics of engraftment, expansion, and interactions of isolated donor cell populations must be comprehensively understood. Here, in preclinical mouse models, we dissect the effects and potency of distinct purified donor cell populations to exert GVL. To enhance GVL we vaccinated donors with a tumor-antigen, WT1. AKR/b or C3H/SW mice were prepared with 4 weekly WT1-peptide/incomplete Freund’s adjuvant vaccinations. Grafts consisted of FACS-purified hematopoietic stem cells (HSC [cKit+Thy1.1loLin−Sca1+]), augmented with CD4+, CD8+, naïve or memory CD4+ and CD8+T cells (TC), or WT1-tetramer selected (WT1)CD8+TC. HSC +/− TC subsets were infused into lethally irradiated, minor-antigen mismatched C57BL/6 mice. A luciferase-labeled, myeloid lineage C57BL/6 leukemia, FBL3, was inoculated on d0 or 14 post-HCT, modeling tumor persistence or early relapse after HCT, respectively. Graft compositions were tested for their strongest GVL potency measured by bioluminescence imaging and survival. When recipients were given tumor cells at the time of HCT (i) HSC+CD4++CD8+ TC grafts from vaccinated donors rescued most recipients, while those from unvaccinated mice resulted in rapid death from leukemia; (ii) recipients of HSC+CD4++CD8+ from unvaccinated mice died faster than recipients of HSC alone, suggesting GVH-related damage to lymphoid organs diminishes the host’s endogenous tumor resistance; (iii) HSC + either CD8+, memory (mem)CD8+, or (WT1)CD8+TC from vaccinated donors provided improved but non-curative protection from leukemia compared to HSC alone; (iv) HSC + CD4+TC grafts (vaccinated or unvaccinated) were associated with rapid death; however, (v) CD4+TC were required to fortify tumor eradication by CD8+TC. To model the clinically relevant scenario of early relapse, we next infused tumor cells on d14 post-HCT. At 1 wk prior to tumor inoculation an in vivo boost WT1-peptide vaccination was applied. (i) Again, HSC+CD4++CD8+ from vaccinated donors achieved better tumor control as compared to HSC +/− unvaccinated CD4++CD8+ TC, but a high proportion of recipients died due to progressive leukemia. We speculate the reason for diminished GVL is a lack of tumor antigens present at the time of TC infusion which resulted in a different donor TC repertoire expanding during lymphopenia. (ii) All subgroups given combinations of naïve/memory CD8+ TC and naïve/memory CD4+ TC had prolonged survival compared with HSC recipients, but no long-term cure. (iii) The only group with improved overall survival received HSC + WT1CD8++ CD4+ TC grafts and a booster vaccine prior to tumor injection. Our findings suggest the benefit was due to an in vivo expansion of tumor-specific TC early post-HCT, driven by the tumor antigen + adjuvant re-exposure. We observed with peptide vaccination that: 1.) ∼2/3 of WT1CD8+TC retained a naïve phenotype, and CD4+TC were required to provide help and promote, suggesting that CD4+TC are required to convert naïve into effector memory CD8+TC. 2.) viral immunizations resulted in robust antigen specific responses compared to more modest development of WT1CD8+TC after the peptide-vaccine. However, the minimal numbers of WT1-specific CD8+TC transferred were highly efficient, whereas recipients of HSC+WT1-depleted TC died rapidly of leukemia, which supports the specificity of the response rather than general alloreactivity. Thus, dynamics of engraftment of donor effector populations are complex, modalities to promote tumor specific CD8+TC expansion early post-HCT are critical to maximize GVL and rely upon the interactions of co-transplanted cell subsets and timing of infusions. Immunogenic maneuvers such as vaccinations are powerful tools to enhance the potency of specific effector populations. Transplant approaches using pure HSC to restore hematopoiesis and manipulated TC populations without deleterious GVHD-inducing effects are the ultimate goal of allogeneic HCT and require informed translation of preclinical models. Disclosures:No relevant conflicts of interest to declare.

