Abstract 2291: On- and off target toxicity profiling for adoptive cell therapy by mass spectrometry-based immunopeptidome analysis of primary human normal tissues

A major constraint for the broad and safe application of Adoptive Cellular Therapy (ACT) is the limited number of validated tumor targets, especially for solid tumors. For T-cell receptor (TCR)-based approaches, presentation of targeted HLA-peptides on normal tissues can lead to on-target toxicity, such as severe inflammatory colitis reported upon re-directing T cells to an HLA-A*02 restricted carcinoembryonic antigen (CEA) epitope. Independently, off-target cross-reactivity of TCRs occurred in previous ACT trials, e.g. when a MAGEA3-directed TCR cross-recognized an HLA-A*01 restricted epitope from titin expressed on heart, which led to fatal cardiac toxicities. Here we present a novel approach allowing the prediction of severe on- and off-target side effects before entering into clinical trials. We used a target discovery engine (XPRESIDENT) combining highly sensitive, quantitative mass spectrometry (LC-MS/MS), RNA-Seq-based differential transcriptomics, immunology and bioinformatics to characterize the human immunopeptidome directly on shock frozen primary human tissues. Over the last years we have built an according database for > 600 tumor samples from > 20 different tumor types and, importantly, > 300 samples from > 40 different normal tissue types, resulting in hundreds of thousands of unique HLA-peptide sequences. These data allow conclusions on which HLA peptides are actually presented on primary normal tissues in a quantitative manner, taking into account relative differences between normal tissues and tumors as well as absolute peptide copy numbers per cell. In order to assess the off-target risk for a TCR, we predict all theoretical HLA- and TCR-binding peptides in the proteome, ideally based on the binding motif of the TCR, and specifically search for actual peptide presentation by normal tissues. When analyzing the above described CEA case, we were able to detect the CEA-derived peptide IMIGVLVGV on HLA-A*02 positive colorectal cancer samples, but importantly also on normal colorectal samples. In the original study describing the titin case tremendous experimental efforts and sophisticated cell culture models were required to retrospectively identify cross-recognition of the peptide on cardiomyocytes as the cause of toxicity. In contrast, with our approach we easily and directly identified the critical peptide ESDPIVAQY as one of the most abundantly presented peptides on an HLA-A*01 positive primary human heart sample. We show that this approa