Abstract LB-C011: Using Organoids and Tumor Infiltrating Lymphocytes to Elucidate the ”Dark Matter” of Human Tumor Antigens

The NIH Surgery Branch conducts clinical trials utilizing tumor-infiltrating lymphocytes (TILs) to treat epithelial malignancies, including colorectal, pancreatic, and breast cancers. Seeking to identify predictors of therapeutic benefit, we established that patient-derived tumor organoids (PDTOs)—which recapitulate the genomic landscape of the original tumor—provide a robust platform for evaluating TIL potency. TIL infusion products from 30 patients (colorectal, n=21; pancreatic, n=4; breast, n=2; other, n=3) were assessed for reactivity against autologous PDTOs. Objective clinical responses were observed in 53% (8/15) of patients whose TILs recognized PDTOs, compared with only one response among patients lacking such recognition. These results established TIL reactivity against autologous organoids as a significant predictor of clinical outcome and prompted an investigation into the antigenic targets recognized by these organoid-reactive T cells. We previously utilized single-cell RNA sequencing of fresh TILs to establish a transcriptional signature that identifies tumor-reactive T cells. While this signature successfully identified neoantigen-reactive TCRs, a substantial number of organoid-reactive clonotypes—termed 'orphan TCRs'—demonstrated no reactivity toward any screened neoantigens. In the current study, using organoid-derived cDNA library expression screening, we successfully identified five distinct targets—including their restricting HLA class I molecules and cognate epitopes—for ten orphan TCR clonotypes derived from patients with non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). Notably, these antigens were derived from non-coding regions, including lncRNA, lincRNA, 5’-UTRs, and gene/non-coding sequence fusions. These findings provide definitive evidence that a natural immune response can target genomic dark matter and support the expansion of the therapeutic landscape to include these previously overlooked genomic regions. Citation Format: Ken-ichi Hanada, Billel Gasmi, Frank J. Lowery, Sri Krishna, Aarushi Bhasin, Brian D. Bui, Alexandra M. Gustafson, Zhiya Yu, Todd D. Prickett, Jared J. Gartner, Sivasish Sindri, Paul F. Robbins, Steven A. Rosenberg, James C. Yang. Using Organoids and Tumor Infiltrating Lymphocytes to Elucidate the ”Dark Matter” of Human Tumor Antigens [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-C011.