Dendritic cells pulsed with multifunctional Wilms' tumor 1 (WT1) peptides combined with multiagent chemotherapy modulate the tumor microenvironment and enable conversion surgery in pancreatic cancer.

BACKGROUND: We aimed to develop a chemoimmunotherapy regimen consisting of a novel Wilms' tumor 1 (WT1) peptide-pulsed dendritic cell (WT1-DC) vaccine and multiagent chemotherapy and to investigate the safety, clinical outcomes, and WT1-specific immune responses of patients with unresectable advanced pancreatic ductal adenocarcinoma (UR-PDAC) who received this treatment. METHODS: Patients with UR-PDAC with stage III disease (locally advanced (LA-PDAC; n=6)), stage IV disease (metastatic (M-PDAC; n=3)), or recurrent disease after surgery (n=1) were enrolled in this phase I study. The patients received one cycle of nab-paclitaxel plus gemcitabine alone followed by 15 doses of the WT1-DC vaccine independent of chemotherapy. The novel WT1 peptide cocktail was composed of a multifunctional helper peptide specific for major histocompatibility complex class II, human leukocyte antigen (HLA)-A*02:01, or HLA-A*02:06 and a killer peptide specific for HLA-A*24:02. RESULTS: The chemoimmunotherapy regimen was well tolerated. In the nine patients for whom a prognostic analysis was feasible, the clinical outcomes of long-term WT1 peptide-specific delayed-type hypersensitivity (WT1-DTH)-positive patients (n=4) were significantly superior to those of short-term WT1-DTH-positive patients (n=5). During chemoimmunotherapy, eight patients were deemed eligible for conversion surgery and underwent R0 resection (four patients with LA-PDAC, one patient with M-PDAC, and one recurrence) or R1 resection (one patient with M-PDAC), and one patient with LA-PDAC was determined to be unresectable. Long-term WT1-DTH positivity was observed in three of the four patients with R0-resected LA-PDAC. These three patients exhibited notable infiltration of T cells and programmed cell death protein-1+ cells within the pancreatic tumor microenvironment (TME). All patients with long-term WT1-DTH positivity were alive for at least 4.5 years after starting therapy. In patients with long-term WT1-DTH positivity, the percentage of WT1-specific circulating CD4+ or CD8+ T cells that produced IFN-γ or TNF-α was significantly greater than that in patients with short-term WT1-DTH positivity after two vaccinations. Moreover, after 12 vaccinations, the percentages of both circulating regulatory T cells and myeloid-derived suppressor cells were significantly lower in patients with long-term WT1-DTH-positive PDAC than in short-term WT1-DTH-positive patients. CONCLUSIONS: Potent activation of WT1-specific immune responses through a combination chemoimmunotherapy regimen including the WT1-DC vaccine in patients with UR-PDAC may modulate the TME and enable conversion surgery, resulting in clinical benefits (Online supplemental file 1). TRIAL REGISTRATION NUMBER: jRCTc030190195.

Enhancing the immunogenicity of Wilms tumor 1 epitope in mesothelioma cells with immunoproteasome inhibitors.

The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit ß5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes.

Enzyme-Linked Immunosorbent Assay for Antibodies Against the Tumor-Associated Antigen-Derived Cytotoxic T-Lymphocyte Epitope.

Antibodies serve as crucial indicators of the immune system in clinical tests. In therapeutic cancer vaccines, IgG antibodies against target antigens are vital for immune monitoring. Additionally, assessing baseline antigen-specific immune responses before cancer vaccine administration is possible by measuring IgM and IgG antibodies against the target antigen. To this end, we have developed an enzyme-linked immunosorbent assay (ELISA) system that detects and quantifies serum levels of IgG and IgM antibodies against the WT1 cytotoxic T-lymphocyte epitope peptide. The assay immobilizes the epitope peptide in a microplate to capture antigen-specific antibodies. Here, this article presents the details of our ELISA system to detect and measure antibodies against a tumor-associated antigen-derived cytotoxic T-lymphocyte epitope with high reproducibility. Detecting these antibodies has novel significance in the context of emerging critical roles of B lineage-cells in tumor immunity.

