T-cell responses directed against multiple HLA-A*0201-restricted epitopes derived from Wilms' tumor 1 protein in patients with leukemia and healthy donors: Identification, quantification, and characterization

University of Oxford, Oxford, England, United Kingdom
Clinical Cancer Research (Impact Factor: 8.72). 01/2006; 11(24 Pt 1):8799-807. DOI: 10.1158/1078-0432.CCR-05-1314
Source: PubMed


Antigens derived from the Wilms' tumor (WT1) protein, which is overexpressed in leukemias, are attractive targets for immunotherapy. Four HLA-A*0201-restricted WT1-derived epitopes have been identified: WT37, WT126, WT187, and WT235. We determined the natural immunogenecity of these antigens in patients with hematologic malignancies and healthy donor.
To detect very low frequencies of WT1-specific CD8+ T cells, we used quantitative reverse transcription-PCR to measure IFN-gamma mRNA production by WT1 peptide-pulsed CD8+ T cells from 12 healthy donors, 8 patients with chronic myelogenous leukemia, 6 patients with acute myelogenous leukemia, and 8 patients with acute lymphoblastic leukemia.
Responses were detected in 5 of 8 chronic myelogenous leukemia patients, 4 of 6 patients with acute myelogenous leukemia, and 7 of 12 healthy donors. No responses were detected in patients with acute lymphoblastic leukemia. The magnitude and extent of these CD8+ T-cell responses was greater in patients with myeloid leukemias than in healthy donors. Clonotypic analysis of WT1-specific CD8+ T cells directly ex vivo in one case showed that this naturally occurring population was oligoclonal. Using fluorescent peptide-MHC class I tetramers incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor, we were able to confirm the presence of high-avidity T-cell clones within the antigen-specific repertoire.
The natural occurrence of high-avidity WT1-specific CD8+ T cells in the periphery could facilitate vaccination strategies to expand immune responses against myeloid leukemias.

