WT1 Vaccination Research Articles

CTIM-17. INDIVIDUALIZED MULTIMODAL IMMUNOTHERAPY (IMI) FOR ADULTS WITH DIFFUSE MIDLINE GLIOMA OR DIFFUSE HEMISPHERIC GLIOMA

Abstract Diffuse Midline Glioma (DMG, H3K27 mutant) and Diffuse Hemispheric Glioma (DHG, H3G34 mutant) are rare malignant CNS tumors with dismal prognosis. We retrospectively searched in our database for adults (>18y) with molecularly define DMG/DHG, treated between May 2015 and 2024 with IMI as part of first-line treatment. Amongst 157 IMI-treated malignant glioma patients, four patients fit these restrictions, pointing to scarce experience of IMI for DMG/DHG adult patients. HM (M, 29y) was diagnosed with a DMG in the pons. He received radiochemotherapy, and continued after 2x TMZm with BRAF-MEK-inhibition therapy. He received one IO-Vac® dendritic cell vaccine but showed fast progression thereafter. He died 9 months after diagnosis. GJE (F, 28y) had a DMG at the corpus callosum. She received radiochemotherapy and 6x TMZm, followed by 2x IO-Vac® and 3x maintenance immunogenic cell death (ICD) immunotherapy (NDV injections, sessions of modulated electrohyperthermia), combined with WT1 and H3K27M long-peptide vaccines. She progressed after 20 months, for which a new IO-Vac® and an H3K27M short- and long-peptide vaccine were given. She died 5 months later. Overall Survival (OS) was 26 months after initial diagnosis. JD (F, 27y) was diagnosed with a DMG in C2-C6. She received radiochemotherapy followed by IMI alone, including 2x IO-Vac®, one ICD immunotherapy, and four doses of anti-PD1. She died 17 months later, making OS 27 months after diagnosis. VJ (F, 21y) had a DHG in the left cerebrum. She received radiochemotherapy and 12 cycles of maintenance TMZ (TMZm), between which ICD immunotherapy was inserted each time. Afterwards, she received two IO-Vac®. Finally, she received ICD immunotherapy (6x) combined with checkpoint inhibitors (4x anti-CTLA4, 10x anti-PD1). Thirty months after diagnosis, she is in remission with a stable Health Utility Index of 0.72. IMI might have contributed to a better OS in three out of four DMG/DHG patients, and should be explored further as part of the treatment for these patients.

WT1 Cancer Vaccine in Advanced Pancreatic Cancer: A Systematic Review.

Advanced pancreatic cancer is one of the prominent contributors to cancer-related mortality globally. Chemotherapy, especially gemcitabine, is generally used for the treatment of advanced pancreatic cancer. Despite the treatment, the fatality rate for advanced pancreatic cancer is alarmingly high. Thus, the dire need for better treatment alternatives has drawn focus to cancer vaccinations. The Wilms tumor gene (WT1), typically associated with Wilms tumor, is found to be excessively expressed in some cancers, such as pancreatic cancer. This characteristic feature is harvested to develop cancer vaccines against WT1. This review aims to systematically summarize the clinical trials investigating the efficacy and safety of WT1 vaccines in patients with advanced pancreatic cancer. An extensive literature search was conducted on databases Medline, Web of Science, ScienceDirect, and Google Scholar using the keywords "Advanced pancreatic cancer," "Cancer vaccines," "WT1 vaccines," and "Pulsed DC vaccines," and the results were exclusively studied to construct this review. WT1 vaccines work by introducing peptides from the WT1 protein to trigger an immune response involving cytotoxic T lymphocytes via antigen-presenting cells. Upon activation, these lymphocytes induce apoptosis in cancer cells by specifically targeting those with increased WT1 levels. WT1 vaccinations, which are usually given in addition to chemotherapy, have demonstrated clinically positive results and minimal side effects. However, there are several challenges to their widespread use, such as the immunosuppressive nature of tumors and heterogeneity in expression. Despite these limitations, the risk-benefit profile of cancer vaccines is encouraging, especially for the WT1 vaccine in the treatment of advanced pancreatic cancer. Considering the fledgling status of their development, large multicentric, variables-matched, extensive analysis across diverse demographics is considered essential.

WT1 and PRAME RNA-loaded dendritic cell vaccine as maintenance therapy in de novo AML after intensive induction chemotherapy.

Intensive induction chemotherapy achieves complete remissions (CR) in >60% of patients with acute myeloid leukemia (AML) but overall survival (OS) is poor for relapsing patients not eligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Oral azacytidine may be used as maintenance treatment in AML in first remission, but can be associated with substantial side effects, and less toxic strategies should be explored. Twenty AML patients in first CR (CR1) ineligible for allo-HSCT were treated with FDC101, an autologous RNA-loaded mature dendritic cell (mDC) vaccine expressing two leukemia-associated antigens (LAAs). Each dose consisted of 2.5-5 × 106 mDCs per antigen, given weekly until week 4, at week 6, and then monthly, during the 2-year study period. Patients were followed for safety and long-term survival. Treatment was well tolerated, with mild and transient injection site reactions. Eleven of 20 patients (55%) remained in CR, while 4 of 6 relapsing patients achieved CR2 after salvage therapy and underwent allo-HSCT. OS at five years was 75% (95% CI: 50-89), with 70% of patients ≥60 years of age being long-term survivors. Maintenance therapy with this DC vaccine was well tolerated in AML patients in CR1 and was accompanied by encouraging 5-year long-term survival.

