WT1 Epitopes Research Articles

Mutation or loss of Wilms' tumor gene 1 (WT1) are not major reasons for immune escape in patients with AML receiving WT1 peptide vaccination

BackgroundEfficacy of cancer vaccines may be limited due to immune escape mechanisms like loss or mutation of target antigens. Here, we analyzed 10 HLA-A2 positive patients with acute myeloid leukemia (AML) for loss or mutations of the WT1 epitope or epitope flanking sequences that may abolish proper T cell recognition or epitope presentation.MethodsAll patients had been enrolled in a WT1 peptide phase II vaccination trial (NCT00153582) and ultimately progressed despite induction of a WT1 specific T cell response. Blood and bone marrow samples prior to vaccination and during progression were analyzed for mRNA expression level of WT1. Base exchanges within the epitope sequence or flanking regions (10 amino acids N- and C-terminal of the epitope) were assessed with melting point analysis and sequencing. HLA class I expression and WT1 protein expression was analyzed by flow cytometry.ResultsOnly in one patient, downregulation of WT1 mRNA by 1 log and loss of WT1 detection on protein level at time of disease progression was observed. No mutation leading to a base exchange within the epitope sequence or epitope flanking sequences could be detected in any patient. Further, no loss of HLA class I expression on leukemic blasts was observed.ConclusionDefects in antigen presentation caused by loss or mutation of WT1 or downregulation of HLA molecules are not the major basis for escape from the immune response induced by WT1 peptide vaccination.

Functional PRAME Specific T Cells Can Be Cultured From CD8+ Cells From Healthy Donors but Not From Patients at First CR: Implications for Immunotherapeutic Strategies in AML.

Abstract 4139 BackgroundTherapeutic vaccination with dendritic cells (DCs) is regarded as an option for the treatment of minimal residual disease in acute myeloid leukemia (AML). In this respect, defined epitope sequences derived from leukemia-associated antigens (LAA) are an attractive source of antigen. WT1 and PRAME are both LAA that are over-expressed in various hematological malignancies and that share a potential for the induction of cellular immune responses. Expression rates in AML of each of these LAA are high, ranging from 60 to 90%. To evaluate the feasibility of using these LAA in DC based vaccine formulations in AML, we investigated the relative cytotoxic T lymphocyte (CTL) priming efficiencies in response to WT and PRAME derived HLA-A2 binding epitopes in healthy donors (HD) and leucapheresis (LF) material or peripheral blood (PB) samples of AML patients in first complete remission (CR). MethodsCD8+ cells were isolated from peripheral blood of HLA-A2+ HD and from G-CSF stimulated HLA-A2+ patients and stimulated with peptide-loaded DCs. DCs were derived from the human HLA-A2 positive AML cell line MUTZ-3 (MUTZ3-DCs) (Santegoets, et al. Cancer Immunol Immunother. 2006). The peptides used were WT1 (126-134) and PRAME (100-108). Irradiated CD8-cells from the same donor were also added as helper cells. CD8+ cells were plated at one million per well in 8 to 30 wells per donor or patient. CD8+ cells were weekly restimulated with peptide-loaded MUTZ3-DCs. HLA-tetramer positivity (Tm+) for the WT1 and PRAME epitopes was weekly screened by flow cytometry. Wells were scored positive if more than 0.02% Tm+, viable CTLs were present for at least 2 weeks. Functionality of Tm+ cells was studied by analysis of IFN-gamma production after stimulation with the JY cell line loaded with relevant peptides or with an irrelevant control peptide. PRAME Tm+ CTLs were isolated by flow cytometric sorting and a CTL clone was derived by limiting dilution. Recognition of HLA-A2 and PRAME positive tumor cell lines and patient samples by the CTL clone was tested in a direct cytotoxicity assay and ELISPOT assay. ResultsOn the whole, frequencies of the induced peptide-specific CTLs were low as shown in the Table below. Priming efficiencies were highest for PRAME. Furthermore, both PRAME and WT1 specific T cells derived from HD produced IFN-gamma in an epitope specific fashion, confirming functionality of the primed T cells. Most notably, no PRAME specific CTLs could be cultured from the patients' blood, whereas a PRAME specific CTL clone, cultured from sorted Tm+ CTLs from HD, recognized and lysed HLA-A2 PRAME positive tumor cell lines and patient-derived AML blastsHealthy Donor or Leucapheresis ProductTm+ rates after co-culture (per no. of tested HD/patients)Total no. of Tm+ co-culturesMedian % Tm+ cells per positive culture (range)Number of restimulations at maximum of Tm +PRAMEHD3/421/500.10% (0.02-1.07)1-5WT1HD2/54/1040.07% (0.03-0.09)1-4PRAMELF0/40/44*0.00%-WT1LF1/41/440.05%4*LF vs HD, p<0.001 by Chi-Square test ConclusionsLAA-specific CD8+ T cell frequencies were generally low. Most consistently successful priming results were obtained for PRAME in HD and functional CTL were derived thereof indicating that this peptide may be an attractive candidate for vaccination strategies. However, priming of patient samples was unsuccessful, implying possible anergy induction or clonal deletion. As a consequence, active immunotherapy with PRAME-loaded DC may require combined therapy with matched allogeneic donor lymphocyte Infusion (DLI). Disclosures:No relevant conflicts of interest to declare.