Integration of the PD-L1 inhibitor atezolizumab and WT1/DC vaccination into standard-of-care first-line treatment for patients with epithelioid malignant pleural mesothelioma-Protocol of the Immuno-MESODEC study.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with a very poor prognosis. Recently, immune checkpoint inhibition (ICI) has taken center stage in the currently ongoing revolution that is changing standard-of-care treatment for several malignancies, including MPM. As multiple arguments and accumulating lines of evidence are in support of the existence of a therapeutic synergism between chemotherapy and immunotherapy, as well as between different classes of immunotherapeutics, we designed a multicenter, single-arm, phase I/II trial in which both programmed-death-ligand 1 (PD-L1) inhibition and dendritic cell (DC) vaccination are integrated in the first-line conventional platinum/pemetrexed-based treatment scheme for epithelioid MPM patients (Immuno-MESODEC, ClinicalTrials.gov identifier NCT05765084). Fifteen treatment-naïve patients with unresectable epithelioid subtype MPM will be treated with four 3-weekly (±3 days) chemo-immunotherapy cycles. Standard-of-care chemotherapy consisting of cisplatinum (75mg/m2) and pemetrexed (500mg/m2) will be supplemented with the anti-PD-L1 antibody atezolizumab (1200 mg) and autologous Wilms' tumor 1 mRNA-electroporated dendritic cell (WT1/DC) vaccination (8-10 x 106 cells/vaccination). Additional atezolizumab (1680 mg) doses and/or WT1/DC vaccinations (8-10 x 106 cells/vaccination) can be administered optionally following completion of the chemo-immunotherapy scheme. Follow-up of patients will last for up to 90 days after final atezolizumab administration and/or WT1/DC vaccination or 24 months after diagnosis, whichever occurs later. The trial's primary endpoints are safety and feasibility, secondary endpoints are clinical efficacy and immunogenicity. This phase I/II trial will evaluate whether addition of atezolizumab and WT1/DC vaccination to frontline standard-of-care chemotherapy for the treatment of epithelioid MPM is feasible and safe. If so, this novel combination strategy should be further investigated as a promising advanced treatment option for this hard-to-treat cancer.

Generation and evaluation of cancer binding capacity of HLA-A2-WT1 complex-targeting antibody.

Wilms' tumor (WT1), a transcription factor highly expressed in various leukemias and solid tumors, is a highly specific intracellular tumor antigen, requiring presentation through complexation with HLA-restricted peptides.. WT1-derived epitopes are able to assemble with MHC-I and thereby be recognized by T cell receptors (TCR). Identification of new targetable epitopes derived from WT1 on solid tumors is a challenge, but meaningful for the development of therapeutics that could in this way target intracellular oncogenic proteins. In this study, we developed and comprehensively describe methods to validate the formation of the complex of WT1126-134 and HLA-A2. Subsequently, we developed an antibody fragment able to recognize the extracellular complex on the surface of cancer cells. The single chain variable fragment (scFv) of an established TCR-mimic antibody, specifically recognizing the WT1-derived peptide presented by the HLA-A2 complex, was expressed, purified, and functionally validated using a T2 cell antigen presentation model. Furthermore, we evaluated the potential of the WT1-derived peptide as a targetable extracellular antigen in multiple solid tumor cell lines. Our study describes methodology for the evaluation of WT1-derived peptides as tumor-specific antigen on solid tumors, and may facilitate the selection of potential candidates for future immunotherapy targeting WT1 epitopes.

WT1-specific TCRs directed against newly identified peptides install antitumor reactivity against acute myeloid leukemia and ovarian carcinoma.

