Identification of anti-TIM-3 based checkpoint inhibitor combinations with activity in immunotherapy refractory melanoma models.

backgroundA significant percentage of melanomas are refractory to immune checkpoint inhibitor (ICI) monotherapies and combinations. As there are currently no effective second-line therapies available for ICI-resistant patients, we sought to identify novel checkpoint inhibitor combinations for future clinical evaluation.

methodsWe used responding and resistant SM1, SW1 and B16-10 melanoma models to explore the mechanism of action of anti-programmed cell death protein 1 (PD-1)+lymphocyte activation gene (LAG-3), anti-PD-1+cytotoxic lymphocyte associated protein 4 (CTLA-4), the anti-PD-1+LAG-3+CTLA-4 triplet, the anti-PD-1+T-cell immunoglobin and mucin domain 3 (TIM-3) doublet and the anti-PD-1+LAG-3+TIM-3 triplet. Flow cytometry, tetramer binding assays, T-cell depletion and analysis of the tumor-draining lymph nodes were used to determine the mechanisms of action of the ICI combinations. Single-cell RNA sequencing (scRNA-Seq) data from patients with melanoma was additionally analyzed to determine immune checkpoint expression across T-cell subsets and their association with ICI resistance.

resultsThe PD-1+CTLA-4+LAG-3 triplet combination had good efficacy in the SM1 and B16-F10 melanoma models and led to a more favorable immune microenvironment (increased CD69+CD8+ T cells and gp-100-tumor specific T cells, reduced exhausted T cells) than either doublet. The ICI-resistant SW1 model did not respond to the PD-1+CTLA-4+LAG-3 triplet and was characterized by an accumulation of TIM-3+terminally exhausted CD8+T cells. Targeting of TIM-3 using the anti-PD-1+LAG-3+TIM-3 triplet led to complete tumor regressions in the SW1 and SM1 models. The TIM-3-based triplet decreased exhausted CD8+T cells and regulatory T cells, increased numbers of gp-100 reactive CD8+T cells and increased progenitor-exhausted CD8+T cells (TCF7+Ki67+ TIM-3-) numbers in tumor-draining lymph nodes. scRNA-Seq analysis demonstrated that TIM-3 levels were higher in the CD8+T cells of patients with melanoma who were non-responsive to immunotherapy. PD-1 and TIM-3 expression was strongly correlated in the CD8+T cells from patients with melanoma, providing the rationale for co-targeting these checkpoints.

conclusionMelanoma models characterized by high levels of terminally exhausted CD8+T cells (TIM-3+) can be successfully treated with the anti-PD-1+LAG-3+TIM-3 combination. The observation that high TIM-3 expression in CD8+T cells is associated with ICI resistance suggests the potential utility of anti-TIM-3-based combinations as a second-line treatment strategy in patients with advanced melanoma.