The Response to Repeated Vaccination with PR1 and WT1 Peptides Admixed in Montanide Adjuvant Is Transient and Fails to Induce Sustained Anti-Leukemia Responses in Patients with Myeloid Malignancies.

Abstract 4096Poster Board III-1031We previously demonstrated the immunogenicity of a combined vaccine approach employing two leukemia-associated antigenic peptides, PR1 and WT1 (Rezvani Blood 2008). Eight patients with myeloid malignancies received one subcutaneous 0.3 mg and 0.5 mg dose each of PR1 and WT1 vaccines in Montanide adjuvant, with 100 μg of granulocyte-macrophage colony-stimulating factor (GM-CSF). CD8+ T-cell responses against PR1 or WT1 were detected in all patients as early as 1 week post-vaccination. However, responses were only sustained for 3-4 weeks. The emergence of PR1 or WT1-specific CD8+ T-cells was associated with a significant but transient reduction in minimal residual disease (MRD) as assessed by WT1 expression, suggesting a vaccine-induced anti-leukemia response. Conversely, loss of response was associated with reappearance of WT1 transcripts. We hypothesized that maintenance of sustained or at least repetitive responses may require frequent boost injections. We therefore initiated a phase 2 study of repeated vaccination with PR1 and WT1 peptides in patients with myeloid malignancies. Five patients with acute myeloid leukemia (AML) and 2 patients with myelodysplastic syndrome (MDS) were recruited to receive 6 injections at 2 week intervals of PR1 and WT1 in Montanide adjuvant, with GM-CSF as previously described. Six of 7 patients completed 6 courses of vaccination and follow-up as per protocol, to monitor toxicity and immunological responses. Responses to PR1 or WT1 vaccine were detected in all patients after only 1 dose of vaccine. However, additional boosting did not further increase the frequency of PR1 or WT1-specific CD8+ T-cell response. In 4/6 patients the vaccine-induced T-cell response was lost after the fourth dose and in all patients after the sixth dose of vaccine. To determine the functional avidity of the vaccine-induced CD8+ T-cell response, the response of CD8+ T-cells to stimulation with 2 concentrations of PR1 and WT1 peptides (0.1 and 10 μM) was measured by IC-IFN-γ staining. Vaccination led to preferential expansion of low avidity PR1 and WT1 specific CD8+ T-cell responses. Three patients (patients 4, 6 and 7) returned 3 months following the 6th dose of PR1 and WT1 peptide injections to receive a booster vaccine. Prior to vaccination we could not detect the presence of PR1 and WT1 specific CD8+ T-cells by direct ex-vivo tetramer and IC-IFN-γ assay or with 1-week cultured IFN-γ ELISPOT assay, suggesting that vaccination with PR1 and WT1 peptides in Montanide adjuvant does not induce memory CD8+ T-cell responses. This observation is in keeping with recent work in a murine model where the injection of minimal MHC class I binding peptides derived from self-antigens mixed with IFA adjuvant resulted in a transient effector CD8+ T cell response with subsequent deletion of these T cells and failure to induce CD8+ T cell memory (Bijker J Immunol 2007). This observation can be partly explained by the slow release of vaccine peptides from the IFA depot without systemic danger signals, leading to presentation of antigen in non-inflammatory lymph nodes by non-professional antigen presenting cells (APCs). An alternative explanation for the transient vaccine-induced immune response may be the lack of CD4+ T cell help. In summary these data support the immunogenicity of PR1 and WT1 peptide vaccines. However new approaches will be needed to induce long-term memory responses against leukemia antigens. To avoid tolerance induction we plan to eliminate Montanide adjuvant and use GM-CSF alone. Supported by observations that the in vivo survival of CD8+ T-effector cells against viral antigens are improved by CD4+ helper cells, we are currently attempting to induce long-lasting CD8+ T-cell responses to antigen by inducing CD8+ and CD4+ T-cell responses against class I and II epitopes of WT1 and PR1. Disclosures:No relevant conflicts of interest to declare.

