Identification of Cancer Immunotherapeutic Candidates Derived from Dysregulated Cell Signaling Pathways by Mass Spectrometry

Cancer is the leading cause of death worldwide, and over the past 60 years the overall survival rate has barely improved due to the lack of effective treatment options even though our understanding of the molecular mechanisms behind the disease have increased dramatically. Understanding how transformation events governed by dysregulated cell signaling are eliminated by the immune system before they result in cancer has driven our search for more effective cancer treatments. We hypothesize that human leukocyte antigen (HLA) class I-associated phosphopeptides are candidates for a cancer immunotherapy that harnesses the ability of the immune system to identify and specifically eliminate transformed cells. We identified HLA-associated phosphopeptides derived from dysregulated cell signaling pathways presented by ovarian and colorectal cancer using optimized iron(III)-immobilized metal affinity chromatography (IMAC) enrichment and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). We then demonstrated that tumor-specific phosphopeptides illicit responses from healthy donor T cells and tumor infiltrating lymphocytes (TILs), and phosphopeptide specific TILs are able to kill tumors in vitro. We were also able to show that HLA molecules protect bound phosphopeptides from dephosphorylation, which provides evidence that phosphopeptides are not transiently presented to the immune system. Collectively, our investigation provided insight into phosphopeptide presentation by HLA and identified biologically active phosphopeptides that are strong immunotherapeutic candidates.

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