Galinpepimut-S (GPS): an investigational agent for the treatment of acute myeloid leukemia

ABSTRACT Introduction: Acute myeloid leukemia (AML) is a disorder wherein clonal expansion of undifferentiated myeloid precursors results in compromised hematopoiesis and bone marrow failure. Even though numerous AML patients respond to induction chemotherapy, relapse is common and hence new therapeutic approaches are needed. Wild-type Wilms tumor gene (WT1) is greatly expressed in numerous blood disorders and so this has led to development of galinpepimut-S, a WT1 vaccine as a modality to maintain remission in patients with AML. Areas covered: We summarize and examine the structure, key features, safety, and efficacy data of galinpepimut-S (GPS) for AML. GPS has been shown to be safe and tolerable in phase 1 and phase 2 studies and is now being evaluated in a phase 3 study. Expert opinion: Given the unmet need in the treatment of relapsed and refractory AML, especially among the elderly and patients with comorbidities who are not fit enough to undergo traditional salvage treatments, GPS could potentially fill the gap for this subset of patients. Future clinical trials utilizing GPS in second complete remission 2 (CR2) compared to best available therapy in AML and in combination with other immunotherapeutic agents (like pembrolizumab) for treatment for various malignancies are underway.

IMT-03 CLINICAL TRIAL FOR NEWLY DIAGNOSED MALIGNANT GLIOMA WITH WT1-W10 VACCINATION

OBJECTWilms’ tumor 1 (WT1) peptide vaccination is considered a potentially effective therapy against malignant glioma. We conducted a Phase I/II study to investigate the safety and feasibility of novel WT1 peptide (W10) vaccination therapy for patients with newly diagnosed malignant glioma.METHODSWT1 vaccination therapy was performed for patients with malignant glioma who have undergone concurrent radiotherapy and temozolomide therapy. A mixture of WT1 peptide with inactivated pertussis whole cell vaccine was injected intradermally once a week for at least 12 weeks.RESULTSTwenty-seven patients (12 men, 15 women; median 65 years) with the following tumors were enrolled: WHO grade IV (15), WHO grade III (12). PFS and OS of glioblastoma cases were 12.7 months 21.9 months, respectively. PFS of the MGMT unmethylated group was shorter than the methylated group. Interestingly enough, overall survival in the MGMT unmethylated group was not significantly different from the methylated group. Analysis of recurrent cases after immunotherapy showed decreased expression of WT1 antigen and increased Treg. They were suggested as a cause of treatment resistance. No serious adverse events were observed except for Grade 1 erythema at the injection sites.CONCLUSIONSThis study of a novel WT1 vaccination therapy demonstrated safety and feasibility in the management of newly diagnosed malignant gliomas.

Immunohistochemical Expression of WT1 in Nasopharyngeal Carcinoma Among Filipino Patients in a Tertiary Hospital

Background. Nasopharyngeal carcinoma (NPC) is endemic in Southeast Asia and the Philippines. Novel treatments are desirable due to the high disease burden and adverse effects of existing modalities. Detection of WT1 expression via immunohistochemistry has been reported in many tumors. Moreover, immunotherapy via WT1 peptide vaccination has shown promising results in a wide range of malignancies. No studies on WT1 expression in NPC have been published in any population. Objective. Documenting WT1 expression in NPC via immunohistochemistry may provide insight into the possibility of using WT1 vaccination for this disease. Methodology. This was a retrospective descriptive study. All newly-diagnosed cases of NPC from 2016 to 2017 with samples stored in the Department of Laboratories of the Philippine General Hospital were considered. Cases were included based on specific criteria. The tumor classification of each case was reviewed and WT1 immunohistochemistry staining was performed. Assessment of the strength of WT1 immunostaining was conducted. The results were analyzed using Chi-square test for association with fisher exact correction. Results. A total of 57 cases were included, all of which were non-keratinizing squamous cell carcinomas (NK-SCCs). Forty-nine were undifferentiated type while eight were differentiated type. The mean age was 48 years. Two thirds were male, one third were female. Seventeen of the 57 cases (29.8%) were positive for WT1 immunostaining, and all were undifferentiated type. The majority (82.32%) of the positive cases showed cytoplasmic expression. There was a significant association between tumor classification and WT1 staining. Conclusion. Similar to studies conducted in other carcinomas, a considerable subset of NPCs express WT1. This finding opens other avenues for exploration, including the feasibility of WT1 peptide vaccination as a treatment option. Further studies on the associations between WT1 and NPC are recommended.

