PROJECT SUMMARY Cytokine immunotherapies, such as IL-2 and more recently IL-15, have been used to stimulate endogenous cytotoxic natural killer (NK) and T cell responses against advanced cancers and to support adoptively transferred lymphokine-activated killer cells. While responses have been achieved in some patients, the use of IL-2 and IL- 15 has been hampered by modest benefits with the risk of severe systemic toxicity, particularly when given intravenously (IV). Distinct from IV delivery, inhaled IL-15 therapy offers the advantages of concentrated delivery to the lungs, a frequent site of gross metastatic disease, while limiting systemic exposure and potential toxicity. Osteosarcoma (OSA) is an aggressive primary bone cancer with a high propensity for lung metastases, and outcomes for patients with lung metastases have been stagnant for the past 4 decades. In our recently completed (manuscript in preparation) first-in-kind trial of inhaled rhIL-15 in dogs with spontaneous lung metastases from OSA and melanoma, we observed clinical benefit in 37% (1 complete response, 1 partial response, and 5 stable disease at 60 days in 19 dogs). Here, we propose to evaluate the immune mechanisms of response to inhaled IL-15 in mouse and human models of metastatic OSA, hypothesizing that a unique tissue resident NK population in the lungs has heightened cytotoxic and proliferative responses consistent with a memory- like phenotype which enables augmented anti-tumor responses. In Aim 1, we will analyze lung resident NK cells (CD3-NKp46+CD49a+) in K7M2 and LM8 models of OSA, hypothesizing that inhaled IL-15 delivered by nebulizer will stimulate NK-mediated anti-tumor responses against OSA lung metastases. Matched human blood, non-tumor-bearing lung, and tumor samples from patients with OSA undergoing metastasectomy will be used to validate these mouse studies, in particular that human lung NK cells show features of heightened cytotoxicity and effector function which can be harnessed to infiltrate immunosuppressive OSA metastases in the setting on inhaled IL-15. In Aim 2, we will evaluate combination therapy. T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) has emerged as a critical marker of NK cell exhaustion which limits anti-tumor responses in multiple cancer types, including sarcomas. Thus, we will combine inhaled IL-15 with TIGIT blockade using anti-TIGIT monoclonal antibodies to assess NK phenotype and function, trafficking to lung tumors, persistence, and tumor regression following treatment in mouse models. Toxicity will also be carefully assessed given the potential for combination immunotherapy to precipitate immunopathology and cytokine storm. Metastatic OSA is a rare and difficult-to-treat cancer. Despite great success in other cancers, immunotherapy targeting PD-1, PD-L1, and CTLA-4 has shown minimal efficacy in OSA, underscoring the need for novel immunotherapies for these aggressive ca...