There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients’ tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients’ tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies.

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Background: There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Memory CD8+ T cell infiltration is now well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) are specifically targeting and killing tumor cells. However, the epitopes that these TILs use to identify cancer cells have not been determined. This has limited the use of immunotherapies in CRC, despite their efficacy in other cancer types. Recently, phosphopeptides have emerged as strong candidates for tumor-specific epitopes, since dysregulation of signaling in cancers leads to aberrant protein phosphorylation. Here, we identify CRC-associated phosphopeptides and assess the tumor-resident immunity against these novel epitopes. Methods: We compared tumor and healthy tissue from CRC patients, to identify tumor-specific MHC class-I associated phosphopeptides. The tissues were lysed, the MHC class-I complexes affinity purified, and the bound peptides eluted. Phosphopeptides were enriched using immobilized metal affinity chromatography, and characterized using mass spectrometry. TILs, from the same tumors, were extracted and expanded, and their responses to the phosphopeptides assessed using multiplexed intracellular cytokine staining. Cytolytic activity was observed by staining for surface mobilization of CD107a. Healthy donor responses were quantified using interferon-γ ELISpot, and functionality assessed using a europium release killing assay. Results: We have identified 125 tumor-associated MHC class-I phosphopeptides from CRC, with different HLA-restrictions. There were, on average, 3.5 times more different phosphopeptides identified on cancer than healthy tissues, at 6.7-fold higher levels. Many of these novel epitopes are attributable to signaling events in well-defined cancer pathways and are therefore markers of malignancy. Through analysis of TIL's cytokine responses to these phosphopeptides, we have established that they are playing a key role in tumor-resident immunity. There were multifunctional TILs present in primary and metastatic tumors that recognized and killed in response to these phosphopeptides. Up to 0.7% of expanded TILs targeted each phosphopeptide, comparable with responses seen to viral epitopes. Thus, immunity to tumor-associated phosphopeptides represents a biological strategy for distinguishing tumor from healthy tissue. Furthermore, we have shown that healthy donors have pre-existing, memory T cell responses to many (58%) of these CRC-associated phosphopeptides. These phosphopeptide-specific T cells are readily expanded ex vivo and can kill CRC cell lines. Therefore, MHC class-I associated phosphopeptides are ideal immunotherapeutic targets, as immunity must spare healthy tissue. Conclusion: The identification of this novel class of MHC class-I epitopes in CRC offers new hope for the future of immunotherapy in this malignancy. Citation Format: Sarah A. Penny, Jennifer G. Abelin, Abu Z. Saeed, Stacy A. Malaker, Paisley D. Trantham, Jeffrey Shabanowitz, Stephen T. Ward, Donald F. Hunt, Mark Cobbold. Phosphopeptides as novel T cell epitopes in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3627. doi:10.1158/1538-7445.AM2014-3627

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