156 Dark immunopeptidome of head and neck squamous cell carcinoma in serial cell lines from a single patient during multimodality treatment time-course

<h3>Background</h3> The rapidly growing field of immunopeptidomics has greatly expanded our understanding of the breadth of targets that T cells can potentially recognize on cancer cells. Non-canonical peptides (NCP) derived from non-coding or untranslated regions of the genome, along with peptides derived from circRNA, aberrant splicing, or transposable elements represent potential shared targetable alternative neoantigens, that are not expressed in the thymus. Recent data highlights that some cryptic peptides are shared and appear to increase malignancy in cancer, solidifying their importance as targets. Utilizing a combination of mass spectrometry and computational based approaches, we sought to characterize the immunopeptidome of head and neck squamous cell cancer (HNSCC) cell lines generated from biopsies from initial surgery, then 14 and 16 months later. Over this period the patient received surgery, radiation, chemotherapy and investigational immunotherapy. <h3>Methods</h3> HNSCC cell lines from 3 time points were lysed, and lysates subject to treatment with detergent with protease inhibitor. HLA complexes were purified by anti-HLA-I antibody (w6/32) and HLA-bound peptides eluted and analyzed by the Orbitrap mass spectrometer (ORBITRAP FUSION LUMOS with FAIMS-Pro interface (Thermofisher Scientific)). Multiple databases containing peptide information derived from UniProt, published Ribo-Seq data, circRNA, and other small open-reading frames were searched against our identified peptides using the PEAKS X software. Concurrently, total RNA-Seq libraries (WatchMaker Genomics) were generated from patient cell lines and full cryptic protein databases were generated computationally using the pipeline described in Scull et al.¹ Full cryptic databases were subject to database searching to identify both canonical and NCP. <h3>Results</h3> Our comprehensive assessment characterized the immunopeptidome of cell lines derived from a single patient prior to and following multiple treatments and identified 26,964 canonical and 1,499 NCP. Importantly, 4,750 canonical and 94 NCP were present at all three time points and could suggest immune targets. <h3>Conclusions</h3> These studies of tumor cell lines generated from serial biopsies from a patient who progressed on multiple conventional and investigative immunotherapy treatments provide insights into the spectrum of antigens that could be targeted. Current efforts seek to identifying whether the source proteins of common HLA-presented epitopes are associated with malignant properties, whether these are shared across other HNSCC specimens, and whether the patient made an immune response to these epitopes. <h3><b>Acknowledgements</b>Support</h3> Shimadzu Corporation, Providence Medical Foundation, The Harder Family, Nancy Lematta, The Chiles Foundation, Robert W. Franz, and Elsie Franz Finley. <h3>Reference</h3> Scull KE, Pandey K, Ramarathinam SH, Purcell AW. Immunopeptidogenomics: Harnessing RNA-seq to illuminate the dark immunopeptidome. <i>Molecular and Cellular Proteomics</i> 2021 Jan 1;<b>20</b>. <h3>Ethics Approval</h3> PSJH IRB# 06-108.