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    • "The relative contribution of alloreactive cells, as compared with TAAs specific T cells is not difficult to quantify, however since most TAAs are aberrantly expressed self-proteins resulting in T cells with low-affinity TCR, it is possible that the alloreactive component is more determinant for GvL. Additionally, although low frequency TAA specific T cells are transferred to patients after allo-HSCT or DLI, they do not persist (Rezvani et al., 2005, 2007), potentially due to activation-induced apoptosis (Molldrem et al., 2003), or terminally differentiated effector memory phenotype (Brenchley et al., 2003). Given the successful abrogation of GVHD in vivo, several ongoing clinical trials have replaced the time consuming in vitro allo-depletion step with in vivo allo-depletion using AP1903 for those developing GVHD in the haploidentical ( "
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    ABSTRACT: Adoptive T-cell therapy can involve donor lymphocyte infusion (DLI) after allogeneic hematopoietic stem cell transplantation, the administration of tumor infiltrating lymphocyte (TILs) expanded ex-vivo, or more recently the use of T cell receptor (TCR) or chimeric antigen receptor (CAR) redirected T cells. However cellular therapies can pose significant risks, including graft-versus-host-disease and other on and off-target effects, and therefore strategies need to be implemented to permanently reverse any sign of toxicity. A suicide gene is a genetically encoded molecule that allows selective destruction of adoptively transferred cells. Suicide gene addition to cellular therapeutic products can lead to selective ablation of gene-modified cells, preventing collateral damage to contiguous cells and/or tissues. The ‘ideal’ suicide gene would ensure the safety of gene modified cellular applications by granting irreversible elimination of ‘all’ and ‘only’ the cells responsible for the unwanted toxicity. This review presents the suicide gene safety systems reported to date, with a focus on the state-of-the-art and potential applications regarding two of the most extensively validated suicide genes, including the clinical setting: herpes-simplex-thymidine-kinase (HSV-TK) and inducible-caspase-9 (iCasp9).
    Frontiers in Pharmacology 10/2014; 5(524). DOI:10.3389/fphar.2014.00254 · 3.80 Impact Factor
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    • "Although WT1 is expressed in normal tissues during embryogenesis, immunological tolerance to WT1 is not complete: WT1-specific CTLs have been detected and expanded following exposure to the peptide both in healthy donors (Rezvani et al, 2003) and in patients with AML (Scheibenbogen et al, 2002). Recent studies have shown immune and clinical responses with peptide vaccinations against single epitopes of WT1 (Oka et al, 2004; Keilholz et al, 2009); however, naturally occurring CD8 + T cell responses against myeloid leukaemias target multiple epitopes , potentially enhancing the strength and diversity of these responses (Gannag e et al, 2005; Rezvani et al, 2005). We have identified two distinct peptide epitopes of WT1 that are presented by HLA-A0201 (A2) and function as targets for leukaemia-reactive CD8 + CTL: pWT126 and pWT235 (Bellantuono et al, 2002). "
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    ABSTRACT: Wilms' Tumour 1 (WT1) is a zinc finger transcription factor that is over-expressed in acute myeloid leukaemia (AML). Its restricted expression in normal tissues makes it a promising target for novel immunotherapies aiming to accentuate the cytotoxic T lymphocyte (CTL) response against AML. Here we report a phase I/II clinical trial of subcutaneous peptide vaccination with two separate HLA-A2-binding peptide epitopes derived from WT1, together with a pan-DR binding peptide epitope (PADRE), in Montanide adjuvant. Eight HLA-A2-positive patients with poor risk AML received five vaccination cycles at 3-weekly intervals. The three cohorts received 0·3, 0·6 and 1 mg of each peptide, respectively. In six patients, WT1-specific CTL responses were detected using enzyme-linked immunosorbent spot assays and pWT126/HLA-A*0201 tetramer staining, after ex vivo stimulation with the relevant WT1 peptides. However, re-stimulation of these WT1-specific T cells failed to elicit secondary expansion in all four patients tested, suggesting that the WT1-specific CD8(+) T cells generated following vaccination may be functionally impaired. No correlation was observed between peptide dose, cellular immune response, reduction in WT1 mRNA expression and clinical response. Larger studies are indicated to confirm these findings.
    British Journal of Haematology 02/2014; 164(3):366-75. DOI:10.1111/bjh.12637 · 4.71 Impact Factor
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    • "Also, WT1 is the most promising potential target of leukemia as it is more abundant in leukemia cells than in normal hematopoietic cells (4,5) and seems to participate in leukemogenesis and may be necessary to maintain the viability of leukemia cells (6). Furthermore, WT1 is apparently immunogenic as shown by spontaneous immune responses in leukemic patients (7,8). A growing body of evidence supports WT1 as a promising target for hematological malignancies, and various clinical trials have demonstrated the feasibility of this approach (3,9-15). "
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    ABSTRACT: A cell line with transfected Wilms' tumor protein 1 (WT1) is has been used for the preclinical evaluation of novel treatment strategies of WT1 immunotherapy for leukemia due to the lack of appropriate murine leukemia cell line with endogenous WT1. However, silencing of the transgene occurs. Regarding the effects of hypomethylating agents (HMAs) on reactivation of silenced genes, HMAs are considered to be immune enhancers. We treated murine WT1- transfected C1498 (mWT1-C1498) with increasing doses of decitabine (DAC) and azacitidine (AZA) to analyze their effects on transgene reactivation. DAC and AZA decreased the number of viable cells in a dose- or time-dependent manner. Quantification of WT1 mRNA level was analyzed by real-time polymerase chain reaction after mWT1-C1498 treated with increasing dose of HMA. DAC treatment for 48 h induced 1.4-, 14.6-, and 15.5-fold increment of WT1 mRNA level, compared to untreated sample, at 0.1, 1, and 10µM, respectively. Further increment of WT1 expression in the presence of 1 and 10µM DAC was evident at 72 h. AZA treatment also induced up-regulation of mRNA, but not to the same degree as with DAC treatment. The correlation between the incremental increases in WT1 mRNA by DAC was confirmed by Western blot and concomitant down-regulation of WT1 promoter methylation was revealed. The in vitro data show that HMA can induce reactivation of WT1 transgene and that DAC is more effective, at least in mWT1-C1498 cells, which suggests that the combination of DAC and mWT1-C1498 can be used for the development of the experimental model of HMA-combined WT1 immunotherapy targeting leukemia.
    Immune Network 04/2012; 12(2):58-65. DOI:10.4110/in.2012.12.2.58
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