Abstract C014: Broadening the repertoire of PDAC-specific targets for immune-based therapy through high-resolution immunopeptidomics
Authors: Ely Zackery A. , Freed-Pastor William A. , Kulstad Zachary J. , Abelin Jennifer G. , Verzani Eva , Kapner Kevin S. , Klaeger Susan , Clauser Karl R. , Agus Miles , Jaeger Alex M. , Pattada Nimisha B. , Bhutkar Arjun , Aguirre Andrew J. , Carr Steven A. , Jacks Tyler 
Published: November 2022

Abstract
Pancreatic adenocarcinoma (PDAC) is among the most lethal cancer types and has been largely recalcitrant to traditional immunotherapy. A large subset of PDAC tumors is computationally predicted to harbor potentially immunogenic peptides for MHC class I (MHC-I) presentation, but the nature, expression, and immunogenicity of these peptides has yet to be determined. By investigating the PDAC immunopeptidome, we can uncover and exploit novel immune-based targets for PDAC and render it vulnerable to immunotherapy. Prior efforts to study the immunopeptidome in PDAC have largely focused on profiling MHC-associated peptides (MAPs) from bulk tumor samples. This approach is severely limited by the contribution of MAPs from the non-malignant compartments, which constitutes most of the cellular mass in PDAC. We can overcome this limitation by using patient-derived organoids (PDOs) to expand a pure cancer cell population for MHC-I immunoprecipitation, followed by LC/MS-MS. We applied this approach and detected 17,000-20,500 unique MAPs per sample, a dramatic increase in depth and resolution over prior efforts. To ascertain which MAPs may be PDAC-restricted, we first analyzed bulk RNA-sequencing data from the Genotype-Tissue Expression Project (767 patients, 30 tissues) to generate a set of genes that are functionally undetectable (Q90 &lt;1 TPM) in healthy somatic tissues. We cross-referenced this list with our set of PDO MAPs, yielding 143 PDAC-restricted MAPs. To further expand our search space, we implemented a tiered tissue-based filter to relax TPM cutoffs in less essential tissues (ex. prostate) while maintaining strict cutoffs in essential tissues (ex. brain), generating 85 additional MAPs. Both approaches uncovered cancer-restricted MAPs present in most PDO lines, which may represent shared therapeutic targets. While somatic mutations are a well-established source of tumor-specific neoantigens, these have yet to be investigated with immunopeptidomics in human PDAC. Despite harboring an intermediate mutational burden, we detected at least one mutation-derived neoepitope in most PDOs, a detection frequency much closer to the detection frequency of cancers with high mutational burden than previously expected. Additionally, non-canonical peptide sources, including retained introns (RI) and novel unannotated open reading frames (nuORF), may represent a source of cancer-restricted MAPs. Interestingly, we detect nuORF- and RI-derived MAPs (n=298) in all PDOs, suggesting that these too may be therapeutically relevant in PDAC. To assess the immunogenicity of candidate MAPs, future studies will utilize established protocols for priming and expanding MAP-specific human T cells with autologous DCs. Functional evaluation of MAP-specific T cells will help prioritize strategies for vaccination, and the generation of T cell receptor sequences for adoptive cell therapy. Collectively, these data deepen our understanding of the PDAC immunopeptidome and provide a novel set of targets for immunotherapy in PDAC.
Citation Format: Zackery A. Ely, William A. Freed-Pastor, Zachary J. Kulstad, Jennifer G. Abelin, Eva Verzani, Kevin S. Kapner, Susan Klaeger, Karl R. Clauser, Miles Agus, Alex M. Jaeger, Nimisha B. Pattada, Arjun Bhutkar, Andrew J. Aguirre, Steven A. Carr, Tyler Jacks. Broadening the repertoire of PDAC-specific targets for immune-based therapy through high-resolution immunopeptidomics [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C014.