Introduction: Localized prostate cancer (PCa) can be successfully treated by androgen deprivation, radiotherapy and surgery, however these may not be sufficient to eradicate cancer stem cells (CSCs). CSCs are more resistant to such treatments than the bulk of the tumor; and can contribute to disease relapse. PCa patients who relapse have a poor prognosis. We hypothesize that CSCs could be killed by T-cells in an antigen specific way, thus preventing the possibility of relapse. In this study we identified novel PCa CSC antigens by HLA ligandome analysis and isolated antigen specific CD8+ T-cells. Methods: We identified CSCs using aldehyde dehydrogenase (ALDH) activity as a CSC marker. ALDH high and low cells from the DU145 PCa cell line and from prostate adenocarcinoma primary tissue were characterized in vitro; DU145 CSCs and non-CSCs were additionally characterized in vivo. We isolated peptide-HLA complexes from DU145 cells by immunoprecipitation and analyzed the eluted peptides by mass spectrometry. We identified CSC antigens based on the gene expression in ALDH high and low DU145 cells (measured by qPCR). To select antigens for further analysis we performed homology modelling of the HLA-peptide interface using COOT software and the YASARA server for energy minimization. The interface interactions were quantified using PISA software. We additionally confirmed antigen expression in the primary cells by fluorescence microscopy and PCR. Tetramers were produced to isolate T-cells which recognized a selection of these antigens. Results: The ligandome analysis identified over 1900 peptides. We selected antigens with low gene expression in healthy tissues ( and high predicted binding to DU145 HLA alleles ( ALDH high DU145 cells were more tumorigenic in vivo than ALDH low cells. ALDH high DU145 and primary prostate cancer cells grew larger colonies and spheres in vitro. We identified 11 CSC antigens by qPCR; 6 upregulated in ALDH high DU145 cells (e.g., TACSTD2) and 5 abundant in both ALDH high and ALDH low DU145 cells (e.g., XPO1). Relevant 9-mer epitopes from three antigens* induced CD8+ T-cell responses in vitro. Antigen-specific CD8+ T-cells were identified by tetramer staining at a frequency of approx. 15 per 100000 cells. These cells are currently being expanded to use in CTL assays. Conclusion: We have identified CSC antigens which could lead to specific targeting by T-cells and prevention of PCa relapse. Further epitopes restricted to the more frequent HLA alleles could additionally be predicted in silico from the novel antigens we identified. We are also investigating prediction of viral epitopes highly aligned with the self-peptides (determined in silico) to boost the immune response against CSCs. *Subject of an ongoing patent application.

Citation Format: Amy S. Codd, Saly Al-Taei, Serina Tokita, Emi Mizushima, Pierre J. Rizkallah, Tom Whalley, Barbara Szomolay, Kristin Ladell, James E. McLaren, Sian Llewellyn-Lacey, David A. Price, Takayuki Kanaseki, Toshihiko Torigoe, Stephen Man, Zsuzsanna Tabi. Identification of prostate cancer stem cell antigens for T-cell immunotherapy by HLA ligandome analysis [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B007.