BACKGROUND: Transcription factor Wilms' tumor gene 1 (WT1) is an ideal tumor target based on its expression in a wide range of tumors, low-level expression in normal tissues and promoting role in cancer progression. In clinical trials, WT1 is targeted using peptide-based or dendritic cell-based vaccines and T-cell receptor (TCR)-based therapies. Antitumor reactivities were reported, but T-cell reactivity is hampered by self-tolerance to WT1 and limited number of WT1 peptides, which were thus far selected based on HLA peptide binding algorithms. METHODS: In this study, we have overcome both limitations by searching in the allogeneic T-cell repertoire of healthy donors for high-avidity WT1-specific T cells, specific for WT1 peptides derived from the HLA class I associated ligandome of primary leukemia and ovarian carcinoma samples. RESULTS: Using broad panels of malignant cells and healthy cell subsets, T-cell clones were selected that demonstrated potent and specific anti-WT1 T-cell reactivity against five of the eight newly identified WT1 peptides. Notably, T-cell clones for WT1 peptides previously used in clinical trials lacked reactivity against tumor cells, suggesting limited processing and presentation of these peptides. The TCR sequences of four T-cell clones were analyzed and TCR gene transfer into CD8+ T cells installed antitumor reactivity against WT1-expressing solid tumor cell lines, primary acute myeloid leukemia (AML) blasts, and ovarian carcinoma patient samples. CONCLUSIONS: Our approach resulted in a set of naturally expressed WT1 peptides and four TCRs that are promising candidates for TCR gene transfer strategies in patients with WT1-expressing tumors, including AML and ovarian carcinoma.

A phase I study of the WT2725 dosing emulsion in patients with advanced malignancies.

WT2725 is a Wilms' tumor gene 1 (WT1)-derived-oligopeptide vaccine designed to induce WT1-specific cytotoxic T-lymphocytes against WT1+ tumors in human leukocyte antigen (HLA)-A*0201+ and/or HLA-A*0206+ patients. Here, we report the results of a phase I study of WT2725. In this phase I, open-label, dose-escalation and expansion two-part study, the WT2725 dosing emulsion was administered as a monotherapy to patients with advanced malignancies known to overexpress WT1, including glioblastoma. In part 1, 44 patients were sequentially allocated to four doses: 0.3 mg (n = 5), 0.9 mg (n = 5), 3 mg (n = 6), and 9 mg (n = 28). In part 2, 18 patients were allocated to two doses: 18 mg (n = 9) and 27 mg (n = 9). No dose-limiting toxicities were observed, so the maximum tolerated dose was not reached. Median progression-free survival was 58 (95% confidence interval [CI] 56-81) days (~ 2 months) across all patients with solid tumors; median overall survival was 394 days (13.0 months) (95% CI 309-648). Overall immune-related response rate in solid tumor patients was 7.5% (95% CI 2.6-19.9); response was most prominent in the glioblastoma subgroup. Overall, 62.3% of patients were considered cytotoxic T-lymphocyte responders; the proportion increased with increasing WT2725 dosing emulsion dose. WT2725 dosing emulsion was well tolerated. Preliminary tumor response and biological marker data suggest that WT2725 dosing emulsion may exert antitumor activity in malignancies known to overexpress the WT1 protein, particularly glioblastoma, and provide a rationale for future clinical development.Trial registration: NCT01621542.

Lenalidomide improves the antitumor activity of CAR-T cells directed toward the intracellular Wilms Tumor 1 antigen.