T-cell Immune Abnormality and Novel Immunotherapeutic Approach by Blocking the B7-H1-PD-1 Pathway Combined with WT1 Tumor Vaccine in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are hematologic malignancies caused by the neoplastic transformation of hematopoietic stem or myeloid progenitor cells and characterized by cytopenias, excessive apoptosis of hematopoietic cells, and a high risk of progression to acute myeloid leukemia. During disease progression, clonal blasts gain a more aggressive nature, whereas nonclonal immune cells become less efficient via an unknown mechanism. Stem cell transplantation is the only curative treatment for MDS, but it is not suitable for elderly patients. B7-H1 (CD274) molecules, which are expressed on antigen-presenting cells and inhibit T-cell responses through programmed death-1 (PD-1) expressed on activated T cells, are expressed on various tumor cells, and B7-H1-expressing tumor cells evade attack by tumor-specific cytotoxic T lymphocytes (CTLs). We reported immune dysfunction associated with T cells in MDS, i.e., an increase in T-cell apoptosis, higher expression levels of PD-1 on circulating T cells, and higher levels of plasma-soluble interleukin-2 receptor. Furthermore, we demonstrated that MDS blasts overexpress Wilms tumor gene WT1 mRNA, suggesting that the anti-WT1 immune response elicited by WT1 peptide vaccine may induce tumor regression in some patients. In the current study, we investigated B7-H1 expression on MDS blasts and analyzed the characteristics and inhibitory effects on T-cell immune responses of B7-H1 blasts. Finally, to develop new strategies, we are now analyzing a novel immunotherapy involving blockade of the B7-H1-PD-1 pathway in combination with WT1 tumor vaccine and or inhibition of regulatory T cells. First, we analyzed B7-H1 expression in 3 MDS cell lines, i.e., F-36P, OIH-1, and SKM-1, and on MDS blasts from 29 MDS patients, 32 patients with acute myeloid leukemia transformed from MDS (AL-MDS), and 10 hematologically normal individuals. A high level of B7-H1 expression at the mRNA and protein levels was detected only in F-36P cells using reverse transcription-PCR and flow cytometry (FCM), respectively. B7-H1 protein was not detectable on OIH-1 and SKM-1. Blasts from patients with high-risk MDS and AL-MDS expressed B7-H1 molecules more often compared with those from low-risk MDS patients. B7-H1 molecules were expressed in fewer than 5% of blasts in normal individuals. Furthermore, we found that the cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which may be associated with MDS pathophysiology, induced B7-H1 expression on SKM-1 cells or blasts from MDS patients, and that B7-H1 induction by these cytokines was mediated by nuclear factor (NF)-κB activation, whose activation was observed in MDS patients, in particular in advanced disease. Second, to investigate the characteristics of B7-H1 blasts, we analyzed proliferative advantage, i.e., cell cycle and colony formation, in B7-H1 and B7-H1 cell fractions in MDS blasts using FCM and the methylcellulose assay, respectively. B7-H1 MDS blasts had greater intrinsic proliferative capacity than B7-H1 MDS blasts when examined in both assays. Third, to investigate the immunomodulatory effects of B7-H1 MDS blasts on T-cells, T-cell apoptosis and

Lymphodepletion is permissive to the development of spontaneous T-cell responses to the self-antigen PR1 early after allogeneic stem cell transplantation and in patients with acute myeloid leukemia undergoing WT1 peptide vaccination following chemotherapy

