ABSTRACT Melanoma is a serious form of skin cancer that develops in skin color cells (melanocytes). When melanomas progress to an advanced stage, treatment options are limited and are associated with significant morbidity and mortality. In this grant application, we propose to develop a combinatorial immunotherapy to treat metastatic melanoma. Several studies have revealed that the cytokine TRAIL (tumor necrosis factor-related apoptosis- inducing ligand) induces apoptosis in a wide variety of tumor cells, but does not cause toxicity to most normal cells. Treating this disease with TRAIL holds great promise, but it is not simple, because the likely agent to treat the disease, recombinant TRAIL, does not last long enough in the body to be effective. We have found a way to have TRAIL reside in another cell such as the natural killer (NK) cell; that cell then secretes TRAIL on a long-term basis. Importantly, NK cells have an additional advantage--they seek out melanoma cells, so they bring TRAIL directly to tumor sites, where it can exert its profound tumor-killing effects. Recent clinical trials have shown that anti-PD-1 (programmed cell death-1) and anti-PD-L1 (programmed cell death-ligand 1) agents have impressive antitumor effects in metastatic melanoma. Thus, we hypothesize that the combination of secretory TRAIL-armed NK cells with anti-PD-1/PD-L1 agents will effectively enhance the tumoricidal efficacy of NK cells. To test the hypothesis, (1) we will assess the biological effect of secretory TRAIL-armed NK cells and anti-PD-1/PD-L1 agents on metastatic melanoma cells, and (2) we will examine the preclinical efficacy of combinatorial immunotherapy with secretory TRAIL-armed NK cells and anti-PD-1/PD-L1 agents in a xenograft mouse model of metastatic melanoma. The proposed studies for the first aim will examine the cytotoxicity of melanoma cells during combined treatment with secretory TRAIL-armed NK cells and anti-PD- 1/PD-L1 agents. In the second aim, we will employ a mouse xenograft model to assess the effect of treatment with secretory TRAIL-armed NK cells in combination with anti-PD-1/PD-L1 agents on growth and regression of metastatic melanoma. We believe that the successful outcome of this study will support the application of this combinatorial immunotherapy to patients with advanced malignant melanoma.