Abstract Introduction: Intra-tumor heterogeneity (ITH) is a major driver of treatment resistance. ITH also affects anti-tumor immunity, with immune cell infiltration, neo-antigen expression and T cell receptor (TCR) profiles differing between separate regions of an individual tumor. However, the extent to which separate tumor subclones differ in their capacity for immune evasion, the tumor-intrinsic mechanisms underlying any such heterogeneity, and its impact on cancer immunosurveillance remain largely unexplored. We have previously developed personalized models of anti-tumor immunity, based on co-cultures of cancer organoids and autologous T-cells. These co-culture systems can be used to evaluate the efficacy of cancer immunosurveillance at the level of an individual patient. Approach: Here, we leverage the multi-region TRACERx lung cancer evolution study to generate a patient-derived study platform that allows the evaluation of T-cell responses to individual cancer subclones. We generated libraries of >20 separate non-small cell lung cancer (NSCLC) organoid lines, based on isolating individual (clonal) organoids established from multiple spatially separated tumor regions. Each organoid subline was co-cultured with autologous tumor infiltrating lymphocytes (TIL) to evaluate how they differ in their capacity to elicit a T-cell response. Results: Our data reveal heterogeneity between individual clonal organoid sublines in their capacity to stimulate TIL. The proportion of TIL being activated by a particular subclone, as measured by 4-1BB (CD137) expression, ranged from 5 to 42%. These differences could not be explained by differences in MHC class I or PD-L1 expression. We are currently using DNA, RNA and TCR sequencing to characterize ‘immune evading’ and ‘non-immune evading’ sublines. Data will be updated on emerging subclonal immune evasion mechanisms inferred through DNA/RNA/TCR sequencing. Conclusion: Individual cancer subclones show differences in the degree of immune evasion. This patient-derived study platform allows moving beyond descriptive analyses of the heterogeneity of anti-tumor immunity, allowing fine-grained functional studies of how ITH affects cancer immunosurveillance. Citation Format: Krijn K. Dijkstra, Roberto Vendramin, Robert E. Hynds, David R. Pearce, Despoina Karagianni, Felipe Gálvez-Cancino, Oriol Pich, Mark S. Hill, Vittorio Barbè, Andrew Rowan, Selvaraju Veeriah, Cristina Naceur-Lombardelli, Antonia Toncheva, Supreet Bola, Mariam Jamal-Hanjani, Crispin Hiley, Kevin Litchfield, James Reading, Sergio A. Quezada, Charles Swanton, TRACERx consortium. Patient-derived co-cultures of TRACERx lung cancer organoids and autologous T-cells reveal heterogeneity in immune evasion between cancer subclones abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 692.