PROJECT SUMMARY Immune checkpoint inhibitors have revolutionized the treatment of cancer because they yield dramatic responses and long-lasting therapeutic benefits in previously untreatable cancers (e.g. metastatic melanoma). Despite this promise, the responses to immune checkpoint inhibitors have been restricted to small subsets of patients and are associated with toxic, and potentially fatal immune-related adverse events. In response to this urgent need to devise novel therapeutic strategies that dramatically increase the response rate to immune checkpoint inhibitors, we have engineered an ensemble nanoimmunotherapy that combine the advantages of nanotechnology and immunotherapy. Specifically, our ensemble comprises: Prussian blue nanoparticles (PBNPs) biofunctionalized with immune adjuvants and administered in combination with systemically administered checkpoint inhibitors. We utilize the PBNPs for photothermal therapy, which ablates tumor cells, releasing tumor antigens, and damage-associated molecular patterns that increase tumor immunogenicity. The loss of immunogenicity is one of the key immune escape mechanisms in cancer. Additionally, the PBNPs are biofunctionalized to serve as a depot for local delivery of exogenous immune adjuvants, particularly toll-like receptor agonists that play an important role in breaking tolerance to tumor antigens and improving tumor antigen presentation, which is impaired in immunosuppressive tumors. These effects are complemented by systemically administered checkpoint inhibitors (anti-PD-1 and anti-CTLA-4) that reverse suppression of immune cell responses (particularly T cells) and unleash their potent antitumor effects. We believe that this ensemble approach of targeting tumor cells, antigen presenting cells, and T cells may hold the key in converting a non-responsive ?cold? tumor to a responsive ?hot? tumor. In Aim 1, we seek to determine whether the PBNPs biofunctionalized with toll-like receptor agonists and used for photothermal therapy elicits immunogenic cell death and improves antigen presentation. In Aim 2, we test the efficacy of the ensemble nanoimmunotherapy on tumor eradication and preventing relapse. In Aim 3, we evaluate the efficacy of the nanoimmunotherapy in treating disseminated cancer. The successful completion of the project will provide critical insight into the use of multifunctional nanoparticles in combination with immunotherapies to overcome tumor immune evasion mechanisms and improve therapeutic outcomes. Importantly, it will provide the impetus for clinical translation of our nanoimmunotherapy, thereby extending its lasting benefits to a larger proportion of cancer patients.