The HLA ligandome of oropharyngeal squamous cell carcinomas reveals shared tumour-exclusive peptides for semi-personalised vaccination

British Journal of Cancer ARTICLE www.nature.com/bjc OPEN Translational Therapeutics The HLA ligandome of oropharyngeal squamous cell carcinomas reveals shared tumour-exclusive peptides for semi-personalised vaccination Lena Mühlenbruch1,2,3,4, Tsima Abou-Kors5, Marissa L. Dubbelaar1,2,3,6, Leon Bichmann1,7, Oliver Kohlbacher 7,8,9,10, Martin Bens11, Jaya Thomas12, Jasmin Ezić5, Johann M. Kraus13, Hans A. Kestler13, Adrian von Witzleben5, Joannis Mytilineos14,15,16, Daniel Fürst14,15, Daphne Engelhardt5, Johannes Doescher5, Jens Greve5, Patrick J. Schuler5, Marie-Nicole Theodoraki 5, Cornelia Brunner5, ✉ Thomas K. Hoffmann5, Hans-Georg Rammensee1,2,3,4, Juliane S. Walz1,2,3,17 and Simon Laban 5 1234567890();,: © The Author(s) 2023 BACKGROUND: The immune peptidome of OPSCC has not previously been studied. Cancer-antigen specific vaccination may improve clinical outcome and efficacy of immune checkpoint inhibitors such as PD1/PD-L1 antibodies. METHODS: Mapping of the OPSCC HLA ligandome was performed by mass spectrometry (MS) based analysis of naturally presented HLA ligands isolated from tumour tissue samples (n = 40) using immunoaffinity purification. The cohort included 22 HPVpositive (primarily HPV-16) and 18 HPV-negative samples. A benign reference dataset comprised of the HLA ligandomes of benign haematological and tissue datasets was used to identify tumour-associated antigens. RESULTS: MS analysis led to the identification of naturally HLA-presented peptides in OPSCC tumour tissue. In total, 22,769 peptides from 9485 source proteins were detected on HLA class I. For HLA class II, 15,203 peptides from 4634 source proteins were discovered. By comparative profiling against the benign HLA ligandomic datasets, 29 OPSCC-associated HLA class I ligands covering 11 different HLA allotypes and nine HLA class II ligands were selected to create a peptide warehouse. CONCLUSION: Tumour-associated peptides are HLA-presented on the cell surfaces of OPSCCs. The established warehouse of OPSCC-associated peptides can be used for downstream immunogenicity testing and peptide-based immunotherapy in (semi) personalised strategies. British Journal of Cancer; https://doi.org/10.1038/s41416-023-02197-y BACKGROUND Oropharyngeal squamous cell carcinoma (OPSCC) is diagnosed in 93,000 patients worldwide per year and 51,000 annual deaths can be attributed to this disease [1]. In OPSCC, human papillomavirus induced (HPV-positive) cancers and non-virally associated, primarily tobacco- and alcohol-associated (HPV-negative) cancers must be discriminated [2, 3]. For different types of curative treatment, a survival advantage for HPV-positive OPSCC has been confirmed [4–6]. As a result, the latest classification of the American Joint Committee on Cancer (AJCC) cancer staging manual version 8 discriminates between HPV-positive and HPVnegative cancers based on the surrogate marker p16 [7, 8]. Immunotherapy targeting the PD1/PD-L1 axis has become a central column of treatment in recurrent and metastatic disease [9–11] and is currently studied intensively in locoregionally advanced disease [12–14]. In recurrent and metastatic disease, 1 Institute for Cell Biology, Department of Immunology, Eberhard Karls University of Tübingen, 72076 Tübingen, Baden-Württemberg, Germany. 2Department of Peptide-based Immunotherapy, Eberhard Karls University and University Hospital Tübingen, 72076 Tübingen, Baden-Württemberg, Germany. 3Cluster of Excellence iFIT (EXC2180) “ImageGuided and Functionally Instructed Tumor Therapies”, University of Tübingen, 72076 Tübingen, Baden-Württemberg, Germany. 4German Cancer Consortium (DKTK), Partner Site Tübingen, 72076 Tübingen, Baden-Württemberg, Germany. 5Department of Otorhinolaryngology and Head & Neck Surgery, Ulm University Medical Center, Head and Neck Cancer Center of the Comprehensive Cancer Center Ulm, Ulm, Germany. 6Quantitative Biology Center (QBiC), Eberhard Karls University Tübingen, 72076 Tübingen, BadenWürttemberg, Germany. 7Applied Bioinformatics, Department of Computer Science, Eberhard Karls University Tübingen, 72076 Tübingen, Baden-Württemberg, Germany. 8 Cluster of Excellence Machine Learning in the Sciences (EXC2064), Eberhard Karls University Tübingen, 72076 Tübingen, Baden-Württemberg, Germany. 9Institute for Translational Bioinformatics, University Hospital Tübingen, 72076 Tübingen, Baden-Württemberg, Germany. 10Institute for Bioinformatics and Medical Informatics, Eberhard Karls University Tübingen, 72076 Tübingen, Baden-Württemberg, Germany. 11Leibniz-Institute on Aging, Fritz-Lipmann-Institute, 07745 Jena, Thüringen, Germany. 12CRUK and NIHR Experimental Cancer Medicine Center & School of Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton SO17 1BJ, UK. 13Ulm University, Institute of Medical Systems Biology, Ulm, Germany. 14Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Transfusion Service, Baden–Württemberg–Hessen, and University Hospital Ulm, Ulm, Germany. 15Institute of Transfusion Medicine, Ulm University, Ulm, Germany. 16German Stem Cell Donor Registry, German Red Cross Blood Transfusion Service, Ulm, Germany. 17Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), Department of Internal Medicine, University Hospital Tübingen, Tübingen, Baden-Württemberg 72076, Germany. ✉email: Received: 13 September 2022 Revised: 24 January 2023 Accepted: 1 February 2023 L. Mühlenbruch et al. 2 the response rates of anti-PD1 antibodies lie below 20% [9–11]. Because the success of PD1/PD-L1 antibodies relies on the presence of pre-existing cancer-antigen specific immunity [15], vaccination against cancer antigens may improve efficacy of such treatments [16]. However, it is currently unclear which antigens should be targeted. Immune responses to viral antigens in HPVpositive disease [17, 18] and immune responses to other cancer antigens [19, 20] including mutation-associated antigens, so called neoantigens, have previously been described [21, 22]. The respective significance of the different types of cancer antigens for immunotherapy is currently unclear. To establish vaccination strategies for OPSCC, it is crucial to understand its antigenic landscape. Tumour-specific immune cells rely on the presentation of peptides from cancer antigens on human leucocyte antigens (HLA)—the immunopeptidome or HLA ligandome. For optimal immune responses against the tumour, these HLA-presented peptides need to be tumour-exclusive. Thus, the analysis of the HLA ligandome can be used to identify promising disease-specific vaccination targets [23–26]. Here we performed the first comprehensive analysis of the natural HLA ligandome of OPSCC by mass spectrometry to guide personalised or semi-personalised vaccine development. MATERIALS AND METHODS Patients Patients with histologically confirmed OPSCC who were treated surgically were included into this non-interventional study except for o (...truncated)