9-mer WT1 Peptide Research Articles

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

Wilms Tumor Gene (WT1) Peptide–based Cancer Vaccine Combined With Gemcitabine for Patients With Advanced Pancreatic Cancer

Wilms tumor gene (WT1) protein is an attractive target for cancer immunotherapy. We aimed to investigate the feasibility of a combination therapy consisting of gemcitabine and WT1 peptide-based vaccine for patients with advanced pancreatic cancer and to make initial assessments of its clinical efficacy and immunologic response. Thirty-two HLA-A*24:02 patients with advanced pancreatic cancer were enrolled. Patients received HLA-A*24:02-restricted, modified 9-mer WT1 peptide (3 mg/body) emulsified with Montanide ISA51 adjuvant (WT1 vaccine) intradermally biweekly and gemcitabine (1000 mg/m) on days 1, 8, and 15 of a 28-day cycle. This combination therapy was well tolerated. The frequencies of grade 3-4 adverse events for this combination therapy were similar to those for gemcitabine alone. Objective response rate was 20.0% (6/30 evaluable patients). Median survival time and 1-year survival rate were 8.1 months and 29%, respectively. The association between longer survival and positive delayed-type hypersensitivity to WT1 peptide was statistically significant, and longer survivors featured a higher frequency of memory-phenotype WT1-specific cytotoxic T lymphocytes both before and after treatment. WT1 vaccine in combination with gemcitabine was well tolerated for patients with advanced pancreatic cancer. Delayed-type hypersensitivity-positivity to WT1 peptide and a higher frequency of memory-phenotype WT1-specific cytotoxic T lymphocytes could be useful prognostic markers for survival in the combination therapy with gemcitabine and WT1 vaccine. Further clinical investigation is warranted to determine the effectiveness of this combination therapy.

SY7-6 - WT1 Peptide-Based Cancer Immunotherapy

ABSTRACT In 2001, a phase I clinical study of cancer immunotherapy targeting the WT1 protein was carried out for the first time for patients with leukemia, MDS, lung cancer, or breast cancer. Patients were intradermally injected with an HLA-A*2402-restricted, natural (CMTWNQMNL) or modified (CYTWNQMNL) 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant at 0.3, 1.0, or 3.0 mg per body at 2-week intervals, with toxicity and clinical and immunological responses. Toxicity consisted only of local erythema at the WT1 vaccine injection sites. Four of eight patients with AML who had minimal residual disease for the assessment of clinical effect responded to three injections of WT1 vaccine in decrease in WT1 mRNA levels in peripheral blood. Three of the four patients are successively being injected WT1 vaccine and are in complete remission over 8 years. A new phase I/II clinical study, in which WT1 vaccination was intensified and repeated every week at a dose of 3.0 mg/body of modified WT1 peptide for three months, were started from 2004, and safety of this study was confirmed. Clinical effect of WT1 vaccination is satisfactory for glioblastoma multiforme with relapse and disease control rate is 47.7%. Five of 28 SD (stable disease) patients are long-term survivers and PFS for the four patients in 364, 312, 189, and 158 weeks, respectively. Adverse effect is only erythema at WT1 vaccine injection sites. WT1 vaccination in combination with gemcitabine for 32 patients with advanced pancreatic cancer resulted in 7 PR (21.9%), 17 SD (53.1%), 7PD (21.9%) and 1 unevaluable. Response rate and disease control rate were 21.9% and 75.0%, respectively. Two patients with PR became operable and operation was carried out. Residual tumor masses were completely resected and pathologically examined. No and minimal residual tumor cells were individually observed. Long-term survival and DTH-positivity to WT1 peptide were correlated both in glioblastoma multiforme and advanced pancreatic cancer. Randomized controlled study has been performing for advanced pancreatic cancer.

