Abstract Head and neck squamous cell carcinomas (HNSCC) are the sixth most common malignancies worldwide. 45% of patients are diagnosed at a late tumor stage associated with poor survival. For metastatic, unresectable or recurrent (m/uR) HNSCC, immune checkpoint inhibition (ICI) was recently approved as a novel therapeutic option showing significant survival benefits compared to standard chemotherapy-based treatment. However, response to ICI is still limited to a small number of patients calling for further improvement of T cell-based immunotherapies. Peptide-based approaches, which rely on the specific immune recognition of tumor-associated human leukocyte antigen (HLA) presented peptides, represent promising and low side effect treatment options. Peptide vaccination has been shown to enhance and induce long-term anti-tumoral immune responses and even clinical responses in HNSCC patients. However, current vaccines are either monovalent, based on patient-individual tumor-specific mutations or restricted to a single HLA allotype and therefore neither widely applicable nor suitable for reliable studies and large-scale production. In this study, using mass spectrometry (MS) -based immunopeptidome analysis of a large cohort of HNSCC patient (n = 30) tumor and adjacent benign samples, we established a tumor-associated off-the-shelf peptide warehouse for broadly applicable personalized therapies. The malignant dataset, comprising 91651 HLA ligands, was compared to adjacent benign and various benign tissues (www.hla-ligand-atlas.org) to identify tumor-exclusive antigens. Further antigen selection was based on allotype-specific high frequent presentation. In total, 23 frequently presented and tumor-exclusive HNSCC-associated peptides were selected for six of the most common HLA class I allotypes (A*01, A*02, A*24, B*15, B*35, B*40) covering >75% of the world population, as well as five HLA class II presented peptides binding various different HLA class II allotypes. Immunogenicity was validated by IFN-γ ELISPOT screening for spontaneous preexisting T cell responses targeting the respective peptides as well as by in vitro priming experiments of naïve T cells in HNSCC patients and healthy volunteers. Furthermore, immunopeptidome analyses identified these antigens in patient plasma samples providing first evidence for “liquid biopsy” immunopeptidome analysis without the need of primary tumor tissue. A phase I study evaluating safety, immunogenicity as well as first efficacy of this warehouse-based vaccine in combination with ICI in HNSCC patients is currently being set up, with personalized peptide selection based on individual HLA-allotype and MS analysis of patient tumor/plasma sample. In conclusion, we here designed a peptide warehouse that enables a polyvalent and widely applicable but still personalized peptide vaccination in HNSCC patients. Citation Format: Sarah Schroeder, Thorben Gross, Annika Nelde, Marcel Wacker, Jens Bauer, Jonas Rieth, Marissa Dubbelaar, Lena Muehlenbruch, Yacine Maringer, Paul-Stefan Mauz, Martin Sailer, Julia Philipp, Sven Becker, Thomas Breuer, Helmut R. Salih, Hans-Georg Rammensee, Hubert Löwenheim, Juliane S. Walz. Immunopeptidomics-guided tumor antigen warehouse design for peptide-based immunotherapy in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3555.