PR1, an HLA-A*0201 epitope shared by proteinase-3 (PR3) and elastase (ELA2) proteins, is expressed in normal neutrophils and overexpressed in myeloid leukemias. PR1-specific T cells have been linked to graft-versus-leukemia (GVL) effect. We hypothesized that lymphopenia induced by chemo-radiotherapy can enhance weak autoimmune responses to self-antigens such as PR1. We measured PR1-specific responses in 27 patients 30-120 days following allogeneic stem cell transplant (SCT) and correlated these with ELA2 and PR3 expression and minimal residual disease (MRD). Post-SCT 10/13 CML, 6/9 ALL, and 4/5 solid tumor patients had PR1 responses correlating with PR3 and ELA2 expression. At day 180 post-SCT, 8/8 CML patients with PR1 responses were BCR-ABL-negative compared with 2/5 BCR-ABL-positive patients (P = 0.025). In contrast, PR1 responses were detected in 2/4 MRD-negative compared with 4/5 MRD-positive ALL patients (P = 0.76). To assess whether the lymphopenic milieu also exaggerates weak T-cell responses in the autologous setting, we measured spontaneous induction of PR1 responses in 3 AML patients vaccinated with WT1-126 peptide following lymphodepletion. In addition to WT1-specific T cells, we detected PR1-specific T cells in 2 patients during hematopoietic recovery. Our findings suggest that lymphopenia induced by chemo-radiotherapy enhances weak autoimmune responses to self-antigens, which may result in GVL if the leukemia expresses the relevant self-antigen.

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

Enhanced tumor immunity of WT1 peptide vaccination by interferon-β administration

To induce and activate tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) for cancer immunity, it is important not only to select potent CTL epitopes but also to combine them with appropriate immunopotentiating agents. Here we investigated whether tumor immunity induced by WT1 peptide vaccination could be enhanced by IFN-β. For the experimental group, C57BL/6 mice were twice pre-treated with WT1 peptide vaccine, implanted with WT1-expressing C1498 cells, and treated four times with WT1 peptide vaccine at one-week intervals. During the vaccination period, IFN-β was injected three times a week. Mice in control groups were treated with WT1 peptide alone, IFN-β alone, or PBS alone. The mice in the experimental group rejected tumor cells and survived significantly longer than mice in the control groups. The overall survival on day 75 was 40% for the mice treated with WT1 peptide+IFN-β, while it was 7, 7, and 0% for those treated with WT1 peptide alone, IFN-β alone or PBS alone, respectively. Induction of WT1-specific CTLs and enhancement of NK activity were detected in splenocytes from mice in the experimental group. Furthermore, administration of IFN-β enhanced expression of MHC class I molecules on the implanted tumor cells. In conclusion, our results showed that co-administration of WT1 peptide+IFN-β enhanced tumor immunity mainly through the induction of WT1-specific CTLs, enhancement of NK activity, and promotion of MHC class I expression on the tumor cells. WT1 peptide vaccination combined with IFN-β administration can thus be expected to enhance the clinical efficacy of WT1 immunotherapy.

9242 POSTER Effective WT1 Peptide Vaccination With Long-lasting Amplification of WT1-specific Cytotoxic T Lymphocytes in a Patient With CML in Chronic Phase

9242 POSTER Effective WT1 Peptide Vaccination With Long-lasting Amplification of WT1-specific Cytotoxic T Lymphocytes in a Patient With CML in Chronic Phase

WT1 peptide vaccination following allogeneic stem cell transplantation in pediatric leukemic patients with high risk for relapse: successful maintenance of durable remission

WT1 peptide vaccination following allogeneic stem cell transplantation in pediatric leukemic patients with high risk for relapse: successful maintenance of durable remission

Clinical effects of Wilms tumor protein (WT) 1 peptide vaccination in patients with WT1-expressing solid tumors as compared to patients with AML/MDS.