Computational Analysis on the Binding of Epitope Peptide to Human Leukocyte Antigen Class I Molecule A*2402 Subtype

Immunological response induced by small amino peptide has attracted much recent attention in the field of immunotherapy. Wilms' tumor (WT1) protein is one of the potent tumor antigens inducing immunological response in mouse and human, because WT1 is over expressed in many types of leukemia and various kinds of solid tumors. A 9-mer peptide encoded in WT1 protein (CMTWNQMNL; amino acid 235-243) is known to serve as antigenic peptide for human leukocyte antigen (HLA)-A*2402 molecule. It was reported that the replacement of the second amino residue, which is deeply responsible for the peptide binding to HLA, induced strong immunological response compared to the natural peptide. In this study, 19 kinds of single amino substitutions were introduced at position 2 of this 9-mer WT1 peptide. We performed molecular dynamics simulation on the complex of each of WT1 epitope peptides and HLA-β2 micro globulin (β2m) molecule, and subsequently estimated the binding affinity using molecular mechanics/generalized-Born surface area method combined with normal mode analysis. Our computation indicated that the peptide containing M2Y or M2W mutation showed high binding affinity to the HLA-β2m molecule as well as the natural peptide. We have also examined the role of the residue at position 2 in peptide binding to HLA-β2m. The calculation showed that van der Waals interaction between the side chain of the residue at position 2 and hydrophobic residues inside B-pocket of HLA are important. These findings will be helpful to search other potent peptides that will enhance strong immunological response specific to HLA-A*2402 molecule.

Long-Term WT1 Peptide Vaccination Over 7 Years for 3 AML Patients with Molecular Relapse

AbstractAbstract 3295WT1 (Wilms' tumor gene 1) encodes a transcription factor, which regulates various types of genes and plays an important role in cell proliferation and differentiation. WT1 is highly expressed in leukemia and various types of solid tumors. In vivo immunization of mice with MHC class I-restricted 9-mer WT1 peptide elicited WT1-specific cytotoxic T lymphocytes (CTLs) and rejected WT1-expressing leukemia cells, demonstrating that WT1 is a tumor rejection antigen. Next, identification of human HLA class I-restricted WT1 peptides was performed, and WT1 235 (235-243 aa) and WT1 126 (126-134 aa) were determined as an HLA-A*2402- or HLA-A*0201- restricted WT1 peptide, respectively, by using in vitro WT1-specific CTL induction assay.On the basis of these fundamental experiments, we performed for the first time a phase I clinical trial of WT1 peptide immunotherapy with the approval of our ethical committee. A total of 26 patients with acute myeloid leukemia (AML), myelodysplastic syndrome, breast or lung cancer were intradermally injected with a HLA-A*2402-restricted natural or modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant at 0.3, 1.0 or 3.0 mg at 2-week intervals. As shown in Table1, 12 AML patients were WT1-vaccinated and eight of the 12 AML patients were evaluable for clinical effect because of the existence of minimal residual disease (MRD) assessed by WT1 mRNA assay. Since four of the eight patients had obvious clinical response with the decrease in WT1 mRNA within three injections of WT1 vaccine, WT1 vaccination was repeated for them afterward (in the case that clinical response was not obvious within three injections of WT1 vaccine, WT1 vaccination was discontinued). However, one patient (No. 20) had re-increase in WT1 mRNA and WT1 vaccination was discontinued. The remaining three patients (Nos. 21, 23 and 24) have been repeatedly receiving WT1 vaccination (intervals were changed from 2 weeks to one month) over 7 years until now. WT1 mRNA levels, which directly reflected the amount of leukemic blast cells, were abnormally high and 102-103 copies/mg RNA in PB (normal level is < 50 copies/mg RNA) before WT1 vaccination in these three AML patients, showing that the patients were at molecular relapse with MRD. In nos. 21 and 23 patients, a decrease and increase in WT1 mRNA levels were repeated during early stages of WT1 vaccination. However, afterward, WT1 mRNA levels gradually decreased and reached normal level approximately three and a half years after the beginning of WT1 vaccination and have been maintained at normal level until now. In patient no. 24, high levels of WT1 mRNA (near 103 copies) gradually decreased by repeated WT1 vaccination and returned to normal level approximately 5 years after the beginning of WT1 vaccination although it was much delayed. Thus, these three patients had achieved continuous complete remission regardless of high risk for clinical relapse. [Display omitted] WT1-specific immune responses were evaluated by WT1 tetramer assay. Frequencies of WT1-specific cytotoxic T lymphocytes (CTLs) (WT1 tetramer+ CD8+ T cells) in the patients were already higher before WT1 vaccination, compared to those in healthy volunteers, which was a frequent phenomenon in WT1-expressing tumor-bearing patients, but the WT1-specific CTLs were a IFN-g− inactivated form. After WT1 vaccination, a IFN- g+ activated from of WT1-specific CTLs appeared and was maintained until now.No adverse effects except local erythema and eruption at the injection sites of WT1 vaccine were observed regardless of repeated long-term WT1 vaccination over 7 years. These finding indicated that WT1-specific CTLs could attack WT1-expressing leukemic blast cells but ignored WT1-expressing normal cells such as podocytes of kidney and progenitor cells of various types of organs.In conclusion, we presented here the first report of promising clinical outcome of very long-term WT1 vaccination for AML patients with high risk for relapse. WT1 peptide immunotherapy should have good prospects as a cure-oriented therapy for AML and could be the first choice of the treatments for AML patients who had still MRD after induction chemotherapy, especially for elder patients who were intolerable to intensive chemotherapy. WT1 peptide immunotherapy may be also a better choice of treatments for AML patients who have high risk for relapse but are intolerable to allogeneic hematopoietic stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.