Comparative Analysis of CD8+ t Cell Priming Efficiencies against a Panel of Leukemia-Associated HLA-A2 Restricted Epitopes Identifies PRAME as a Possible Vaccination Target in AML

Background Therapeutic vaccination with dendritic cells (DCs) is regarded as an option for the treatment of minimal residual disease in acute myeloid leukemia (AML). In this respect, defined epitope sequences derived from Leukemia-Associated Antigens (LAA) are an attractive source of antigen. WT1, PRAME, hTERT, and survivin are all LAA that are expressed in various hematological malignancies and that share a potential for the induction of cellular immune responses. Expression rates in AML of each of these LAA are high, ranging from 60 to 90%. To evaluate the feasibility of using these LAA in DC based vaccine formulations in AML, we investigated the relative Cytotoxic T Lymphocyte (CTL) priming efficiencies in response to a panel of LAA-derived HLA-A2 binding epitopes.Methods CD8+ cells were isolated from peripheral blood of healthy donors (HD) and stimulated with peptide-loaded DCs. DCs were derived from the human AML cell line MUTZ-3 (MUTZ3-DCs).1 The peptides used were WT1 (126–134), PRAME (100–108), hTERT (540–548), and survivin (96–104). Irradiated CD8− cells from the same donor were also added. CD8+ cells were plated at one million per well in 8 to 30 wells. Weekly CD8+ cells were restimulated with peptide-loaded MUTZ3-DCs. HLA-tetramer positivity (Tm+) for WT1, PRAME, hTERT or survivin epitopes was weekly screened by flow cytometry. Wells were scored positive if more than 0.02% Tm+, viable CTLs were present for at least 2 weeks. Functionality of Tm+ cells was studied by analysis of IFN-gamma production after stimulation with the JY cell line loaded with relevant LAA peptides or with an irrelevant control peptide (BCR-ABL).Results Percentages of the induced peptide-specific CTLs were low as shown in the table below. Priming efficiencies were highest for PRAME. Furthermore, PRAME and WT1 specific CTLs produced IFN-gamma in an epitope specific fashion, confirming functionality of the primed T cells.Number of HD that were Tm+ after co-cultureNumber of wells that were Tm+ after co-cultureMedian % Tm+ cells per positive well (range)Number of restimulations at maximum of Tm+PRAME3/428/600.10% (0.02–1.07)1–5WT12/54/1040.07% (0.03–0.09)1–4hTERT1/21/240.25%5Survivin1/12/180.17% (0.12–0.21)8Conclusions On the whole, the detected LAA-specific CD8+ T cell frequencies were low. Most consistently successful priming results were obtained for PRAME, indicating that this peptide may be an attractive candidate for inclusion in AML-specific DC-based vaccine regimens.

Leukemia-Reactive Cytotoxic CD8+ and CD4+ T-Cells Specific for Novel WT-1 Epitopes Are Generated In Vitro by Sensitization with Overlapping Pentadecapeptides (15-mers) Spanning the Wilms Tumor Protein.