OBJECTIVES: CAR-based immunotherapies represent a potentially curative strategy for hematological malignancies. However, there are a number of intracellular antigens that CAR-T cells are unable to target. Furthermore, CAR-T cells often suffer from insufficient expansion in part because of the immunosuppressive mechanisms. Lenalidomide (LEN), an immunomodulatory drug, can potentiate T cell functionality. Therefore, it is necessary to investigate combinatorial therapy using CAR-T cells and LEN for enhancing function. METHODS: We redirected T cells to express HLA-A*2402+-restricted-CAR capable of recognizing WT1235-243 peptide and adoptively transferred them into tumor-bearing mice to test their anti-tumor activity. Then we assessed the combinatorial efficacy using CAR-T cells and LEN in vitro and in vivo. RESULTS: Using an anti-WT1 CAR-T, we showed that LEN enhances CAR-T cell function in a concentration-dependent manner. Our data demonstrated that LEN improved the anti-tumor activity of CAR-T cells in vivo by increasing the infiltration of tumors with CD3+ and CD8+ T cells. Proteomics studies supported LEN enhanced the efficacy of CAR-T cells, including T-cell activation, mitochondrial activity and immune synapse formation. CONCLUSION: These results demonstrate that lenalidomide potentiates WT1 CAR-T activity and paves the way to evaluate the combination of LEN with CAR-T for a planned clinical trial.

Targeting intracellular WT1 in AML with a novel RMF-peptide-MHC-specific T-cell bispecific antibody.

Antibody-based immunotherapy is a promising strategy for targeting chemoresistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated by using CrossMAb and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms tumor protein (WT1) in the context of HLA-A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary acute myeloid leukemia (AML) cells was mediated in ex vivo long-term cocultures by using allogeneic (mean ± standard error of the mean [SEM] specific lysis, 67 ± 6% after 13-14 days; n = 18) or autologous, patient-derived T cells (mean ± SEM specific lysis, 54 ± 12% after 11-14 days; n = 8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean ± SEM specific lysis on days 3-4, 45.4 ± 9.0% vs 70.8 ± 8.3%; P = .015; n = 9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors exhibited a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo, and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase 1 trial in patients with relapsed/refractory AML (#NCT04580121).

Identification of mouse helper epitopes for WT1-specific CD4+ T cells.

Helper T lymphocytes (HTLs) play a central role in cancer immunity because they can not only help the induction and proliferation of cytotoxic T lymphocytes (CTLs) but also their differentiation into cytotoxic CD4+ T cells and directly kill the target cells.This study describes the identification of three novel mouse Th epitope peptides, WT135-52, WT186-102 and WT1294-312, derived from WT1 protein, which is the most potent tumor-associated antigen. Compared to immunization with WT1 CTL peptide alone, immunization with the addition of these WT1-specific Th peptides strongly induced WT1-specific CTLs, continued to maintain them, and efficiently rejected the challenge of WT1-expressing tumor cells. Importantly, the majority of WT1-specific CTLs induced by the co-immunization with WT1 CTL and the WT1-specific Th peptides were CD44+CD62L- effector memory CD8+ T cells, which played a central role in tumor rejection. Establishment of mouse models suitable for the analysis of the detailed mechanism of these functions of HTLs is very important. These results clearly showed that WT1-specific HTLs perform an essential function in WT1-specific tumor immunity. Therefore, the WT1-specific Th peptides identified here should make a major contribution to elucidation of the mutual roles of WT1-specific CTLs and HTLs in cancer immunity in in vivo mouse models.

Overexpression of Wilms tumor 1 promotes IL-1ß expression by upregulating histone acetylation in keratinocytes.

