Abstract PR-07: Discovery and therapeutic potential of novel cryptic peptides in pancreatic cancer
Authors: Ely Zackery A , Kulstad Zachary J , Gunaydin Gurcan , Addepalli Sudarsana , Verzani Eva K , Abelin Jennifer G , Casarrubios Marta , Clauser Karl R , Wang Xilin , Lippincott Isabelle , Louvet Cedric , Schmitt Tom , Agus Miles , Kapner Kevin S , Hennessey Connor J , Cleary James , Hadrup Sine R , Klaeger Susan , Su Jennifer , Jaeger Alex M , Wolpin Brian M , Raghavan Srivatsan , Smith Eric , Greenberg Philip D , Aguirre Andrew J , Carr Steven A , Jacks Tyler , Freed-Pastor William A 
Published: September 2024

Abstract
While neoantigen-directed therapies have primarily focused on tumor-specific somatic mutations, emerging evidence suggests that some malignancies aberrantly translate regions of the genome outside of annotated open reading frames (ORFs), leading to HLA-I presentation of cryptic peptides. Non-canonical HLA-I bound peptides (ncHLAp) can arise from aberrant translation of genomic elements like 5’ and 3’ untranslated regions (UTRs), long noncoding RNAs (lncRNAs), retained introns, and translation in alternative reading frames. Pancreatic cancer has a low-to-intermediate mutational burden; hence, efforts to broaden the landscape targetable antigens in this disease are greatly needed. Here, we leveraged twelve pancreatic cancer (PDAC) patient-derived organoids (PDOs) to purify and enrich the malignant compartment from low tumor cellularity tumor specimens. PDOs were subjected to extensive genomic (whole genome sequencing) and transcriptomic (RNA-sequencing) profiling to enable mutation calling and HLA typing. We then employed a personalized proteogenomic platform coupled with high-depth immunopeptidomics and empirically identified &gt;90,000 unique PDAC HLA-I-bound peptides (HLAp). We detected HLAp arising from somatic mutations (both missense and frameshift) in a subset of PDAC patients; however, we did not detect any shared mutation-derived neoepitopes in our patient cohort. Moreover, we empirically identified &gt;1,700 ncHLAp, primarily arising from translation of novel unannotated open reading frames (nuORFs), and a substantial proportion of ncHLAp were shared amongst PDAC patients with the appropriate HLA haplotype. We developed a highly stringent pipeline using immunopeptidomics and ribosome-sequencing to investigate translation of nuORFs across a range of healthy tissues, including healthy thymus, and found that &gt;500 ncHLAp exhibit highly-specific cancer-restricted translation. We next investigated the immunogenicity of PDAC-restricted ncHLAp and mutation-derived HLAp using an ex vivo T cell priming and expansion platform. Here we demonstrate that a subset of both mutation-derived and PDAC-restricted ncHLAp harbor robust immunogenicity. ncHLAp-specific T cell receptors (TCRs) were identified from reactive cytotoxic T lymphocytes (CTLs) via single-cell TCR-sequencing, and antigen specificity was confirmed after TCR reconstruction and cloning. Finally, using a newly developed organoid: T cell co-culture platform, we demonstrate that ncHLAp-reactive TCR-redirected T cells exhibit robust cytotoxicity and tumoricidal activity in PDAC, underscoring the translational potential for this novel class of antigens. Collectively, we have shown that cryptic epitopes, arising from aberrant translation in PDAC, represent a particularly promising class of antigens for next generation immune-based therapies.
Citation Format: Zackery A Ely, Zachary J Kulstad, Gurcan Gunaydin, Sudarsana Addepalli, Eva K Verzani, Jennifer G Abelin, Marta Casarrubios, Karl R Clauser, Xilin Wang, Isabelle Lippincott, Cedric Louvet, Tom Schmitt, Miles Agus, Kevin S Kapner, Connor J Hennessey, James Cleary, Sine R Hadrup, Susan Klaeger, Jennifer Su, Alex M Jaeger, Brian M Wolpin, Srivatsan Raghavan, Eric Smith, Philip D Greenberg, Andrew J Aguirre, Steven A Carr, Tyler Jacks, William A Freed-Pastor. Discovery and therapeutic potential of novel cryptic peptides in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-07.