TRLS-12 Pd-1 and Lag3 Combination Blockade Therapy Reduces Melanoma Brain Metastatic Tumor Growth in Preclinical Models and Is Abrogated by Neutrophil Infiltration

Abstract BACKGROUND Previous clinical trials with PD-1 and LAG3 combination blockade showed significantly improved survival in patients with advanced melanoma. However, the efficacy of the immunotherapy combination has not been evaluated in melanoma patients with brain metastasis, which is frequently observed in melanoma. METHODS Intracranial bolting of mice with syngeneic mouse melanoma cell lines were used to develop melanoma brain metastatic tumors in an immunocompetent system. Mice were treated with antibodies targeting mouse PD-1 and LAG3. CyTOF, multiplex immunofluorescence (mIF), scRNAseq, and spatial transcriptomics were used to assess changes in tumor immune microenvironment across responder and resistant models. RESULTS Treatment of mice with PD-1 and LAG3 combination blockade significantly reduced melanoma brain metastatic tumor growth which was dependent on increased infiltration of pro-inflammatory immune cells including T cells, NK cells, and dendritic cells. In resistant models, tumors exhibited an increase in anti-inflammatory Ly6G+ neutrophils and FOXP3+ regulatory T cells. Addition of anti-Ly6G, anti-CTLA4, or radiation therapy further enhanced melanoma tumor response to PD-1 and LAG3 combination blockade. Tissue analysis of a melanoma patient with brain metastasis treated with PD-1 and LAG3 blockade showed increased necrotic tissue with high infiltration of granzyme-B+ CD8 T cells and dendritic cells, suggesting patient response to combination immunotherapy. CONCLUSIONS PD-1 and LAG3 immune checkpoint blockade significantly reduces melanoma brain metastatic tumor growth. Increased neutrophil and regulatory T cells are associated with resistance to the combination immunotherapy and treatments targeting these immune populations can sensitize resistant tumors to PD-1 and LAG3 blockade. Future studies aim to identify the mechanism of neutrophil recruitment and the functional role at promoting immune suppression.