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1408 Non-canonical peptide sources broaden the landscape of targetable antigens in pancreatic cancer
  1. Zackery A Ely1,
  2. Zachary J Kulstad2,
  3. Jennifer G Abelin3,
  4. Eva K Verzani3,
  5. Sudarsana Addepalli2,
  6. Karl Clauser3,
  7. Kevin S Kapner2,
  8. Connor J Hennessey2,
  9. Miles Agus1,
  10. Susan Klaeger3,
  11. Alex M Jaeger4,
  12. Jennifer Su1,
  13. Philip D Greenberg5,
  14. Andrew J Aguirre2,6,
  15. Steven Carr3,
  16. Tyler Jacks1 and
  17. William Freed-Pastor2,6
  1. 1Koch Institute at MIT, Cambridge, MA, USA
  2. 2Dana-Farber Cancer Institute, Boston, MA, USA
  3. 3Broad Institute, Cambridge, MA, USA
  4. 4Moffitt Cancer Center, Tampa, FL, USA
  5. 5Fred Hutchinson Cancer Research Center, Seattle, WA, USA
  6. 6Harvard Medical School, Boston, MA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Aberrant translation of the non-coding genome in cancer can generate novel peptides capable of presentation by major histocompatibility complex class I (MHC-I; HLA-I in humans) and these non-canonical peptide sources can broaden the landscape of potentially targetable antigens in low-to-intermediate mutational burden malignancies. While emerging evidence suggests that translation of unannotated open reading frames (uORFs) can give rise to MHC class I-associated peptides (MAPs) across a range of malignancies, it is currently unknown to what extent these translation products are truly cancer-restricted and how effectively the resulting non-canonical MAPs (ncMAPs) can elicit a T cell response.

Methods We leveraged twelve pancreatic cancer (PDAC) patient-derived organoids (PDOs) to purify the malignant compartment from low tumor cellularity tumor specimens. We developed a cutting-edge proteogenomics pipeline, coupled with high-depth immunopeptidomics to identify pancreatic cancer MAPs derived from somatic mutations, retained introns, and uORFs. To investigate the cancer-specificity of ncMAPs, we developed a translation-centric analysis pipeline that examines translation of uORFs encoding ncMAPs across a range of healthy tissues, including healthy thymus. To evaluate for immunogenicity, we employed a highly sensitive ex vivo platform to prime and expand ncMAP-specific cytotoxic T lymphocytes (CTLs) and evaluate cytolytic potential.

Results We demonstrate that ncMAPs are abundant and predominate over mutation-derived peptides in the pancreatic cancer immunopeptidome, establishing a novel class of recurrent cancer-restricted epitopes available for immune recognition. We observed widespread translation and MHC-I presentation of numerous ncMAPs across many healthy tissues, highlighting the importance of our translation-centric approach to assess cancer-restriction. Excitingly, we nominated over 500 ncMAPs that exhibit cancer-specific translation patterns. Approximately 30% of ncMAPs exhibited bona fide cancer-restricted translation patterns, and a substantial subset of these were shared among patients. We next interrogated immunogenicity using a highly sensitive ex vivo vaccination platform and demonstrated that the majority of cancer-restricted ncMAPs evaluated were highly immunogenic. Remarkably, the proportion of ncMAPs harboring immunogenic potential was substantially higher than mutation-derived neoepitopes and tumor-associated antigens, underscoring their therapeutic potential relative to traditional immunotherapy targets.

Conclusions These findings demonstrate that aberrant translation in pancreatic cancer can give rise to recurrent cancer-restricted ncMAPs capable of recognition by cytotoxic T lymphocytes. Collectively, our findings furnish a novel set of recurrent, cancer-restricted immunotherapy targets not subject to central tolerance. We believe these findings will prompt translation-centric investigations in other solid tumors. We envision that these novel antigens will augment ongoing efforts to treat pancreatic cancer patients with vaccines and cell-based therapies.

Ethics Approval Informed consent was obtained from patients at least 18 years old with pancreatic cancer under Dana-Farber/Harvard Cancer Center Institutional Review Board (IRB)-approved protocols 11–104, 17–000, 03–189, and/or 14–408 for tissue collection, molecular analysis, and organoid generation.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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