The Wilms Tumor protein, WT-1 is differentially expressed in 60–80% of acute leukemias and CML. Evidence also suggests WT-1 expression in clonogenic leukemia stem cells, making WT-1 an attractive target for T-cell therapy. However, to date, only a limited number of immunogenic epitopes have been identified. To address this limitation, we employed a pool of 15-mers with 10 amino acid overlaps spanning the complete sequence of WT-1 loaded on autologous monocyte derived dendritic cells or EBV transformed BLCL to sensitize T-cells from normal donors of varied HLA genotypes. Specific 15-mers eliciting WT-1 specific responses were identified by quantitating IFNγ+ CD8+ or CD4+ T-cell responses to secondary stimulation with 15-mer subpools organized in a mapping grid with single overlaps. Phenotypes of T-cells producing IFNγ+ in response to identified specific 15-mers were determined by FACS analysis. We identified the HLA class I or II allele presenting each epitope by assessing the cytolytic activity and/or cytokine production of the pooled-peptide sensitized T-cells in response to 2° stimulation with a panel of PHA blasts loaded with the identified 15-mer, each sharing a single HLA allele with the T-cell donor. T-cells from each of 15 donors tested generated WT-1 peptide reactive T-cells in response to the WT-1 peptide pool. Of 23 epitopes identified that elicited T-cell responses, 19 were heretofore unrecognized epitopes presented by HLA A0201(N=4), A0301(N=2), A2402 (N=2), B3501(N=3), B0701(N=1), B4402(N=2), B3808(N=1), DRB, 0401(N=2), DRB10402(N=1) and DRB1 0701(N=1). Each of the 15 new epitopes presented by class I alleles elicited IFNγ+ CD8+ T-cells that were also cytotoxic against leukemic blasts sharing the restricting allele. All four epitopes presented by class II HLA alleles elicited IFNγ+ CD4+ T-cell responses, of which 2 were also cytotoxic against HLADR+ leukemia blasts expressing the restricting allele. Responses to these newly identified epitopes were comparable to or exceeded those elicited by the known immunogenic peptide epitope 126–134RMFPNAPYL presented by HLAA0201. Like RMF-specific T-cells, T-cells specific for 4 of the new epitopes tested to date do not exhibit activity against normal hematopoietic progenitor cells. Thus, by sensitizing T-cells from unselected normal donors, with autologous DCs loaded with a pool of overlapping 15-mers spanning WT-1, we have generated T-cells specific for a series of new epitopes of WT-1, each, presented by a distinct class I or II HLA allele, which also exhibit HLA-restricted cytotoxic against WT-1+ leukemic cells. Clinical trials of adoptive therapy with WT-1 specific T-cells generated by this approach are being initiated for adoptive therapy.

Peptide Epitopes from the Wilms' Tumor 1 Oncoprotein Stimulate CD4+ and CD8+ T Cells That Recognize and Kill Human Malignant Mesothelioma Tumor Cells

Wilms' tumor 1 protein (WT1), a transcription factor overexpressed in malignant mesothelioma, leukemias, and other solid tumors, is an ideal target for immunotherapy. WT1 class I peptide epitopes that were identified and shown to stimulate CD8(+) T cells are being tested as vaccine candidates in several clinical trials. The induction and maintenance of a robust memory CD8(+) cytotoxic T-cell response requires CD4(+) T-cell help. Three HLA class II peptide epitopes of WT1 with high predictive affinities to multiple HLA-DRB1 molecules were identified using the SYFPEITHI algorithm. Due to the highly polymorphic nature of the HLA class II alleles, such reactivity is critical in the development of a broadly useful therapeutic. One of the WT1 CD4(+) peptide epitopes, 122-140, comprises a previously identified CD8(+) peptide epitope (126-134). By mutating residue 126 from an arginine to a tyrosine, we embedded a synthetic immunogenic analogue CD8(+) epitope (126-134) inside the longer peptide (122-140). This analogue was previously designed to improve immunogenicity and induce a potent CD8(+) response. WT1 peptides 328-349 and 423-441 are able to stimulate a peptide-specific CD4(+) response that can recognize WT1(+) tumor cells in multiple HLA-DRB1 settings as determined by IFN-gamma enzyme-linked immunospot assays. The mutated WT1 peptide epitope 122-140 is able to induce CD4(+) and cytotoxic CD8(+) WT1-specific T-cell responses that can recognize the native WT1 epitopes on the surface of human WT1(+) cancer cells. Cross-priming experiments showed that antigen-presenting cells pulsed with either mesothelioma or leukemia tumor lysates can process and present each of the CD4(+) peptides identified. These studies provide the rationale for using the WT1 CD4(+) peptides in conjunction with CD8(+) peptide epitopes to vaccinate patients with WT1-expressing cancers.