2560 Background: WT1 holds great promise for cancer immunotherapy, is expressed in many hematologic and solid malignancies, and is essential for tumor proliferation. After demonstrating good immunological and clinical efficacy of WT1 peptide vaccination in patients with active AML and MDS, we have initiated a vaccination trial for patients with WT1 expressing carcinomas. We here compare immunogenicity and clinical efficacy among carcinoma patients (CP) and AML/MDS patients. Methods: Patients with ovarian (n=8), thyroid (n=2), breast (n=1), gastric (n=1), and larynx cancer (n=1), astrocytoma (n=1) and mesothelioma (n=3) received 0.2mg HLA-A0201-restricted WT1.126-134 peptide admixed with 1 mg KLH (day 3), and 62.5µg GM-CSF (day 1-4). Quantitation of WT1 specific T cells was performed in peripheral blood (PB) of CP by tetramer staining and compared with analogue analyses in 25 AML/MDS patients. Results: Seventeen CP received a median of 11 vaccinations (range 1-35) and 12 of 17 are evaluable for T cell response. None of the 12 had WT1-specific T cells at baseline, in contrast to 28% of AML/MDS patients. After 4 vaccinations WT1-specific T cells were detectable in 67% of CP and in 54% of AML/MDS patients. After 6 vaccinations WT1-specific T cells were present in 40% of CP and in 78% of AML/MDS patients. Thus, the overall conversion rate was 75% in CP and 56% in AML/MDS. WT1 specific CD8+ T cells in most AML/MDS patients remained detectable during the course of vaccination, whereas in CP WT1 specific CD8+ T cells were only transient, despite of continuation of vaccination and ongoing clinical efficacy, which was demonstrated with 1 partial remission and 6 disease stabilisations between 2 and 22+ months. Conclusions: These results demonstrate profound differences in spontaneous and vaccine induced T cell response among patients with haematological and solid malignancies, suggesting considerably higher spontaneous immunogenicity of AML/MDS, but a higher conversion rate in CP. In contrast to AML/MDS WT1 specific T cells in PB of CP were despite ongoing clinical efficacy only transient, possibly due to migration into the tumor.

Randomized, double-blinded, phase II trial of a WT1 peptide vaccine as adjuvant therapy in patients with malignant pleural mesothelioma (MPM).

TPS139 Background: The transcription factor, WT1, is commonly over-expressed in MPM and immunohistochemical tests for WT1 are used diagnostically. Certain WT1 peptides are processed and presented to the immune system and are thus an attractive target for T cell based immunotherapy. Using computer prediction analysis we designed analog peptides derived from WT1 sequences by substituting amino acids at key HLA-A0201 binding positions. We tested the safety and immunogenicity of a WT1 vaccine comprised of four peptides in patients with thoracic neoplasms expressing WT1 (Krug et al, Cancer Immunol Immunother, 2010). Six out of nine patients tested demonstrated CD4 T-cell proliferation to WT1 specific peptides, and all five HLA-A0201 patients tested mounted a CD8 T-cell response. One patient remains without progression >30 months after the start of the study. The median survival (measured from the date of the first vaccinations) was 13 months. Based on this pilot trial, we proposed a randomized phase II trial in a MPM patient population with minimal disease burden. Methods: Eligible patients will have MPM-expressing WT1 treated with combined modality therapy including surgery. Patients will be randomized to receive WT1 peptides plus Montanide adjuvant and GM-CSF, or Montanide and GM-CSF alone. The primary endpoint is progression free survival (PFS). 39 patients will be enrolled in each arm, which will provide 90% power to detect an improvement in PFS at one year from 50% to 70%. Immune responses will be evaluated using T cell proliferation assays, MHC tetramer staining and interferon-γ ELISPOT. Supported by grants from the Department of Defense (PR093640), NIH (Ca 23766,) and the Meso Foundation (MARF).

Repeated PR1 and WT1 peptide vaccination in Montanide-adjuvant fails to induce sustained high-avidity, epitope-specific CD8+ T cells in myeloid malignancies