A phase I clinical study of a cocktail vaccine of Wilms\u2019 tumor 1 (WT1) HLA class I and II peptides for recurrent malignant glioma

PurposeThe safety and clinical efficacy of WT1 human leukocyte antigen (HLA) class I peptide vaccine have been established, but the safety of a cocktail vaccine of WT1 HLA class I and II peptides has not. To verify its safety, we performed a phase I clinical trial for patients with recurrent malignant gliomas and assessed the immunological responses and survival data.Patients and methodsFourteen HLA-A*24:02-positive patients with recurrent malignant glioma (2 with grade 3, 12 with grade 4) were enrolled. Every week, the patients received alternately a vaccine containing 3 mg of WT1 HLA-A*24:02-restricted (HLA class I) peptide and a cocktail vaccine of the HLA class I peptide and one of 0.75, 1.5 or 3 mg of the WT1 HLA class II peptide. For patients who showed no significant adverse effects within 6 weeks, the WT1 vaccine was continued at 2–4-week intervals.ResultsEleven of the 14 patients completed WT1 vaccination for 6 weeks, while 3 patients dropped out earlier due to disease progression. All patients showed grade I level of skin disorders at the injection sites. No grade III/IV toxicity or dose-limiting toxicity was observed for any dose of WT1 HLA class II peptide. Six of the 14 patients had stable disease at 6 weeks. Median OS and 1-year OS rates were 24.7 weeks and 36%, respectively.ConclusionThe safety of a cocktail vaccine of WT1 HLA class I and II peptides for malignant gliomas was verified. This vaccine is, therefore, considered promising for patients with recurrent malignant glioma.

WT1 peptide vaccine in Montanide in contrast to poly ICLC, is able to induce WT1-specific immune response with TCR clonal enrichment in myeloid leukemia

BackgroundThe optimal strategy for vaccination to induce CD8+ T cell responses against WT1 is not known.MethodsA pilot randomized study in HLA-A02+ patients to receive vaccination with WT1 in Montanide or in poly ICLC, a TLR3 agonist, to explore the novel immune adjuvant was conducted. Seven patients were randomized. Four patients received WT1 in Montanide, and three with WT1 in poly ICLC. Five patients were in morphologic remission and two had residual morphologic disease at the study entry.ResultsAll patients finished the induction phase without any major toxicity except mild transient local injection reaction. One patient on the Montanide arm developed aseptic ulceration at two vaccine sites which healed without antibiotics. Three of 4 patients on the Montanide arm had a decreased expression of WT1 after WT1 vaccination, and two of them demonstrated generation of WT1-specific cytotoxic CD8+ T cell responses with biased TCR beta chain enrichment. In contrast, no obvious WT1-specific immune responses were detected in two patients on the poly ICLC arm, nor was there clonal enrichment by TCR alpha/beta sequencing; however, these patients did also have decreased WT1 expression and remained in remission several years after the initiation of treatment.ConclusionsWT1 peptide vaccine with Montanide as an adjuvant induces detectable WT1-specific CD8+ T cell responses with clonal TCR enrichment, which may be capable of controlling leukemia recurrence in the setting of minimal residual disease. Poly ICLC may induce anti-leukemic activity in the absence of detectable WT1 specific CD8+ T cell responses.Trial registration NCT01842139, 7/3/2012 retrospectively registered; https://clinicaltrials.gov/ct2/show/NCT01842139.

Antitumor effect of oral cancer vaccine with Bifidobacterium delivering WT1 protein to gut immune system is superior to WT1 peptide vaccine

ABSTRACTDespite the revolutionary progress of immune checkpoint inhibitors (CPIs) for cancer immunotherapy, CPIs are effective only in a subset of patients. Combining CPIs and cancer vaccines to achieve better clinical outcomes is a reasonable approach since CPI enhances cancer vaccine-induced tumor-associated antigen (TAA) specific CTL. Among the various TAAs so far identified, WT1 protein is one of the most promising TAAs as a cancer vaccine target. Until now clinical trials of WT1 vaccine have demonstrated only modest clinical efficacy. These WT1 vaccines were based on peptides or dendritic cells (DCs), and there was no oral cancer vaccine. Recently, we developed a WT1 oral cancer vaccine using a recombinant Bifidobacterium displaying WT1 protein, which can efficiently deliver WT1 protein to the gut immune system, and we demonstrated that this oral cancer vaccine had a significant anti-tumor effect in a C1498-WT1 murine leukemia syngeneic tumor model. The WT1 protein displayed in this vaccine consists of about 70% of the WT1 amino acid sequence including multiple known CD4 and CD8 T-cell epitopes of WT1. In this commentary, we introduce our recent data indicating the superior anti-tumor effect of a WT1 oral cancer vaccine delivering WT1 protein to the gut immune system compared to a peptide vaccine.

WT1 Peptide Vaccine Is Able to Induce WT1-Specifc Immune Response with TCR Clonal Enrichment to Control Minimal Residual Disease in Patients with Myeloid Leukemia