Dendritic Cell Immunization Induces Nonprotective WT1-specific CTL Responses in Mouse

In the present article we describe the immunogenicity in the mouse of 2 epitopes from the tumor-associated antigen Wilms tumor 1 antigen (WT1). The newly described K-restricted pWT330 epitope stimulates high-avidity allo-major histocompatibility complex restricted cytotoxic T lymphocyte (CTL) capable of killing WT1-expressing tumor cell lines. The epitope pWT126 has been previously described as a D-restricted CTL epitope. Both epitopes are weakly immunogenic as immunization with incomplete Freund adjuvant induced poor CTL responses. In contrast, when coated onto dendritic cells (DCs) both peptides readily induced CTL responses. However, these peptide-specific CTL were of low avidity and unable to recognize WT1-expressing tumor cells in vitro and to protect against tumor challenge in vivo. In contrast, vaccination with DCs coated with peptides derived from the nonself antigen ovalbumin (OVA) induced CTL that recognized OVA-expressing tumor cells and protected against tumor growth in vivo. These data show that although DC vaccination readily stimulated CTL against WT1 peptides, these CTL did not display antitumor activity in vitro and in vivo. This suggests that tolerance to the 2 WT1 epitopes interferes with the generation of protective CTL immunity in mice.

Cytotoxic CD8 T Cell Immune Responses to Wilms Tumor Protein (WT-1) Characterizes Immunosuppression-Responsive Myelodysplasia (MDS).

Clinical observations and experimental evidence link bone marrow failure in myelodysplastic syndrome (MDS) with a T cell-dominated immunologically-mediated pathophysiology in some patients. Indeed, among 129 patients treated with immunosuppression at NIH, trisomy 8 was associated with hematologic response to immunosuppression. We have demonstrated that trisomy 8 patients have oligoclonal CD8 T cell expansion, as determined by increased proportions of one or more T cell receptor (TCR) V beta subfamilies (Sloand et al. Blood 106:841; 2005) which had cytotoxic activity toward trisomy 8 cell progenitors. Here we examine the response of cytotoxic T cells to cyclin D1 and Wilms tumor protein (WT-1); these candidate autoantigens had earlier been observed to be over-expressed in trisomy 8 CD34 cell in our microarray analysis (Chen et al. Blood 104:4210, 2004 ). Bone marrow mononuclear cells from 19 trisomy 8 patients (defined by cytogenetics and FISH) had significantly increased levels of WT-1 mRNA and protein expression compared to normal CD34 cells by real time PCR and immunoblot, respectively (p<0.001). When patient and control CD8 cells were cultured in the presence of WT-1- and cyclin D1-loaded HLA-A2-restricted antigen-presenting cells (using three different HLA-A0201- restricted epitopes of WT-1 and four different epitopes for CD1), eight patients' CD8 cell tested positive for interferon gamma, indicating a cytotoxic response. Staining with labeled MHC class I A2-restricted tetramers used to measure WT-1-specific cells showed concordant results in all eight patients. Tetramer staining was increased in 12 of 14 trisomy 8 patients examined. Cytotoxic T cells specific for WT-1 were present within the expanded V β subfamilies previously found to suppress trisomy 8 cell proliferation in vitro. CD8 cells from another eight MDS patients without trisomy 8 but who had responded to immunosuppression, sampled prior to treatment, also demonstrated WT1-specific cytotoxic activity, but only two of 18 non-responders showed measurable levels of WT-1-specific cytotoxic T cells (entire treated cohort seen in Fig 1). We infer from these data an autoimmune pathophysiology for trisomy 8 MDS with: 1) over-expression of WT-1 by the aneuploid clone; 2) a specific cytotoxic CD8 T cell immune response to WT-1; 3) subsequent cytotoxic T cell marrow suppression by apoptosis, either by cross reactivity or a bystander effect; and 4) improvement in hematopoiesis after immunosuppression. Other MDS which is responsive to immunosuppression may also be mediated by WT1. [Display omitted]

Identification of Novel MHC Class I and Class II Epitopes of WT1 Using a Peptide Library Screen.