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

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

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.

Tumor immunotherapy targeting WT1 (Wilms’ tumor gene) peptide for bone and soft-tissue sarcomas: A preliminary report

10042 Background: WT1 (Wilms’ tumor gene) is originally isolated as a tumor-suppressor gene in a subset of Wilms’ tumors, however, recent studies have indicated its oncogenic activity in various types of cancers, and demonstrated that WT1-specific cytotoxic T lymphocytes specifically kill WT1-expressing cancer cells, thus WT1 protein can be a molecular target for cancer immunotherapy as an attractive tumor rejection antigen. However, WT1 expression in human bone and soft-tissue sarcomas has been poorly understood. Methods: 1) The expression levels of WT1 gene were examined in 36 cases of various types of human bone and soft-tissue sarcomas using quantitative RT-PCR method. They included 12 MFH (11 soft-tissue, 1 bone), 9 osteosarcomas, 6 synovial sarcomas, 4 myxoid liposarcomas, 3 MPNST, one each case of angiosarcoma and clear cell sarcoma. 2) Then, we conducted a phase I/II clinical trial of tumor-specific immunotherapy targeting WT1 peptide for various kinds of cancers including 12 patients with locally advanced and/or metastatic sarcoma (bone 2, soft-tissue 10) which showed WT1 expression and HLA -A*2402. Patients were intracutaneously injected with 3.0mg of HLA- A*2402-restricted natural/or modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant weekly. After 12-times injection, clinical response was evaluated by RECIST. Results: 1) 28 (78%) out of 36 cases of various types of bone and soft-tissue sarcomas overexpressed the WT1 gene, and its expression at the protein level was confirmed by immunohistochemical analysis, suggesting a possibility of immunotherapy using WT1 peptide vaccine for sarcomas. 2) The clinical responses included 4 SD and 7 PD with a case of long-standing SD over 1.5 years. One patient discontinued treatment with patient's decision. There were no patients with objective response (PR/CR). Conclusion: The present preliminary study suggested that tumor immunotherapy using WT1 peptide vaccine can be a new treatment strategy for patients with bone and soft-tissue sarcomas. No significant financial relationships to disclose.

A Phase I/II Trial of a WT1 (Wilms' Tumor Gene) Peptide Vaccine in Patients with Solid Malignancy: Safety Assessment Based on the Phase I Data

We conducted a phase I study to investigate the safety of a weekly WT1 tumor vaccine therapy in patients with solid tumors that had been refractory to all other anti-cancer therapies. Skin-test-negative patients were intradermally injected weekly for 12 weeks with 3.0 mg of an HLA-A*2402-restricted modified 9-mer WT1 peptide emulsified in Montanide ISA51 adjuvant. We estimated the Bayesian posterior probability of the occurrence of grade 3 or 4 toxicity when receiving the weekly WT1 vaccination. This analysis provided the basis for making a decision to terminate the phase I study and switch to phase II. Moreover, we performed an exploratory assessment of the anti-tumor effects of WT1 treatment. Ten patients received 114 vaccinations with WT1 on a weekly schedule. No grade 3 or 4 toxicities were observed. Based on the Bayesian approach, it was highly likely that the probability of grade 3 or 4 toxicity was below 20% (the posterior probability = 0.914). Fifteen grade 2 and two grade 1 toxicities were observed; all of these incidents, however, were determined by the Independent Data and Safety Monitoring Committee to be unrelated to the WT1 treatment. One patient exhibited a partial response; five additional patients had stable disease while receiving weekly WT1 treatment. This paper confirms that the potential toxicities of the treatment schedule of weekly WT1 vaccination are acceptable and suggested a potential anti-tumor effect. Consequently, we validated the decision to continue to the phase II trial.

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

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