Psoriasis is a common inflammatory skin disease. Infiltration of inflammatory cells and excessive proliferation of keratinocytes are the histopathological markers of psoriasis. The transcription factor Wilms Tumor 1 (WT1) is overexpressed in several tumor types, and plays an important part in the proliferation and apoptosis of cells. Studies have found that, compared with normal skin, WT1expression in the skin lesions of patients with psoriasis are increased significantly. Knockdown of WT1 inhibited the proliferation of a human epidermal keratinocyte cell line (HaCaT cells) and promoted their apoptosis, whereas WT1 overexpression exhibited the opposite effect. WT1 was overexpressed or inhibited in HaCaT cells by transfection with the WT1 plasmid or WT1 small interferring RNA (siRNA) using Lipofectamine 2000. Transcriptome sequencing and bioinformatics analysis revealed significant differences in IL-1ß expression between the experimental group and control group. Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assays showed that expression of IL-1ß and WT1 were consistent. Subsequently, IL-1ß was demonstrated to be a target of WT1 by chromatin immunoprecipitation (ChIP)-sequencing and luciferase reporter assay. ChIP-qPCR showed that WT1 regulated IL-1ß expression by altering acetylation. Expression of WT1 mRNA was positively correlated with expression of IL-1ß mRNA in psoriatic skin lesions. Our study suggested that WT1 likely promotes psoriasis development by regulating its target gene IL-1ß, which shows high expression in psoriatic lesions and is involved in psoriasis development. These findings provide a new target for psoriasis treatment.

Non-transplantable cord blood units as a source for adoptive immunotherapy of leukaemia and a paradigm of circular economy in medicine.

Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.

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

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.

Wilms tumor gent 1 (WT1)-specific adoptive immunotherapy in hematologic diseases.

As an attractive tumor-associated antigen (TAA), Wilms tumor gene 1 (WT1) is usually overexpressed in malignant hematological diseases. In recent years, WT1-specific adoptive immunotherapy has been the "hot spot" for tumor treatment. The main immunotherapeutic techniques associated with WT1 include WT1-specific cytotoxic T lymphocytes (CTLs), vaccine, and T cell receptor (TCR) gene therapy. WT1-based adoptive immunotherapy exhibited promising anti-tumorous effect with tolerable safety. There are still many limitations needed to be improved including the weak immunogenetics of WT1, immune tolerance, and short persistence of the immune response. In this review, we summarized the progress of productive technologies and the clinical or preclinical investigations of WT1-specific immunotherapy in hematological diseases.

WT1-specific CD8 + cytotoxic T cells with the capacity for antigen-specific expansion accumulate in the bone marrow in MDS.

Wilms' tumor 1 (WT1) is a tumor-associated antigen and immunotherapy target in myelodysplastic syndrome (MDS). Further information is needed on the characteristics of WT1-specific CD8 + T cells to develop immunotherapeutic strategies for MDS. To clarify the frequency, distribution, and phenotype of WT1-specific CD8 + T cells, which occur innately in MDS patients, we analyzed paired peripheral blood (PB) and bone marrow (BM) samples from 39 patients with MDS or acute myeloid leukemia with myelodysplasia-related changes. The median frequency of WT1 tetramer-binding CD8 + T cells in the CD8 + T cell population was 0.11% in PB and 0.18% in BM. A further tetramer assay combined with mixed lymphocyte peptide culture (MLPC assay) was used to detect functional WT1-specific CD8 + T cells that could respond to the WT1 peptide. Functional WT1-specific CD8 + T cells were detected in BM in 61% of patients, which was significantly higher than in PB (23%, p = 0.001). The frequency of these cells estimated by the MLPC assay was tenfold higher in BM than in PB. The majority of WT1 tetramer-binding CD8 + T cells in BM had a unique phenotype with co-expression of CD39 and CXCR4. These findings will facilitate the development of novel immunotherapeutic strategies for MDS.

Identification of two distinct populations of WT1-specific cytotoxic T lymphocytes in co-vaccination of WT1 killer and helper peptides.

