PROJECT SUMMARY/ABSTRACT: PROJECT 1 Melanoma is the most aggressive skin cancer and causes over 10,000 deaths per year in the US. While great strides have been made in the treatment of melanoma, many patients are still non-responsive to immunotherapy. Understanding the function of PD1 and LAG3 on the immune response in both the tumor and periphery of patients is critical to the progress of immunotherapy in melanoma. Although LAG3 is the third ‘checkpoint’ to be targeted with >10 agents in clinical trials, we still know very little about how LAG3 blockade, alone or with anti- PD1, impacts the immune response to melanoma. We have demonstrated synergistic PD1/LAG3 combinatorial immunotherapy in mouse models of cancer and clear evidence of clinical benefit from dual inhibitory receptor (IR) blockade has stimulated anti-PD1 and anti-LAG3 trials in cancer patients failing previous immunotherapies. Our overarching hypothesis is that LAG3 and PD1 single-agent blockades differentially regulate, and synergize to promote, CD8+ T cell function in the tumor and peripheral blood of MPs and that a LAG3-dominant IR module in peripheral CD8+ T cells downregulates patient response to PD1-targeted therapy. Specific Aim 1. What is the mechanism of anti-PD1 (nivo) and anti-LAG3 (rela), alone and in combination, on the phenotype and function of CD8+ T cells? We have initiated accrual in a unique biomarker-focused phase II randomized clinical trial to evaluate the combination of anti-PD1 and/or anti-LAG3 therapy in treatment naïve melanoma patients. We hypothesize that T cell activation and proliferation pathways are differentially regulated in CD8+ T cells by anti-PD1 vs. anti-PD1/LAG3 and that unique synergistic molecular programs will be revealed by this immunotherapeutic combination in treatment-naïve patients with metastatic melanoma. Our novel trial design will assess the mechanistic impact of anti-PD1 and/or LAG3 immunotherapy in peripheral and tumor CD8+ T cells. Specific Aim 2. Does IL6 drive a LAG3-dominant IR module in peripheral CD8+ T cells, and does it predict responsiveness to immunotherapy? We have recently made a series of novel findings regarding IR expression in peripheral CD8+ T cells that implies a novel mechanism of immune resistance in melanoma patients. We hypothesize that IL6-driven systemic immune dysfunction and subsequent resistance to anti-PD1 therapy is driven by a LAG3-dominant IR module and that this dysfunction can be ameliorated by anti-LAG3/PD1 blockade. This project will [i] define new biomarkers of response and/or resistance to nivo, rela, and the combination; [ii] potentially identify a patient population that will optimally benefit from LAG3-based therapies; and [iii] develop novel combinatorial immunotherapies of increased efficacy in melanoma.