Background: Immunotherapy has the potential for clinical efficacy in patients with myeloid leukemia, especially in the setting of minimal residual disease. WT-1, aberrantly expressed in both myeloid and lymphoid leukemia, is associated with adverse risk in AML. WT1 is highly antigenic and is an attractive target for immunotherapy. The optimal strategy for vaccination to induce CD8+ T cell responses against WT1 is not known.Methods: We performed a pilot randomized study of HLA-A02+ patients to receive vaccination with WT1 126-134 peptide (RMFPNAPYL) in Montanide or in poly ICLC (Hiltonol from Oncovir, a TLR3 agonist) to explore the novel immune adjuvant in patients with myeloid leukemia (NCT01842139). The vaccine was administered q 2 weeks X 6 during the induction phase followed by monthly booster vaccinations X 6 months.Enrollment: Seven patients (4 males, 3 female ages 39 to 73) were randomized. Four patients received WT1 in Montanide (3 AML, 1 CML myeloid blast phase, 2 s/p allo-SCT), and three with WT1 in poly ICLC (2 AML, one MDS RAEB2 s/p allo-SCT). Five patients were in morphologic remission (3 in CR1) and two had very low burden of residual morphologic disease at study entry.Toxicities: All patients finished the induction phase without any major toxicity except mild transient local injection reaction. One patient post allo-SCT on the Montanide arm developed transverse myelitis with evidence of bacterial meningitis following the first monthly booster vaccination. Another patient on the Montanide arm developed aseptic ulceration at the 12th vaccine site followed by inflammation at the 11th WT1 vaccine site, and persistent erythema at the 1st induction vaccine site about 4 weeks after the completion of all 12 WT1 vaccinations. The aseptic ulcers eventually healed with wound care without antibiotics.Efficacy: Three of 4 patients on the Montanide arm had decease of WT1 qRT-PCR levels after WT1 vaccination, and two of them demonstrated generation of WT1-specific cytotoxic CD8+ T cell responses with biased TCR beta chain enrichment. Three patients from who cells were available for TCR alpha and beta CDR3 sequencing had TCR clonal enrichment after WT1 vaccination. In contrast, no obvious WT1-specific immune responses were detected in 2 patients on the poly ICLC arm, nor was there clonal enrichment by TCR alpha/beta sequencing; however, these patients did have a decrease in WT1 qRT-PCR levels and remained in remission 3 years after the initiation of WT1 vaccination. Thus, WT1 peptide in poly ICLC may induce anti-leukemia immune response not detected by our current assays. The third patient on the poly ICLC arm was later found to have A0202 instead of A0201, and thus could serve as negative control. Not surprisingly, this patient did not have a decrease of WT1 qRT-PCR levels nor TCR clonal evolution during vaccination. The patient tolerated the vaccine well without injection reactions and had stable AML for 12 weeks, but the disease progressed before the first monthly WT1 vaccination.Conclusions: WT1 peptide vaccine with Montanide as an adjuvant induces WT1-specific CD8+ T cell responses with TCR clonal and specific TCR beta CDR3 enrichment, which may be capable of controlling leukemia recurrence in the setting of minimal residual disease. Future investigation to combine checkpoint inhibitors with peptide vaccination might further enhance efficacy in patients with myeloid leukemia. DisclosuresLiu:Karyopharm: Research Funding; BMS: Research Funding. Salazar:Oncovir Inc: Employment. Odenike:Suneisis: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Algeta: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Spectrum: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Geron: Research Funding; CTI/Baxter: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sanofi-Aventis: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Gajewski:Abbvie: Consultancy; Celldex: Consultancy, Research Funding; Jounce: Consultancy; Incyte: Consultancy, Research Funding; Evelo: Patents & Royalties: Patent application; BMS: Research Funding; Merck: Consultancy, Research Funding; Roche/Genentech: Consultancy, Research Funding; Bayer: Consultancy; Aduro: Patents & Royalties: Patent application.

DSP-7888, a Novel Cocktail Design of WT1 Peptide Vaccine, and Its Combinational Immunotherapy with Immune Checkpoint-Blocking Antibody Against PD-1

Background:Wilms' tumor gene 1 (WT1), broadly expressed in hematological malignancies and solid tumors, is a promising candidate for development as a cancer vaccine. WT4869, one of our WT1 vaccine candidates demonstrated early signs of clinical activity in Azacitidine (AZA)-resistant higher-risk MDS patients with a median survival (OS) of 13.0 months in a phase 1/2 study (Suzuki T, Ueda Y, Ogura M, et al. A Phase 1/2 Study of WT1 Peptide Cancer Vaccine WT4869 in Patients with Myelodysplastic Syndromes (MDS). Blood. 2015;126:2868). In this study, we report the characterization of DSP-7888 and the potential synergic effect with anti-PD-1 antibody. DSP-7888, which is a novel cocktail peptide vaccine designed to induce cytotoxic T lymphocytes (CTLs) that recognize WT1 antigen in an HLA-A*02:01, HLA-A*02:06 or HLA-A*24:02 restricted manner, has entered early clinical trials in patients with MDS and pediatric brain cancer in Japan and solid tumors in the U.S.Methods:WT1-specific T cells were measured by WT1 tetramer staining following stimulation of human peripheral blood mononuclear cells (PBMCs) with DSP-7888. In vivo CTL inducing activity of DSP-7888 was evaluated by ELISPOT assay and 51Cr-release assay after immunization of HLA-A2.1/DR1 transgenic (Tg) mice or HLA-A24.2 Tg mice. The response of induced CTLs against the WT1 epitope was measured by IFN-γ ELISA assay following co-culture with irradiated cancer cells. The frequency of PD-1 positive CTLs in spleen and tumor was analyzed by flow cytometry. The anti-tumor effect of DSP-7888 with or without anti-PD-1 antibody was evaluated using HLA-A24.2 Tg mice bearing mouse EL4 cells expressing both HLA-A24.2 and WT1.Results:HLA-A*02:01- or HLA-A*24:02-restricted and WT1-specific CTLs were induced in HLA-A2.1/DR1 Tg mice, HLA-A24.2 Tg mice and human PBMCs. Two groups of DSP-7888-induced CTLs recognized WT1 in an HLA-A*02:01-restricted manner and one in an HLA-A*24:02-restricted manner. Compared to its component vaccine without helper peptide, DSP-7888 induced a large number of WT1-specific CTLs in HLA-A2.1/DR1 Tg mice. The activity of DSP-7888-induced CTLs was relatively maintained in the environment including cancer cells. In addition, it was further activated by treatment with anti-PD-1 antibody. On the other hand, the activity of the component vaccine-induced CTLs was very low even when treated with anti-PD-1 antibody. In DSP-7888-administered tumor-bearing mice, half of WT1-specific CTLs in spleen expressed PD-1; however, in tumor more than 90% of the CTLs expressed this protein. Combination therapy of DSP-7888 and anti-PD-1 antibody showed a higher anti-tumor effect than each monotherapy alone in this model.Conclusions:In this study, DSP-7888 induced CTLs that recognize multiple WT1 epitopes. The helper peptide included in DSP-7888 enhanced the response of the CTL induction, and contributed to the lasting cytotoxic activities even in the tumor immunosuppressive environment, suggesting potential of DSP-7888 as a cancer vaccine. In addition, the in vitro treatment with anti-PD-1 antibody furthermore enhanced the activity of DSP-7888-induced CTLs. The combination of DSP-7888 and anti-PD-1 antibody may induce multiple WT1 epitope-specific CTLs and maintain the intratumoral cytotoxic activity. Further evaluations of DSP-7888 are warranted. DisclosuresGoto:Sumitomo Dainippon Pharma Co.,Ltd: Employment. Nakamura:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Suginobe:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Takasu:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Takanashi:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Ban:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Li:Sumitomo Dainippon Pharma Co.,Ltd.: Employment.