Although several HLA-A*0201-restricted immunodominant peptides from the leukemia-restricted protein WT-1 are characterized, T cell responses to peptide sequences binding to other common class I and II epitopes of WT-1 remain almost completely unexplored. A more comprehensive definition of the WT-1 antigen would extend peptide antigen vaccines to individuals lacking HLA-A*0201 and *2402 and improve vaccine potency by recruiting both CD4+ and CD8+ T cell responses. Here we used a WT1 peptide library to identify WT-1 peptide sequences inducing CD4+ and CD8+ T cell responses in normal individuals and patients with AML and other myeloid leukemias. Six cases were studied. The library consisted of 110 15mer peptides overlapping by 11aa covering the entire WT-1 protein in 21 pools. Monocytes were isolated by plastic adherence and pulsed with peptide pools for 3 hours. Autologous CD8+ and CD4+ T cells were then added. Pools of peptides were prepared in such a way that each peptide was represented in two different peptide pools, allowing the identification of the respective peptide by responses in the two corresponding pools. Cells were harvested for RNA extraction and reverse transcription. Real time PCR (RQ-PCR) was used to identify peptide-specific induction of IFN-γ and IL-2 in CD8+ and CD4+ T cells. The SYFPEITHI binding motif software was then used to predict the probable HLA restriction for the candidate epitopes. To confirm candidate peptide immunogenecity and HLA restriction, selected peptides were synthesized and tested individually. In addition to the known HLA-A*0201 peptides WT37, WT126, WT187 and WT235 we identified 20 new MHC class I and II epitopes of WT1. Four were restricted by more than one HLA allele, demonstrating the promiscuity of epitope binding. One epitope (VPGVAPTLV) was restricted to HLA-A*0201 and HLA-B*5101. One epitope (SGQFTGTAGACRYGP) was restricted by a class I HLA allele, namely HLA-*6801 and a class II HLA allele, DR*1501. Two epitopes (YGPFGPPPPSQASGQ and QKKFARSDELVRHHN) were restricted by multiple MHC class II alleles. The proliferative response of CD4+ and CD8+ T cells to candidate peptides was confirmed using CFSE labeling. We now plan to characterize the antileukemic effects of CD4+ and CD8+ T cells induced by these peptides with a view to designing broad-spectrum vaccines inducing leukemia-reactive T cells across a wide range of HLA types.

Identification of novel WT1 epitopes by sensitization of human T cells with overlapping pentadecapeptides spanning the WT1 protein for the targeted adoptive immunotherapy of leukemia

Identification of novel WT1 epitopes by sensitization of human T cells with overlapping pentadecapeptides spanning the WT1 protein for the targeted adoptive immunotherapy of leukemia

T Cell Responses Directed Against HLA A*0201-Restricted Epitopes Derived from WT1 Are Readily Identifiable in Patients with AML and CML, but Not in Patients with ALL.

The WT1 gene contributes to leukemogenesis and all adult ALL, AML and CML express WT1 RNA by quantitative real-time reverse transcription polymerase chain reaction (qPCR) techniques. WT1 may therefore be a useful antigenic target for immunotherapy. Four HLA-A*0201-restricted WT1 T cell epitopes have been identified: Db126 (RMFPNAPYL), WH187 (SLGEQQYSV), WT37-45 (VLDFAPPGA) and WT235 (CMTWNQMNL), but only Db126 has been extensively studied in myeloid leukemias. Here, we sought CD8+ T cells directed against these epitopes in 12 healthy SCT donors, 6 patients with AML, 8 with, CML and 6 with ALL prior to SCT. All patients tested with myeloid or lymphoid leukemias expressed MHC class I, B7.1 and WT1. To detect very low frequencies of WT1-specific CD8+ T cells, we used qPCR for interferon-g (IFN-g) mRNA. After isolation, 106 CD8+ T cells were stimulated with C1R-A2 cells (MHC class I-defective LCL cells transfected with HLA-A*0201) loaded with test peptides at concentrations of 0.1, 1 and 10 mM to determine their functional avidity. CD8+ T cells were also stimulated with CMV pp65 (positive control) and gp100 (209-2M) (negative control) peptides. After 3 hr coculture, cells were harvested for RNA extraction and cDNA synthesis. qPCR was performed for IFN-g mRNA and normalized to copies of CD8 mRNA from the same sample. Parallel assays using tetramers demonstrated that the IFN-g copy number was linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. A positive response was defined as a threshold of 100 or more IFN-g mRNA copies/104 CD8 copies and a stimulation index (SI) of 2 or more, where SI = IFN-g mRNA copies/104 CD8 copies in peptide pulsed/unpulsed cultures. Responses to at least one WT1 peptide were detected in 5/8 CML patients, 4/6 patients with AML and 7/12 healthy donors. Notably, a response was not elicited to WT1 in any of the 6 patients with ALL, despite evidence of immune competence as shown by a normal CMV response. Five of five CML responders and 3/4 AML responders recognized 2 or more WT1 epitopes, while the 7 healthy donors recognized only one WT1 epitope. The range of IFN-g mRNA copies/104 CD8 copies was 289–13584, 418–45891 and 160–2683 for CML, AML and healthy donors respectively. WT1-specific tetramer-positive CD8+ T cells displayed both central memory (CD45RO+CD27+CD57−) and terminally differentiated effector memory phenotypes (CD45RO-CD27−CD57+). As multiple WT1-derived epitopes can be targeted simultaneously, it is likely that T cell response to WT1 is polyclonal. These results are important because the presence of memory WT1 responses in patients with myeloid leukemias and healthy individuals should favor vaccination as a means to expand immune responses to leukemia in the autologous and allogeneic transplant setting. Furthermore, the presence of CD8+ T cell responses to multiple WT1 epitopes should favor robust polyclonal immune responses to leukemia. However, failure to detect CD8+ T cell responses to WT1 in ALL patients suggests that WT1 may not be a useful antigen to target for immunotherapeutic purposes in this patient group.

