SUMMARY This proposal aims to translate our engineered extracellular vesicles (EVs) into an effective cancer therapy. Major barriers to effective chimeric antigen receptor (CAR)-T cell therapy in solid cancers include limited CAR-T cell tumor infiltration, loss of their function in the tumor microenvironment (TME), and severe life-threatening toxicities. Designer EVs from cytotoxic immune cells have the potential to overcome these crucial issues and become effective cell-free immunotherapy for solid cancers. In response to PAR-22-071, we will optimize our designer EVs derived from engineered Nature Killer (NK) cell line NK92 to treat melanoma, the deadliest skin cancer. Death Receptor 5 (DR5) is highly expressed in cancer cells, myeloid-derived suppressor cells (MDSCs), and cancer-associated fibroblasts (CAFs). MDSCs and CAFs are the major components of the immunosuppressive TME. We fortuitously discovered that DR5-agonistic single-chain variable fragments (scFvs) were highly effective in killing DR5+ tumor cells when expressed on non-cytotoxic lymphoblastic lymphoma Sup-T1 cells. We developed a new lentiviral vector to deliver more DR5-scFvs to the surface of EVs and infected NK92 cells with the vectors. EVs from the engineered cells (DR5-scFv EVs) were more effective in killing DR5+ melanoma than DR5 antibodies and DR5-CAR-T cells. Systemic delivery of DR5-scFv EVs significantly inhibited DR5+ tumor growth in vivo in multiple cancer models. A unique feature of DR5-scFv EVs is that they significantly inhibited MDSCs and CAFs in vitro and in vivo and increased CD8+ T cell functions in patient-derived organotypic cultures. Loading siRNA to mutant NRAS into DR5-scFv EVs further enhanced their tumor-killing effect on melanoma with mutant NRAS. We will further develop DR5-scFv EVs for clinical translation. In Aim 1, we will optimize cytotoxicity and immune stimulatory functions of DR5-scFv EVs using different approaches and test these designer EVs in state-of-the-art models, such as patient-derived organotypic melanoma cultures and genetically annotated melanoma patient-derived xenografts (MPDXs). Aim 2, we will test the designer EVs in mouse clinical trials using genetically annotated MPDXs in humanized mice. In Aim 3, we will optimize bioprocessing and standardize workflow for the engineered EVs production to increase DR5-scFv-EVs yields and develop Standard Operating Procedures (SOPs) for upstream production, downstream process, and quality controls. This proposal will prepare our designer EVs for GMP production and to be explored as an alternative therapy for CAR- T cells. We expect that our designer EVs will be qualified for NCI translational programs to continue a path toward the clinic upon completing the proposal.