Wilms tumor protein (317-327)

Purpose: 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. Experimental design: 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. Results: 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. Conclusions: These studies provide the rationale for using the WT1 CD4(+) peptides in conjunction with CD8(+) peptide epitopes to vaccinate patients with WT1-expressing cancers.

PAX2 is a transcription factor and member of the highly conserved family of paired box genes. PAX2 is aberrantly expressed in a variety of solid and hematologic malignancies. PAX2 regulates the transcription factor Wilms tumor gene 1, which is a promising target of cancer immunotherapy. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of PAX2 which could be useful for cancer immunotherapy. Thirteen potential HLA-A*0201 epitopes were predicted by a major histocompatibility complex binding algorithm (SYFPEITHI) and a proteasome cleavage algorithm (PAProC) and screened for recognition by T cells from HLA-A*02-positive cancer patients using intracellular cytokine cytometry. Epitope-specific T cells were generated from CD4CD25 regulatory T-cell-depleted peripheral blood mononuclear cell. Nine of 20 colorectal cancer patients, 1 of 13 renal cell carcinoma patients, and 2 of 17 lymphoma patients had a spontaneous CD8 T-cell response toward at least 1 of 6 PAX2 peptide pools. None of the 20 healthy subjects showed reactivity toward PAX2. PAX2.337-345 (TLPGYPPHV)-specific T cells could repeatedly be generated, which specifically lysed the PAX2 expressing colorectal tumor cell line SW480. In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*0201 restricted T-cell epitope and natural ligand of the tumor antigen PAX2. Thus, PAX2 is another embryonic transcription factor, which is of potential interest as immunotherapy target antigen.

Effective tumor vaccine may be required to induce both cytotoxic T lymphocyte (CTL) and CD4+ helper T-cell responses against tumor-associated antigens. CD4+ helper T cells that recognize HLA class II-restricted epitopes play a central role in the initiation and maintenance of antitumor immune responses. The Wilms tumor gene WT1 is overexpressed in both leukemias and solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for cancer immunotherapy. In this study, we identified a WT1 protein-derived 16-mer peptide, WT1(332)(KRYFKLSHLQMHSRKH), which was restricted with HLA-DRB1*0405, one of the most common HLA class II types in Japanese, as a helper epitope that could elicit WT1-specific CD4+ T-cell responses. We established a WT1(332)-specific CD4+ helper T-cell clone (E04.1), which could respond to both HLA-DRB1*0405-positive, WT1-expressing transformed hematopoietic cells and autologous dendritic cells pulsed with apoptosis-induced WT1-expressing cells, indicating that the WT1(332) was a naturally processed helper epitope. Stimulation of peripheral blood mononuclear cells with both the CTL epitope (WT1(235)) and the helper epitope (WT1(332)) in the presence of WT1(332)-specific TH1-type CD4+ T cell clone strikingly enhanced the induction and the functional activity of WT1(235)-specific CTLs compared with that of peripheral blood mononuclear cells with the WT1(235) alone. These results indicated that a helper epitope, WT1(332) should be useful for improvement of the efficacy of CTL epitope-based cancer vaccine targeting WT1 in the clinical setting.