Immunotargetting of the Wilms' Tumor WT1 Antigen for Dendritic Cell and B-Cell-Based Vaccination of Leukemia.

The Wilms' tumor antigen (WT1) is overexpressed in almost all leukemias and in several solid tumors. Overexpression of WT1 blocks the normal differentiation and enhances proliferation of hematopoietic progenitor cells. WT1 is also used in the detection of minimal residual disease. Using WT1-specific MHC class I tetramers, we were able to detect ex vivo low numbers of WT1-specific CD8+ T cells in the peripheral blood or bone marrow of leukemia patients, but not of healthy donors. In one particular donor we could detect up to 24% WT1 tetramer positive cells at the time of diagnosis. WT1 tetramer positive cells were present in all types of leukemia, except for CLL, and also in patients with MDS. Because WT1 plays an important role in leukemogenesis, it could serve as an antigenic target for dendritic cell-based immunotherapy. We used the mRNA electroporation strategy that allows presentation of multiple WT1 epitopes by MHC class I molecules, irrespective of the HLA haplotype. Monocyte-derived DC (Mo-DC) were electroporated with in vitro transcribed WT1 mRNA. RT-PCR and Western blot analysis showed that WT1 RNA and protein, respectively, was present for up to 5 days in WT1-electroporated DC, but not in mock- or EGFP mRNA-electroporated Mo-DC. Importantly, using a CD8+ T cell clone that secretes IFN-gamma upon recognizing the HLA-A2 immunodominant WT1126–134 epitope, we showed that WT1 mRNA-electroporated Mo-DC processed the WT1 protein via the MHC class I pathway and presented the WT1 epitope to the T cells in an HLA- and antigen-specific manner. Since Mo-DCs are a non-expandable source of antigen-presenting cells, we also used proliferating CD40-activated B (CD40-B) cells as inducers for WT1-specific T cell immunity. CD40-B cells were expanded to high numbers from a limited amount of peripheral blood and subsequently electroporated with WT1 mRNA. In T cell clone activation experiments, WT1 mRNA-electroporated CD40-B cells were as efficient as Mo-DC in presenting the WT1 epitope in a MHC class I-restricted manner. Based on these results, we are currently focusing on the in vitro (re)activation of autologous WT1-specific cytotoxic T cells of leukemia patients using WT1-loaded autologous Mo-DC or CD40-B cells and on the immunological parameters to break immune tolerance against the WT1 tumor self antigen.

Sensitization of Human T Cells with Overlapping Pentadecapeptides Spanning the Wt1 Protein Induces Expansion of Leukemocidal T Cells Specific for Both Previously Identified and Novel WT1 Epitopes.

