Deep Exploration of the Immunopeptidome of a Pancreatic Cancer Cell Line: Implications for Clinical Immunopeptidomics and Immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC), with its devastating prognosis and limited treatment options, demands innovative therapeutic strategies. T-cell-based immunotherapy has shown promise for many cancers, including PDAC, but is limited by our knowledge of the breadth of cancer-specific T-cell epitopes available. Thus, a comprehensive assessment of the immunopeptidome of PDAC is essential to pave the way for the effective design of immunotherapy and related interventions. In this study, we immunoaffinity purified Human Leukocyte Antigen (HLA) class I-bound peptides from the Panc1 cell line grown in the absence and presence of cytokine stimulation. These peptides were subjected to an off-line high-pH reversed-phase (HPH-RP) fractionation prior to data acquisition. We demonstrate that HPH-RP fractionation followed by data-dependent acquisition (DDA) is a relatively simple and reliable technique that expands the depth of coverage of the PDAC immunopeptidome, allowing the identification of over 22,500 canonical HLA-bound peptides. In addition, the complementary separation by HPH-RP improved the identification confidence, particularly in the case of co-fragmenting precursors in data-independent acquisition (DIA) workflows. This strategy facilitated the identification of a high number of cancer-testis antigen (CTA)-derived immunopeptides. However, given that fractionation is typically associated with an adsorptive loss, it is impractical to apply HPH-RP on often minuscule clinical specimens and biopsies. Thus, we explored the feasibility of immunopeptidome analysis with cellular inputs as low as 1 million cells (equivalent to approximately 1 mg of tissue) using either a ZenoSWATH DIA interpreted using a spectral library derived from the HPH-RP strategy, or an optimized DDA workflow on the SCIEX ZenoToF 7600 system. Both approaches enabled robust detection of CTA-derived and other potentially clinically actionable immunopeptides even at the lowest cellular inputs. We discuss the relative merits of both acquisition strategies and how they can form the basis for future clinical translational immunopeptidomics approaches to screen tumor antigen presentation in low cellular input PDAC biopsies and provide new opportunities for target identification in immune-based therapies. Data are available via ProteomeXchange with identifiers PXD054360 and PXD054417.