Randomized phase II study of adjuvant WT1 vaccine (SLS-001) for malignant pleural mesothelioma (MPM) after multimodality therapy.

8519Background: WT1 is an ideal candidate for a tumor selective cancer vaccine in malignancies that express WT1, such as MPM. Using native and synthetic WT1 peptide sequences, a multivalent peptide...

Wilms’ tumour antigen 1 Immunity via DNA fusion gene vaccination in haematological malignancies by intramuscular injection followed by intramuscular electroporation: a Phase II non-randomised clinical trial (WIN)

BackgroundIn the UK almost 7000 people are diagnosed with leukaemia each year, but despite continuing advances in diagnosis and treatment with new drugs, such as the tyrosine kinase inhibitors, the majority of these patients will eventually die from their disease. Until quite recently, the only treatment to offer the possibility of long-term disease-free survival was allogeneic stem cell transplantation. However, this carries a substantial risk of mortality and is available to only a minority of patients.ObjectivesThe aim of the study was to test the hypothesis that molecular and clinical responses, induced by T lymphocytes (T cells), can be predicted by increases in the number of CD8+ (cluster of differentiation 8-positive) T cells specific for the vaccine-encoded T-cell epitopes. This project also aimed to build on the established programme of deoxyribonucleic acid (DNA) fusion-gene vaccination delivered by intramuscular injection, exploiting a unique experience with electroporation, to induce durable immune responses with the aim of controlling disease by precision attack of the tumour by CD8+ T cells.MethodA non-randomised, open-label, single-dose-level Phase II clinical trial in two patient groups [chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML)] on stable doses of imatinib. Human leucocyte antigen A2-positive (HLA A2+) patients were vaccinated with two DNA vaccines: (1) p.DOM–WT1-37 (epitope sequence: VLDFAPPGA); and (2) p.DOM–WT1-126 (epitope sequence: RMFPNAPYL). The HLA A2-negative patients formed an unvaccinated control group. The sample size for the HLA A2+ group was originally determined following Simon’s optimal Phase II trial design (Simon R. Optimal two-stage designs for phase II clinical trials.Control Clin Trials1989;10:1–10). This was changed to A’Hern’s single-stage design during the course of the trial (A’Hern RP. Sample size tables for single-stage phase II designs.Stat Med2001;20:859–66), which was endorsed by the trial’s independent oversight committees.ResultsThe study included 12 patients with CML who were vaccinated and nine patients with CML who were unvaccinated as the control group. Both the vaccines and the electroporation were safe, with no new or unexpected toxicities. The evaluation adverse events of special interest (heart, bone marrow, renal) did not reveal safety concerns. TwoBCR–ABL(breakpoint cluster region–Abelson murine leukaemia viral oncogene homolog 1) responses were observed, both of which were defined as a major response, with one in each group. Two Wilms’ tumour antigen 1 (WT1) molecular responses were observed in the vaccinated group and one was observed in the control group. At an immunological level, the vaccine performed as expected.ConclusionsThe study met its primary decision-making target with one major molecular response inBCR–ABLtranscript levels. Overall, the data showed, in this clinical setting, the immunogenicity and safety of the vaccine.LimitationsThe study did not complete recruitment and there were multiple hurdles that contributed to this failure. This is disappointing given the robust induction immune responses againstWT1T-cell responses in 7 out of 10 evaluable patients.Future workEvaluation of the p.DOM–WT1 vaccines in AML remains attractive clinically, but it is unlikely to be feasible at this time. Combination of the DNA vaccine approach with strategies to expand T-cell responses with immunomodulatory antibodies is in development.Funding detailsThis project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council (MRC) and National Institute for Health Research (NIHR) partnership, and Bloodwise.

