The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex ( IntroductionDespite major advances in chemotherapy and hematopoietic stem cell transplantation, many patients with leukemia will relapse because of reemergence of tumor. However, complete molecular remissions have been achieved by infusion of donor lymphocytes into patients, demonstrating that reactive T cells are able to eradicate leukemic cells in vivo. 1 Recently, various leukemia-associated antigens that are recognized by cytotoxic T lymphocytes (CTLs) in the context of major histocompatibility complex (MHC) class I have been identified. 2 One promising candidate is the Wilms tumor antigen, WT1, which is expressed by the majority of acute leukemias (both acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML)), blast crisis of chronic myeloid leukemia (CML), and many solid tumors, including those of lung, breast, and colon. [3][4][5][6] The finding that blockade of WT1 function in primary leukemic cells significantly slows cell growth in vitro 7 additionally suggests that WT1 is of critical importance to the tumor phenotype and that tumor escape by simple WT1 down-modulation is unlikely to occur. In healthy adults, despite ubiquitous expression during embryogenesis, WT1 expression is limited to renal podocytes, gonadal cells, and a low frequency of hematopoietic precursor CD34 ϩ cells, 4,[8][9][10][11] where it is expressed at significantly lower levels than those described in tumors (10-to 100-fold). 4 These features indicate that WT1 could be a useful target for therapeutic vaccination.Antibodies to WT1 12-14 and WT1-peptide specific CD8 ϩ T cells [15][16][17] have been detected in AML and CML patients, confirming that tolerance is incomplete. Furthermore, the presence of WT1-specific T cells correlates with graft-versus-leukemia (GvL) effects after allogeneic stem cell transplantation in ALL patients, 18 implying that expansion of the WT1-responsive repertoire in leukemic patients may help to mediate tumor clearance.Facilitating the study of WT1-specific immunotherapeutic approaches, several MHC class I-restricted peptides have been identified, including the HLA-A*0201-restricted WT1.126 (WT1 [126][127][128][129][130][131][132][133][134] ; RMFPNAPYL) and the HLA-A*0201 and A*24-restricted WT1.235 (WT1 [235][236][237][238][239][240][241][242][243] ; CMTWNQMNL) epitopes. 19-21 T cells with specificity for WT1.126 or WT1.235 have been isolated from the peripheral blood of HLA-A2 ϩ leukemic patients, 15,22,23 although evaluation of lytic ability has been limited to HLA-A2 ϩ WT1.126-specific cells, which were shown to lyse tumor cell lines. 22 Recently, a novel HLA-A2-binding epitope, WT1.37 (WT1 37-45 ; VLDFAPPGA), was identified. 24 High-avidity T cells with WT1.37-specificity were expanded An Inside Blood analysis of this article appear...

For long-term attack on tumor cells in patients with prostate cancer, induction of cytolytic T cells is desirable. Several lineage-specific target proteins are known and algorithms have identified candidate MHC class I-binding peptides, particularly for HLA-A*0201. We have designed tolerance-breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to candidate tumor-derived peptide sequences. Using three separate peptide sequences from prostate-specific membrane antigen (PSMA) (peptides PSMA 27 , PSMA 663 , and PSMA 711 ), this vaccine design induced high levels of CD8 1 T cells against each peptide in a HLA-A Ã 0201 preclinical model. In contrast, the full-length PSMA sequence containing all three epitopes was poorly immunogenic. Induced T cells were cytotoxic against peptide-loaded tumor cells, but only those against PSMA 27 or PSMA 663 peptides, and not PSMA 711 , were able to kill tumor cells expressing endogenous PSMA. Cytotoxicity was also evident in vivo. The preclinical model provides a powerful tool for generating CD8 1 T cells able to predict whether target cells can process and present peptides, essential for planning peptide vaccine-based clinical trials.