Immunogenicity of non-canonical HLA-I tumor ligands identified through proteogenomics

Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from non-canonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the immunogenicity of nonC tumor HLA-I ligands (nonC-TL) to better understand their contribution to cancer immunosurveillance and their therapeutic applicability. Using proteogenomics, we identified 517 nonC-TL from 9 patients with melanoma, gynecological, and head and neck cancer. We found no recognition of the 507 nonC-TL tested by autologous ex vivo expanded tumor reactive T-cell cultures while the same cultures demonstrated reactivity to mutated, cancer-germline, or melanocyte differentiation antigens. However, in vitro sensitization of donor peripheral blood lymphocytes against 170 selected nonC-TL, led to the identification of T-cell receptors (TCRs) specific to three nonC-TL, two of which mapped to the 5’ UTR regions of HOXC13 and ZKSCAN1, and one mapping to a non-coding spliced variant of C5orf22C. T cells targeting these nonC-TL recognized cancer cell lines naturally presenting their corresponding antigens. Expression of the three immunogenic nonC-TL was shared across tumor types and barely or not detected in normal cells. Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they may be attractive novel targets for widely applicable immunotherapies. ### Competing Interest Statement A.G. is member of the scientific advisory board (SAB) of Achilles Therapeutics plc, SingulaBIO, RootPath, Inc., and BioNTech SE, and consults for PACT Pharma, Inc., and Instil BIo. AG is co-inventor of patents licensed and with royalties E-059-2013/0 E-085-2013/0, E-149-2015/0. J.M.L. has received lecture fees from Astellas, Bristol-Myers Squibb, MSD, Novartis, Pierre Fabre, Pfizer, Roche, Sanofi; advisory fees from Bristol-Myers Squibb, Highlight Therapeutics, Novartis, Pierre Fabre, Roche, Sanofi; research grants from Sanofi; and travel grants from Bristol-Myers Squibb, MSD, Novartis, Pierre Fabre, Pfizer, Roche, Ipsen. E. M. is a member of the advisory board of Amgen, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Pierre Fabre, Roche, Sanofi. Honoraria:Amgen, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Pierre Fabre, Roche. Clinical trial participation (principal investigator): Amgen, Bristol-Myers Squibb, GlaxoSmithKline, Merck Sharp & Dohme, Novartis, Pierre Fabre, Roche, Sanofi. J.M.P is consultant for IMMUNOCORE, MSD, BMS, Roche. J.M.P has received research grants from BeiGene, MSD, BMS, Pfizer and Janssen. E.G. is a consultant advisor of Roche/Genentech, F.Hoffmann/La Roche, Ellipses Pharma, Neomed Therapeutics1 Inc, Boehringer Ingelheim, Janssen Global Services, SeaGen, TFS, Alkermes, Thermo Fisher-Bristol-Mayers Squibb, MabDiscovery, Anaveon, F-Star Therapeutics, Hengrui. Remaining authors declare that no other conflict of interest exists