High Post-Remission WT1 Expression Is a Predictive Marker for Subsequent Molecular Relapse and Poor Survival Outcome in Adult Patients with Acute Promyelocytic Leukemia (APL)

Background: Acute promyelocytic leukemia (APL) is classified into favorable-risk group and long-term overall survival (OS) is estimated at around 80%. Relapse rate of APL is relatively lower than another acute myeloid leukemia (AML) subtypes, but we still confront relapse in 10-20% and some relapsed cases are hardly cured even after hematopoietic cell transplantation (HCT). Therefore, it is important to find out patients with high-risk of relapse and early intervention should be considered. In APL, PML-RARa RQ PCR is used as a marker for residual disease, but the marker is not useful for pre-emptive management for relapse prevention because its positivity directly indicates relapse of disease. WT1 expression is a well-known marker in AML, and the expression is higher in APL than the other AML subtypes (Cilloni et al., leukemia, 2002). We monitored WT1 decrement along the treatment courses to identify its significant role as a marker for relapse prediction.Methods: In this study, 117 APL patients with a median follow-up duration of 38.5 months (range, 9.7-81.3) from 2008 to 2014 were analyzed. APL was diagnosed by RT-PCR method for detection of PML-RARa and all patients were available with cytogenetic results. All except 5 with normal karyotype was identified with t(15;17)(q22;q21) and 33 (28.2%) showed combination of additional chromosomal abnormalities. Our treatment protocol was based on LPA99 trial using ATRA and idarubicin monotherapy (Sanz et al., Blood, 2004). In relapsed patients, we applied ATO and some high-risk patients were treated with HCT (n=3). PML-RARa and WT1 expression in the BM samples were quantified by RQ-PCR method, and we used WT1 ProfileQuant¢â kit (Ipsogen) for WT1 monitoring. We measured RQ-PCR levels at diagnosis, post-induction and each of the 3 post-consolidation chemotherapies, and 3 months interval after starting maintenance therapy. FLT3- ITD/TKD mutation was evaluated by multiplex allele-specific PCR and concomitantly analyzed. Significant cut-off level of PML-RARa and WT1 expression was calculated by ROC curve analysis. According to the level, we calculated OS, disease free survival (DFS), and cumulative incidence of relapse (CIR).Results: Hematological complete remission (CR) was identified in 117 (100.0%) patients but complete molecular remission (CMR) was identified in 68 (42.7%) after induction. Among 49 patients who failed to achieve CMR, 44 patients achieved CMR after 1st consolidation and 5 patients after 2nd consolidation. Three-year OS and EFS was 92.5% and 82.0%, and CIR rate was 14.7% (n=13). Three patients showed clonal evolution to therapy-related AML and 1 patient died in CR due to lung cancer. FLT3 -TKD and FLT3 -ITD mutation was identified in 6 (5.1%) and 25 (21.4%) patients, and PML-RARa BCR3 and BCR1 subtype was identified in 32 (27.4%) and 85 (72.6%) patients, respectively. For relapse prediction, we analyzed WT1 expression at several time points in association with CMR after induction, FLT3 -ITD/TKD mutation, BCR subtype, and hyperleukocytosis at diagnosis and during first chemotherapy. High WT1 expression (>120 copies/104ABL1) in early period (3 months) after maintenance therapy significantly predicted subsequent relapse. All paired PML-RARa RQ-PCR was not detected except one sample with early relapse after 3rd consolidation. Patients with high post-maintenance WT1 expression (n=40) showed significantly higher CIR rate (30.7% vs. 4.2%, p=0.0003) and inferior 3-year OS (86.1% vs. 97.9%, p =.0103) and DFS (62.8% vs. 94.1%, p<.0001). Multivariate analysis revealed high leukocyte counts [HR=9.2, 95%CI=2.2-38.5, p =.002], WT1 at 3 months post-maintenance [HR=8.7, 95%CI=1.9-38.9, p =.0051], and FLT3 mutation [HR=5.4, 95%CI=1.5-19.4, p =.0092] were significant factors for relapse prediction. However, even in the BCR3 or FLT3 -positive subgroup (n=48), low WT1 at post-maintenance 3 months was associated with lower CIR rate (13.4% vs. 50.8%, p<.0001) and better DFS (95.2% vs. 49.2%, p<.0001).Conclusion: High post-remission WT1 expression was a reliable marker for the prediction of subsequent relapse in APL, even when PML-RARa was not detected at 3 months post-maintenance. In this high-risk group, early intervention with ATRA±ATO, WT1 vaccination or WT1 -specific cytotoxic cell therapy may be used for relapse prevention. The role of WT1 expression needs to be validated by prospective studies in a large cohort. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IMCT-09WT1 PEPTIDE VACCINATION FOR GLIOMAS; SURVIVALS, BIOMARKERS AND RESPONSE ASSESSMENT

