Luisa Weisbrod ^1* Luigi Capriotti ² Marco Hofmann ¹ Valerie Spieler ¹ Herbert Dersch ¹ Bernd Voedisch ¹ Peter Schmidt ³ Susanne Knake ⁴

• ¹ CSL Behring Innovation GmbH, Marburg, Germany
• ² CSL Behring AG, Bern, Bern, Switzerland
• ³ CSL Parkville, Parkville, Victoria, Australia
• ⁴ Department of Neurology, Philipps University Marburg, Marburg, DE, Germany

The study of peptide repertoires presented by major histocompatibility complex (MHC) molecules and the identification of potential T cell epitopes contribute to a multitude of immunopeptidomebased treatment approaches. Epitope mapping is essential for the development of promising epitopebased approaches in vaccination as well as for innovative therapeutics for autoimmune diseases, infectious diseases and cancer. It also plays a critical role in the immunogenicity assessment of protein therapeutics with regards to safety and efficacy concerns. The main obstacle challenge emerges from the highly polymorphic nature of the human leukocyte antigen (HLA) molecules leading to the requirement of a peptide mapping strategy for a single HLA allele. As many autoimmune diseases are linked to at least one specific antigen, we established FASTMAP, an innovative strategy to transiently co-transfect a single HLA allele combined with a disease-specific antigen into a human cell line. This approach allows the specific identification of HLA-bound peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using FASTMAP we found a comparable spectrum of endogenous peptides presented by the most frequently expressed HLA alleles in the world's population compared to what has been described in literature. To ensure a reliable peptide mapping workflow, we combined the HLA alleles with wellknown human model antigens like coagulation factor VIII, acetylcholine receptor subunit alpha, protein structures of the SARS-CoV-2 virus, and myelin basic protein. Using these model antigens, we have been able to identify a broad range of peptides that are in line with already published and in silico predicted T cell epitopes of the specific HLA/model antigen combination. The transient co-expression of a single affinity-tagged MHC molecule combined with a disease- specific antigen in a human cell line in our FASTMAP pipeline provides the opportunity to identify potential T cell epitopes/endogenously processed MHC-bound peptides in a very cost-effective, fast, and customizable system with high-throughput potential.