The oncofetal protein, WT1, is differentially expressed in 60–80% of acute leukemias and CML. Previously, four peptide epitopes presented by HLA-A0201 or HLA-A2402 have been identified. We used a pool of 15-mers with 10 amino acid overlaps spanning the full sequence of WT1, loaded on autologous monocyte-derived dendritic cells or EBV-transformed BLCL to sensitize and raise T cell lines from a series of normal donors expressing or not expressing HLA-A0201 or HLA-A2402. Specific 15-mers eliciting responses were identified by quantitating IFNg+ CD8+ or CD4+ T cell responses to secondary stimulation with 15-mer subpools organized in a mapping grid with single overlaps. Responses against targets loaded with the identified stimulating 15-mer and sharing single HLA class I or II alleles permitted identification of restricting HLA alleles. Patients inheriting HLA-A0201 have generated CD8+ T cells in response to the complete pool which are selectively reactive against single 15-mers containing the known immunogen 252–260RMFPNAPYL. However, in certain donors, responses to 15-mer 28, containing a heretofore unreported epitope 136–144ALLPAVPSL, (which has a high predicted affinity for HLA-A0201) have been dominant. By mapping responses of T cells sensitized with the complete pool, defining the presenting HLA alleles and delineating immunogenic sequences within specific 15-mers, additional novel epitopes presented by HLA- B0801 and DRB10301 have also been defined. T cells generated by this approach also lyse both peptide-loaded targets as well as primary WT1+ leukemic cells expressing the restricting HLA allele. Thus, sensitization with pools of overlapping synthetic 15-mers spanning WT1 may selectively stimulate WT1-specific CD4+ and CD8+ T cell lines restricted by HLA alleles other than HLA- A0201 or A2402 that are leukemocidal and therefore of potential use for adoptive immunotherapy.

Escherichia Coli Expressing Recombinant WT1 Protein and Listeriolysin O Stimulate Antigen-Specific T Cells Following Vaccination in C57BL/6 Mice.

The Wilm's Tumor (WT1) gene is expressed at high levels in most types of leukemia, but also in various types of solid tumors. We tested the efficacy of recombinant Escherichia Coli (E. Coli) expressing Listeriolysin O (LLO) as an antigen delivery vector for tumor specific immunotherapy targeting WT1 gene products. C57BL/6 (B6) mice were immunised with E. Coli expressing a form of WT1 protein truncated of 70 amino acids at the N-Terminus portion, and then challenged subcutaneously with 5X106 WT1-expressing TRAMP-C tumor cells. A significant inhibitory effect on TRAMP-C tumor growth was observed in vivo in vaccinated mice, with no signs of toxicity in tissues such as liver and kidney on histopathlogic examination. Furthermore, splenocytes from immunised mice were stimulated in vitro either with peptides that have been shown to contain major histocompatibility (MHC) class I binding anchor motifs (P126: RMFPNAPYL and P330: CNKRYFKL), or with irradiated TRAMP-C tumor cells. Specific CD8-positive cytotoxic T lymphocytes (CTLs) responses against TRAMP-C tumor cells were seen in cultures stimulated with irradiated tumor cells. However, no CTL activity was found in cultures stimulated with the relevant WT1 peptides that specifically lyse TRAMP-C tumor cells or the leukemia cell line RMA-S cells pulsed with each of the peptides. These results suggest that P126 and P330 might not be naturally processed WT1 epitopes for B6 mice and supports the possibility of other B6 CTLs epitopes. We are currently using a WT1 peptide library to screen immunised animals to look for novel peptides. Immunisation with E. Coli expressing WT1 protein but not LLO did not show any effect on TRAMP-C tumor growth in vivo. In this latter group, western blot analysis revealed WT1 specific antibodies directed against the N-terminus portion of the WT1 protein in the sera of immunised mice. Altogether the results presented here showed that E. Coli expressing WT1 could stimulated antigen-specific T cells dependant on LLO co-expression and suggest possible vaccine strategies for generating CD8+ T cell responses against tumors.

WT1-targeted immunotherapy of leukaemia

Since malignant cells are derived from normal cells, many tumour-associated antigens are also expressed in normal tissues. For examples, WT1 is expressed at elevated levels in most leukaemias, but it is also expressed at reduced levels in normal CD34+ haematopoietic stem cells and in progenitor cells of other tissues. Antigen expression in normal tissues is likely to trigger immunological tolerance and thus blunt T cell responses. This could explain the observation that WT1 vaccination in mice frequently fails to stimulate high avidity cytotoxic T cell responses. In order to circumvent tolerance, we have isolated from HLA-A2-negative donors high avidity CTL specific for HLA-A2-presented peptide epitopes of WT1. These allorestricted CTL efficiently kill HLA-A2-positive leukaemia cells but not normal CD34+ haematopoietic stem cells. However, adoptive cellular therapy with allorestricted CTL could only be performed in leukaemia patients rendered tolerant to the infused CTL by prior allogeneic stem cell transplantation. In order to circumvent this limitation, we propose to exploit the TCR of allorestricted CTL as therapeutic tool. TCR gene transfer can be used to take advantage of the specificity of allorestricted CTL and transfer it to patient CTL, while avoiding the transfer of immunogenic alloantigens from the donor CTL to the patient.