We have completed two clinical trials on WT1 peptide vaccination against gliomas; phase II trial for recurrent ones (Izumoto, Hashimoto et al., J Neurosurg, 2008) and phase I trial for newly diagnosed glioblastomas focusing on the safe combination with temozolomide (Hashimoto et al., Cancer Immunol Immunother, 2015). Although patients' survival data have been shown in the past SNO meetings, recently updated data for longer follow-up period will be presented. Especially, WT1 vaccination combined with Stupp regimen in the latter trial against glioblastomas produced progression free survival over 48 months in 5 of 7 patients recruited. Subsequent analyses from those trials revealed some immunological and non-immunological biomarkers on response to the vaccination. High level of WT1 protein and HLA class I expression on tumor cells were predictive of longer survival. Immunologically, increased WT1-specific T cells and WT1 antibody in peripheral blood after the vaccination were also predictive, and positive change of delayed type hypersensitivity (DTH) within 3 months from the start of vaccination was also a biomarker; those should rather be thought as “proof of concept” of this immunotherapy. Samples from 20 non-responders who required surgical intervention after vaccination revealed that tumor cells might have escaped from immunological attack by decreasing WT1 and HLA expression, by comparing with pre-vaccination samples in the same patients. On the response assessment, we reported that methionine PET was more accurate than enhancing MRI, when assuming survivals (Chiba, Hashimoto et al., J Neurosurg, 2012). Among MRI-based response assessments of RECIST, Macdonald, immune-related response criteria (irRC) and RANO, our analyses using 60 patients have revealed that RANO was the most appropriate surrogate of survivals in early phase, although more accurate criteria specific to immunotherapy on brain tumors were warranted.

Two distinct effector memory cell populations of WT1 (Wilms' tumor gene 1)-specific cytotoxic T lymphocytes in acute myeloid leukemia patients.

Wilms' tumor gene 1 (WT1) protein is a promising tumor-associated antigen for cancer immunotherapy. We have been performing WT1 peptide vaccination with good clinical responses in over 750 patients with leukemia or solid cancers. In this study, we generated single-cell gene-expression profiles of the effector memory (EM) subset of WT1-specific cytotoxic T lymphocytes (CTLs) in peripheral blood of nine acute myeloid leukemia patients treated with WT1 peptide vaccine, in order to discriminate responders (WT1 mRNA levels in peripheral blood decreased to undetectable levels, decreased but stayed at abnormal levels, were stable at undetectable levels, or remained unchanged from the initial abnormal levels more than 6 months after WT1 vaccination) from non-responders (leukemic blast cells and/or WT1 mRNA levels increased relative to the initial state within 6 months of WT1 vaccination) prior to WT1 vaccination. Cluster and principal component analyses performed using 83 genes did not discriminate between responders and non-responders prior to WT1 vaccination. However, these analyses revealed that EM subset of WT1-specific CTLs could be divided into two groups: the "activated" and "quiescent" states; in responders, EM subset of the CTLs shifted to the "quiescent" state, whereas in non-responders, those shifted to the "activated" state following WT1 vaccination. These results demonstrate for the first time the existence of two distinct EM states, each of which was characteristic of responders or non-responders, of WT1-specific CTLs in AML patients, and raises the possibility of using advanced gene-expression profile analysis to clearly discriminate between responders and non-responders prior to WT1 vaccination.

P4.06 - Long-term molecular remission with an increase of WT1-specific CTLs after cessation of WT1 peptide vaccination in a patient with CML

ABSTRACT Background: 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 patient with CML. Materials and Methods: A 51 years-old male with CML in CP had been treated with 400 mg imatinib for 2.5 years. Bcr-abl transcripts decreased transiently but gradually increased to more than 1, 000 copies thereafter. HLA-A*2402-restricted 9mer mutant 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. The vaccination was undertaken 22 times totally. The appearance of WT1-specific CTLs in PB was confirmed by evaluating the frequency of MHC/WT1 tetramer + CD8+ T cells by using mixed lymphocyte peptide culture (MLPC). Results: Although bcr-abl transcripts increased up to more than 2,000 copies after the the initiation of WT1 vaccination every 2 weeks, the transcripts have decreased to less than 500 copies by the administration of WT1 peptides every 4 weeks. After seven months from the cessation of WT1 peptide vaccination bcr-abl transcripts decreased to the level of major molecular response (MMR), which is lasting thereafter for nearly 4 years. While WT1-specific CTLs were not detected in PB before WT1 peptide vaccination, the CTLs appeared after the second vaccination and remained 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. Conclusions: The present study showed that WT1 peptide vaccination for an imatinib-pretreated CML patient is feasible and effective, which is due to the long-lasting amplification of WT1-specific CTLs with cytotoxicity against WT1-expressing leukemia cells.

