Abstract 7289: High CD8+ T cell TIM-3 expression predicts for sensitivity to TIM-3 based triplet checkpoint inhibitor combinations

Abstract Background: Melanoma response to the immune checkpoint inhibitor (ICI) combinations such as anti-PD-1, anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 is not durable. The triplet combination of anti-PD-1+CTLA-4+LAG-3 is being studied clinically in multiple cancers, including advanced melanoma. This study comprehensively describes the mechanism of action of this ICI triplet, and a second triplet of anti-PD-1+LAG-3+TIM-3, in 3 mouse melanoma models with varying levels of sensitivity to ICI. Methods: Syngeneic mouse models of SM1, SW1 and B16-10 melanoma flank tumors were used to explore the response to anti-PD-1+LAG-3, anti-PD-1+CTLA-4, anti-PD-1+LAG-3+CTLA-4 triplet and anti-PD-1+TIM-3 doublet and anti-PD-1+LAG-3+TIM-3 triplet. We performed flow cytometry on the responding and resistant tumors, tetramer bindings assays, T-cell depletion and IHC to determine the mechanisms of action of these ICI combinations. Additionally, we analyzed scRNA-Seq data from sensitive and ICI resistant human melanoma to determine the overlap in immune checkpoint expression across T -cell subsets. Results: The PD-1+CTLA-4+LAG-3 triplet combination demonstrated good efficacy in the SM1 and B16-F10 models and was associated with a more favorable immune response such as increased CD69+ CD8+ T cells, reduced exhausted T cells and increased GP100-tumor specific T cells than either doublet. The ICI-resistant SW1 model, did not respond to the PD-1+CTLA-4+LAG-3 triplet and exhibited accumulation of TIM-3+ exhausted CD8+ T cells. Targeting elevated TIM-3 on CD8+ T cells, using the anti-PD-1+LAG-3+TIM-3 triplet, but not the PD-1+TIM3 doublet, restored T cell activity and led to a remarkable anti-tumor activity in SW1 model, with some mice showing complete tumor regression. The PD-1+LAG-3+TIM-3 triplet was also highly effective in the SM1 model showing increased percentage of CD69+ CD8+ and tumor specific T cells and reduced exhausted T cells. scRNA-Seq analysis showed that TIM-3 levels were significantly higher in the CD8+ T cells of melanoma patients failing PD-1 therapy. We also observed a good correlation between LAG-3 and TIM-3 expression in the CD8+ T cells. Hence, co-targeting these two checkpoints may possibly reverse the T cell exhaustion. Conclusion: In conclusion, our results demonstrate that ICI resistance associated with high TIM-3 expression in the CD8+ T cells can be overcome through use of a PD-1+LAG-3+TIM-3 combination. The observation that high TIM-3 expression in CD8+ T cells is associated with PD-1 resistance suggests the potential to use anti-TIM3 based ICI combinations as a second-line treatment strategy in patients with advanced melanoma. Citation Format: Manali S. Phadke, Jiannong Li, Sathya Sriramareddy, Paulo Rodriguez, Brian Ruffel, Vincent Luca, Ann Chen, Keiran S.M. Smalley. High CD8+ T cell TIM-3 expression predicts for sensitivity to TIM-3 based triplet checkpoint inhibitor combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7289.