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.