Simultaneous induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) is required for an optimal anti-tumor immune response. WT1332, a 16-mer WT1-derived helper peptide, induce HTLs in an HLA class II-restricted manner and enhance the induction of WT1-specific CTLs in vitro. However, in vivo immune reaction to WT1332 vaccination in tumor-bearing patients remained unclear. Here, a striking difference in WT1-specific T cell responses was shown between WT1 CTL + WT1 helper peptide and WT1 CTL peptide vaccines in patients with recurrent glioma. WT1-specific CTLs were more strongly induced in the patients who were immunized with WT1 CTL + WT1 helper peptide vaccine, compared to those who were immunized with WT1 CTL vaccine alone. Importantly, a clear correlation was demonstrated between WT1-specific CTL and WT1332-specific HTL responses. Interestingly, two novel distinct populations of WT1-tetramerlow WT1-TCRlow CD5low and WT1-tetramerhigh WT1-TCRhigh CD5high CTLs were dominantly detected in WT1 CTL + WT1 helper peptide vaccine. Although natural WT1 peptide-reactive CTLs in the latter population were evidently less than those in the former population, the latter population showed natural WT1 peptide-specific proliferation capacity comparable to the former population, suggesting that the latter population highly expressing CD5, a marker of resistance to activation-induced cell death, should strongly expand and persist for a long time in patients. These results demonstrated the advantage of WT1 helper peptide vaccine for the enhancement of WT1-specific CTL induction by WT1 CTL peptide vaccine.

Clinical Grade Production of Wilms' Tumor-1 Loaded Cord Blood-Derived Dendritic Cells to Prevent Relapse in Pediatric AML After Cord Blood Transplantation.

Hematopoietic cell transplantation (HCT) is a last resort, potentially curative treatment option for pediatric patients with refractory acute myeloid leukemia (AML). Cord blood transplantation (CBT) results in less relapses and less graft-versus-host disease when compared to other sources. Nevertheless, still more than half of the children die from relapses. We therefore designed a strategy to prevent relapses by inducing anti-AML immunity after CBT, using a CB-derived dendritic cell (CBDC) vaccine generated from CD34+ CB cells from the same graft. We here describe the optimization and validation of good manufacturing practice (GMP)-grade production of the CBDC vaccine. We show the feasibility of expanding low amounts of CD34+ cells in a closed bag system to sufficient DCs per patient for at least three rounds of vaccinations. The CBDCs showed upregulated costimulatory molecules after maturation and showed enhanced CCR7-dependent migration toward CCL19 in a trans-well migrations assay. CBDCs expressed Wilms' tumor 1 (WT1) protein after electroporation with WT1-mRNA, but were not as potent as CBDCs loaded with synthetic long peptides (peptivator). The WT1-peptivator loaded CBDCs were able to stimulate T-cells both in a mixed lymphocyte reaction as well as in an antigen-specific (autologous) setting. The autologous stimulated T-cells lysed not only the WT1+ cell line, but most importantly, also primary pediatric AML cells. Altogether, we provide a GMP-protocol of a highly mature CBDC vaccine, loaded with WT1 peptivator and able to stimulate autologous T-cells in an antigen-specific manner. Finally, these T-cells lysed primary pediatric AML demonstrating the competence of the CBDC vaccine strategy.

Impact of mature dendritic cells pulsed with a novel WT1 helper peptide on the induction of HLA­A2­restricted WT1­reactive CD8+ T cells.

The proliferation and activation of CD4+ T helper 1 (Th1) cells and CD8+ cytotoxic T lymphocytes (CTLs) that produce interferon­Î³ (IFN­Î³) is an essential action of effective cancer vaccines. Recently, a novel Wilms' tumor 1 (WT1) helper peptide (WT1 HP34­51; amino acid sequence, WAPVLDFAPPGASAYGSL) applicable for various human leukocyte antigen (HLA) subtypes (HLA­DR, HLA­DP and HLA­DQ) was reported to increase peptide immunogenicity; however, the function of WT1 HP34­51 remains unclear. In the present study, mature dendritic cells (mDCs) pulsed with WT1 HP34­51 (mDC/WT1 HP34­51) activated not only WT1­specific CD4+ T cells but also CD8+ T cells that produced IFN­Î³ following stimulation with immature dendritic cells (imDCs) pulsed with WT1 killer peptide (imDC/WT1 KP37­45) in an HLA­A*02:01­ or HLA­A*02:06­restricted manner. Furthermore, the activated WT1­reactive CD4+ Th1 cells were predominantly effector memory (EM) T cells. In 5 of 12 (41.7%) patients with cancer carrying the HLA­A*02:01 or HLA­A*02:06 allele, WT1­reactive CD8+ T cells stimulated with mDC/WT1 HP34­51 enhanced their levels of WT1 KP37­45­specific IFN­Î³ production, with an increase >10%. Simultaneous activation of CD4+ and CD8+ T cells occurred more often when stimulation with mDC/WT1 HP34­51 was combined with imDC/WT1 KP37­45 restimulation. These results indicated that the novel mDC/WT1 HP34­51 combination induced responses by WT1­specific EM CD4+ Th1 cells and HLA­A*02:01­ or HLA­A*02:06­restricted CD8+ CTLs, suggesting its potential as a WT1­targeting cancer vaccine.

