Abstract NG08: Cancer-restricted cryptic antigens: Novel targets for next-generation T cell-based therapies

Abstract Emerging evidence suggests that cancer cells aberrantly translate regions of the genome outside of annotated open reading frames (ORFs), leading to human leukocyte antigen class I (HLA-I) presentation of cryptic peptides. These non-canonical HLA-I bound peptides (ncHLAp) derived from the dark proteome may represent potentially targetable antigens in low mutational burden cancers like pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States and novel therapeutic strategies are urgently needed. To comprehensively map the PDAC antigen landscape, we employed a personalized proteogenomic platform coupled with high-depth HLA-I immunopeptidomics to empirically identify HLA-I bound peptides (HLAp) from PDAC bulk tumors and patient-derived organoids (PDOs). PDOs were utilized to enrich the malignant compartment from low neoplastic cellularity tumor specimens. PDAC PDOs and bulk tumors were subjected to extensive genomic and transcriptomic profiling to enable high resolution HLA typing and mutation calling. We detected somatic mutation-derived HLAp (mutHLAp), arising from both missense mutations and frameshift mutations, in approximately one third of PDAC patient samples. Beyond somatic mutations, accumulating evidence suggests that cancer cells can translate novel unannotated ORFs (nuORFs) within the dark genome. We augmented our proteomic search spaces to encompass nuORFs, previously defined through ribosome profiling (Ribo-seq), and empirically identified thousands of nuORF-derived ncHLAp from PDAC organoids and bulk tumors. We next assessed tumor-restricted expression of ncHLAp using a newly developed ‘translation-centric’ approach. This pipeline leverages HLA-I immunopeptidomics and Ribo-seq across 275 healthy tissue samples, spanning more than 28 diverse organ systems, including healthy thymus. We identified over 500 ncHLAp derived from noncanonical ORFs with zero evidence of translation in any healthy organ/tissue analyzed. We termed this subset cancer-restricted (CR) ncHLAp. A substantial proportion of CR ncHLAp were shared by multiple PDAC patients with the appropriate HLA, highlighting the potential for ‘off-the-shelf’ therapeutic strategies. To directly assess the immunogenic potential of candidate antigens (including mutHLAp and ncHLAp), we employed a sensitive ex vivo platform for priming and expansion of (neo) antigen-specific T cells using HLA-matched healthy donor peripheral blood mononuclear cells (PBMCs). A substantial subset of both mutation-derived HLAp and cancer-restricted ncHLAp exhibited robust immunogenicity, nominating a set of high priority cancer-restricted cryptic antigens for downstream investigation. Cryptic antigen-specific T cell receptors (TCRs) were then identified using custom DNA-barcoded peptide: HLA multimers (compatible with the 10X Genomics workflow). This approach enabled full-length TCR-alpha/beta sequencing with paired antigen specificity. CRISPR-based TCR redirection was used to generate cryptic antigen-directed TCR engineered T (TCR-T) cells. To evaluate whether cryptic antigen-directed TCR-T cells could recognize and kill pancreatic cancer organoids expressing endogenous levels of their cognate antigens, we developed an organoid: TCR-T cell co-culture platform to enable simultaneous measurement of antigen recognition and cytotoxicity. We demonstrated robust ex vivo cytotoxic potential for several cryptic antigen-specific TCRs. Finally, we explored our highest performing TCRs in vivo using preclinical adoptive cell therapy studies. Cryptic antigen-directed TCR-T cells exerted robust in vivo tumoricidal activity against patient-derived PDAC organoids transplanted into immunocompromised mice, demonstrating that cancer-restricted cryptic antigens (CR ncHLAp) can mediate recognition by cytotoxic T cells and enable tumor cell killing. Collectively, this work nominates cancer-restricted cryptic antigens as tractable targets for next generation immune-based therapies for pancreatic cancer and potentially other tumor types. Citation Format: William Freed-Pastor. Cancer-restricted cryptic antigens: Novel targets for next-generation T cell-based therapies abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr NG08.