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Dysregulated phosphorylation in cancer cells can lead to the generation of novel tumor antigens modified by phosphorylation, which can be targeted for immunotherapy. Using immobilized metal affinity chromatography and mass spectrometry, we have previously identified phosphorylated peptides presented by MHC molecules on cancer cells. Several of the phosphopeptides are presented by melanoma but not by cultured melanocytes. A significant number of the source proteins for these phosphopeptides are involved in cellular processes known to promote tumor growth and metastasis. These antigens are particularly appealing as immunotherapeutic targets because mutations in or downregulation of the source proteins as a means of immune escape may compromise malignancy. We have also shown that the presence of the phosphate creates new antigenic targets for T cell recognition by creating direct interactions with the T cell receptor leading to specific T cell recognition of the phosphate and peptide sequence. Additionally, the presence of the phosphate can improve HLA-A2 binding affinities of some of the phosphopeptides with suboptimal anchor residues. A phosphopeptide derived from β-catenin was of particular interest as an immunotherapeutic target because its source protein has been shown to cooperate with activated N-Ras in immortalizing melanocytes. β-catenin/LEF signaling can also induce cell growth, motility, and transendothelial migration in melanoma cells. Additionally, nuclear expression of phosphorylated β-catenin has been shown to be associated with poor outcomes in melanoma patients. When pulsed onto bone marrow-derived dendritic cells and used to immunize HLA-A2 transgenic mice, we found that βcatpS3330–39 was only weakly immunogenic. We modified βcatpS3330–39 by replacing a suboptimal Ala at the P10 anchor position with a Val (βcatpS33(V)30–39). This modification enhanced the HLA-A2 binding affinity by 10 fold and substantially enhanced immunogenicity. Intravenous immunization of HLA-A2 transgenic mice with βcatpS33(V)30–39 generated high avidity CD8+ T cells that were highly crossreactive to the natural βcatpS3330–39 phosphopeptide. These high avidity βcatpS33-specific T cells also recognized endogenously processed βcatpS3330–39 on several melanoma cells. However the magnitude of the response to βcatpS33(V)30–39 was considerably less than the response induced using another antigen with a comparable HLA-A2 binding affinity. A 10-fold increase in antigen dose resulted in a sizeable increase in the magnitude of the response and a modest decrease in the average avidity for βcatpS3330–39. The level of crossreactivity on βcatpS3330–39 was not adversely impacted by increasing antigen dose. Our results demonstrate that βcatpS33(V)30–39 is more efficient than βcatpS3330–39 at inducing a βcatpS3330–39–specific immune response. The use of βcatpS33(V)30–39 for peptide vaccination is therefore most likely to be more advantageous for the efficient control of tumors. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B14