Enhanced immune reaction resulting from co-vaccination of WT1 helper peptide assessed on PET-CT.

It has become evident that positron emission tomography/computed tomography (PET-CT) using 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) (FDG PET-CT) can detect anti-tumor immune response induced by various immunotherapies. To evaluate whether FDG PET-CT could detect anti-cancer immune response caused by cancer vaccine therapy, we performed a retrospective analysis of FDG PET-CT imaging of patients who were treated with Wilms Tumor 1 (WT1) vaccine therapy in Osaka University during July 2008 and June 2018. Increased FDG uptakes were detected in WT1-vaccinated skin and their draining lymph nodes during the repeated vaccination. While the FDG uptakes seemed to decrease with time after the cessation of WT1 peptide vaccinations, persistence of FDG uptakes for years in WT1-vaccinated skin were also observed in 2 cases who showed good clinical course. Moreover, the FDG uptakes of patients treated with the combination vaccine of WT1 specific cytotoxic T cell (CTL) and helper peptides were significantly stronger than of those treated with the WT1 CTL peptide alone. Since it is evident that the combination vaccine can induce a more robust anti-tumor immunity than can CTL peptide vaccine alone, the FDG uptakes in WT1-vaccinated skin might reflect the degree of immune response. These results suggest that PET-CT might be a good tool for prediction of anti-tumor immune response induced by WT1 vaccine therapy. Larger scale prospective studies therefore seem to be warranted.

Wilms' tumor 1 antigen immunoreactivity in epithelial ovarian cancer - diagnostic and prognostic value.

OBJECTIVES: Ovarian cancer is a heterogeneous disease, with a number of different histological subtypes with various responses to treatment. Wilms' tumor 1 (WT1) immunoreactivity is used to distinguish between OC's various subtypes. However, little is known about the protein's role as a prognostic factor. Thus, the main aim of our study was to evaluate the relationship between WT1 expression and patient overall survival (OS) and lymph node metastases. MATERIALS AND METHODS: Study group consisted of 164 women aged 22-84, diagnosed with epithelial ovarian cancer (EOC). WT1 expression in histological slides was assessed by immunohistochemistry. RESULTS: Serous tumors were the most common subtype among EOC (n = 126; 76.8%), followed by endometrioid (n = 20; 12.2%), clear-cell (n = 14; 8.5%) and mucinous cancer (n = 4; 2.4%). Of all serous EOC, WT1-positive tumors accounted for 75.6% of cases and this number was significantly higher than in other histological subtypes (p < 0.0001). Patients with lymph node metastases were more likely to have WT1-positive than WT1-negative tumors (p = 0.006). There was no significant correlation between WT1 immunoreactivity and OS across the whole study group of EOC patients (p = 0.6); however, in the group of non-serous (mucinous, endometrioid and clear-cell) EOC subjects, WT1 immunoreactivity was associated with shorter OS (p = 0.046). CONCLUSIONS: WT1 immunoreactivity may be helpful in differentiating primary epithelial serous carcinomas from non-serous ovarian cancers; however, its prognostic role in EOC is rather uncertain.