Abstract B005: Identification of tumor-reactive TILs from lung cancer organoids co-culture through ruthenium-based proximity labeling

Abstract Tumor-reactive T cells are crucial for the success of tumor-infiltrating lymphocyte (TIL) therapies. Currently, identifying tumor-reactive T cells and their corresponding TCRs typically relies on pre-characterized tumor antigens. Many solid tumors, including lung cancer, exhibit considerable heterogeneity and consequently possess diverse tumor antigenic profiles. Markers such as PD-1 and CD39 are commonly used to identify tumor-reactive TILs, yet these empirically derived markers can produce false positives or fail to capture rare tumor-reactive populations. We here used an unbiased ruthenium-based photocatalytic proximity labeling approach to do co-culture of TILs derived from lung cancer and autologous tumor organoids to identify and validate tumor-specific T cells and their TCRs in six NSCLC samples. We found that labeled TILs were significantly enriched for tumor antigen-reactive T cells, which could be readily separated from bystander T cells via flow cytometry. Single-cell TCR and RNA sequencing revealed that labeled TILs, in contrast to bystander TILs, exhibited a distinct TCR repertoire and transcriptional profile, characterized by effector cytotoxic and activation signatures with partial expression of exhausted T cell markers. We selected the top 10 CD8+ TCR clones with the highest labeling intensity and assessed the reactivity of their corresponding TCR-transduced, CD8-overexpressing Jurkat cells against autologous organoids. We identified 5 novel tumor antigen-reactive TCRs that demonstrated tumor responsiveness and these T cells displayed a mixed transcriptional profile, including hallmark markers of exhaustion. Additionally, we identified clonally expanded, cytotoxic CD4+ T cell states within the labeled TILs that could kill autologous tumors in an MHC class II-dependent and/or Fas-FasL-dependent manner. The ruthenium-based photocatalytic proximity labeling approach, which requires no gene editing, enables identification of tumor-reactive TILs and TCRs independent of specific antigen/HLA contexts. This platform, especially when combined with organoid technology, has the potential to streamline the development of personalized TIL and TCR-T cell therapies. Citation Format: Weilei Hu, Yifei Bian, Zihan Zhao, Shian Ouyang, Jie P. Li, Hongbin Ji. Identification of tumor-reactive TILs from lung cancer organoids co-culture through ruthenium-based proximity labeling [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B005.