We previously showed that vaccination with one dose of PR1 and WT1 peptides induces transient anti-leukemia immunity. We hypothesized that maintenance of a sustained anti-leukemia response may require frequent boost injections. Eight patients with myeloid malignancies were enrolled in this phase II study, and 6 completed 6 injections of PR1 and WT1 peptides in Montanide-adjuvant with GM-CSF, every two weeks. Both high- and low-avidity PR1 or WT1-specific CD8(+) T cells were detected in all evaluable patients after the first vaccine dose. Repeated vaccination led to selective deletion of high avidity PR1- and WT1-specific CD8(+) T cells and was not associated with significant reduction in WT1-expression. Additional boosting failed to increase vaccine-induced CD8(+) T-cell frequencies further and in all patients the response was lost before the 6(th) dose. PR1- or WT1-specific CD8(+) T cells were not detected in bone marrow samples, excluding their preferential localization to this site. Following a booster injection three months after the 6(th) vaccine dose, no high-avidity PR1 or WT1-specific CD8(+) T cells could be detected, whereas low-avidity T cells were readily expanded. These data support the immunogenicity of PR1 and WT1 peptide vaccines. However, repeated delivery of peptides with Montanide-adjuvant and GM-CSF leads to rapid loss of high-avidity peptide-specific CD8(+) T cells. These results may offer an explanation for the lack of correlation between immune and clinical responses observed in a number of clinical trials of peptide vaccination. New approaches are needed to induce long-term high-avidity memory responses against leukemia antigens.

Minimal Residual Disease following Allogeneic Hematopoietic Stem Cell Transplantation

Minimal residual disease (MRD), both before and after transplantation, is a clinically important yet relatively poorly defined aspect of allogeneic hematopoietic stem cell transplantation (alloHSCT). The clinical relevance of MRD in the context of alloHSCT has been demonstrated by its association with the development of clinical relapse. However, with the possible exception of chronic myeloid leukemia (CML), the specific techniques, timing, frequency, and clinical utility, relative to improvement in patient outcomes, for monitoring MRD in the setting of alloHSCT has yet to be clearly defined. A concise overview of monitoring techniques for detecting MRD, as well as treatment strategies and biological and clinical research initiatives for MRD suggested by the National Cancer Institute First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation, is covered in this article.

Wt1 Peptide Vaccine, one of the most Promising Cancer Vaccines: Its Present Status and the Future Prospects

Wt1 Peptide Vaccine, one of the most Promising Cancer Vaccines: Its Present Status and the Future Prospects

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.

Clinical trials of WT1 peptide vaccine to gynecologic malignancies that are resistant to conventional therapies.

5081 Background: Wilms' tumor gene WT1, which has an oncogenic function, is expressed in various kinds of hematological malignancies and solid cancers. The WT1 protein is highly immunogenic and is considered to be a promising target of cancer treatment. On the clinical trials of WT1 peptide vaccine, the decrease of leukemic cells, shrinkage of solid tumor and long-term dormancy of malignancies are reported. We intend to report the clinical trials of WT1 peptide vaccine to gynecologic malignancies which are resistant to conventional therapies. Methods: Twenty one cases of gynecologic malignancy (12 cases of ovarian cancer, 4 of cervical cancer, 1 of endometrial cancer and 4 cases of other malignant tumor), which are resistant to first-/second-line therapy, were included to the trial. After obtainment of informed consent, modified WT1235 type peptide is injected intracutaneously to the HLA-A*2402- positive patients, whose tumor tissues expressed WT1 protein. The injection was repeated weekly to total of 12 times. Immune response to WT1 peptide was monitored with delayed type hypersensitivity (DTH). Side effect due tothe injection was also monitored. The antitumor effect was assessed based on Response Evaluation Criteria in Solid Tumors (RECIST). If any clinical effect is observed, the injection of the vaccine was continued biweekly. Results: In 15 (71%) of 21 patients, the 12 times of injection are completed. In remaining 6 cases, injection had been stopped due to worsening of performance status. Only 1 patient showed grade 3 vesicle at the injection site, however, no patient was cancelled the injection due to severe side effect. The SD status in RECIST criteria is observed in 5 (24%) patients and the injection is continued for these 5 patients, with the maximum of 29 times. Patients who is positive to DTH reaction during therapy, showed significantly longer overall survival than patients who are negative to DTH through the treatment. Conclusions: It was considered that the induction of WT1-specific immunological responses contributed to the occurrence of clinical responses. Some patients reached long-term dormancy of the tumor. We recognized the WT1 peptide vaccine is a new strategy for gynecologic malignancies. No significant financial relationships to disclose.