IT-12 * WT1 PEPTIDE VACCINATION AGAINST GLIOMAS; COMBINATION WITH TEMOZOLOMIDE, BIOMARKERS AND IMMUNOLOGICAL RESPONSE

We report the long-term outcome on phase I study of combined WT1 peptide vaccination/temozolomide against newly diagnosed glioblastomas. Immunological biomarker and response were investigated in trials targeting newly diagnosed and recurrent cases. The survival analysis was performed on 7 patients in the phase I study. Peripheral blood mononuclear cells were obtained from 7 patients as well as 30 patients who had WT1 vaccination alone against the recurrent glioblastomas. FACS analyses were conduced on total of 37 samples; WT1-specific T cells were indentified using tetramer assay and sub-classified with CCR7 and CD45RA expression. The results were compared between responder and non-responder group. Median PFS of the 7 patients were 43.5 months and 5 patients showed OS longer than 3 years. In responder group, the frequencies of CD4 + central memory (p < 0.01), CD8 + effector memory (EM) (p < 0.01) and WT1-specific EM (p < 0.05) were higher statistically significantly before the vaccination, and those of CD4 + effector (E) (p < 0.01), CD8 + E (p < 0.05) and WT1-specific E (p < 0.01) were lower. The frequencies of WT1-specific T cell (p < 0.001) and WT1-specific EM (p < 0.001) significantly increased after vaccination. Biomarkers were identified, as well as possible immunological response that might present a proof of concept.

Abstract CT218: Wilms′ tumor gene 1 (WT1) peptide-based cancer vaccine combined with gemcitabine for patients with advanced pancreatic cancer

Abstract Wilms′ tumor gene 1 (WT1) protein is an attractive target for cancer immunotherapy. To investigate the feasibility of a combination therapy consisting of gemcitabine and WT1 peptide for patients with advanced pancreatic cancer, 32 HLA-A*24:02-positive patients were enrolled. HLA-A*24:02-restricted, modified 9-mer WT1 peptide (3 mg) emulsified with Montanide ISA51 adjuvant (WT1 vaccine) was injected intradermally biweekly. Moreover, gemcitabine (1,000 mg/m2) was administrated on days 1, 8, and 15 of a 28-day cycle. Median survival time and one-year survival rate were 8.1 months and 29%, respectively. The association between longer survival and positive delayed-type hypersensitivity (DTH) to WT1 peptide alone was statistically significant, and longer survivors featured a higher frequency of memory-phenotype WT1-specific cytotoxic T-lymphocytes (CTLs) before and during treatment. In conclusion, the combination therapy was well-tolerated and promising treatment for advanced pancreatic cancer. DTH-positivity to WT1 peptide alone and a higher frequency of memory-phenotype WT1-specific CTLs could be useful predictive markers for clinical efficacy. Citation Format: Shigeo Koido, Sumiyuki Nishida, Sadamu Homma, Yutaka Takeda, Hideo Komita, Satoshi Morita, Toshinori Ito, Soyoko Morimoto, Yoshihiro Oka, Satoru Yanagisawa, Yoichi Toyama, Masahiro Ikegami, Hiroaki Nagano, Toshifumi Ohkusa, Hisao Tajiri, Haruo Sugiyama. Wilms′ tumor gene 1 (WT1) peptide-based cancer vaccine combined with gemcitabine for patients with advanced pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT218. doi:10.1158/1538-7445.AM2014-CT218

WT1 PEPTIDE VACCINATION FOR MALIGNANT GLIOMAS AS A PART OF MULTI-MODAL THERAPY

BACKGROUND: We have been conducting clinical trials of peptide vaccination targeting WT1 gene product against malignant gliomas. Given the results of phase II trial for recurrent malignant gliomas (Izumoto, Hashimoto et al., J Neurosurg 2008), we performed recursive partitioning analyses (RPA) (Carson et al., J Clin Oncol, 2007). We have also completed phase I trial of combined temozolomide (TMZ) and WT1 vaccination for newly diagnosed glioblastomas (GBM). METHODS: After the Kaplan-Meier analyses giving median PFS and OS, 70 patients with recurrent gliomas who entered into the phase II trial were classified into RPA classes, following Carson et al. Seven patients were enrolled into phase I study for newly diagnosed GBM. They underwent combined TMZ and WT1 vaccination just after the initial RT/TMZ. In both studies, immunological response was analyzed on WT1-specific T cells induction in peripheral blood, delayed type hypersensitivity as well as intra-tumoral immune cell analysis when surgery was indicated after vaccination. In the phase II study, predictive or prognostic indicators were extensively investigated. Although we have originally used RECIST criteria for MRI evaluation, we tried to directly compare RECIST, Macdonald, RANO and irRC using datasets from 50 GBM patients in terms of survival prediction. RESULTS: Median OS in 7 classes (I to VII) obtained by RPA showed the same tendency for OS ranking, and 6 of 7 classes (class II to VII) presented longer OS as compared with Carsons. Hazard ratio calculated for WT1 vaccination was 0.372, which was better than those with other agents used in previously published phase I/II studies. Seven patients with combined TMZ and WT1 vaccination tolerated well and showed no adverse events above CTCAE grade III, except for grade III lymphocytopenia in 71.4% of patients possibly due to preceded RT/TMZ therapy. PFS ranged from 5.2 to 37.1 months (median 31.5 months). We have confirmed the induction of WT1 specific T cells in the majority of patients of both studies. Patients showed longer survival statistically, who had stronger expression of WT1 protein on the tumor cells, DTH turning to be positive and methionine uptake decreased within 3 months. An evaluation by RANO was statistically correlated best with OS among those 4 criteria. CONCLUSIONS: Multivariate analysis with RPA showed the efficacy of WT1 vaccination with some predictive indicators. The following phase I study revealed the safe combination of TMZ and WT1, and throw light on the new multi-modal strategy. SECONDARY CATEGORY: Clinical Neuro-Oncology.