Aberrant phosphorylation in cancer cells results in the selective presentation of phosphorylated peptides by MHC molecules. Peptides bound to HLA-A02:01 demonstrate greater stability compared to their nonphosphorylated forms, which may enhance immune recognition. However, it remains uncertain if phosphopeptides displayed by other common alleles share similar structural or immunogenic traits. To investigate this, we analyzed the identity, structure, and immunogenic potential of phosphopeptides presented by HLA-A03:01, -A11:01, -C07:01, and -C*07:02. We performed immunoprecipitation to isolate peptide-MHC complexes from 10 healthy and tumor tissue samples and analyzed them via mass spectrometry. The resulting dataset was merged with publicly available immunopeptidomics data to form a curated set of phosphopeptides from 20 different healthy and cancerous tissue types. Selected phosphopeptides were assessed for biochemical characteristics using in vitro binding assays and computational docking, and their ability to elicit T cell responses was tested through multimer binding and cytokine analysis in T cells from healthy donors. We found phosphopeptides presented by HLA-A03:01, -A11:01, -C07:01, and -C07:02 across multiple tumor types, especially leukemias and lymphomas, but not in normal tissues. These peptides originated from genes critical for tumor cell survival. Phosphopeptides generally bound HLA-A03:01 as well as or worse than their unmodified counterparts, while HLA-C07:01 selectively presented phosphorylated peptides but rarely their unmodified forms. Binding to HLA-C07:01 relied on interactions in the B-pocket, which were missing in HLA-C07:02. T cells recognizing HLA-A02:01 and -A11:01 phosphopeptides were detectable in autologous settings even in the presence of the nonphosphorylated peptide, whereas HLA-A03:01 and -C07:01 phosphopeptides required allogeneic T cells for T cell activation. Phosphopeptides presented on multiple alleles that are tumor-specific could serve as immunotherapy targets, but not all are inherently immunogenic. Peptides presented by HLA-A02:01 and -A11:01 consistently triggered T cell responses, whereas those associated with HLA-A03:01 and -C07:01, though presented, did not. Therefore, allele-specific differences should be considered when designing phosphopeptide-targeted therapies for broader patient coverage.
