Abstract Over the last decade Chimeric Antigen Receptor based T cell therapy (CAR-T) has developed into an effective immunotherapy for some cancers. However, CAR-T cell therapies have several shortcomings and clinical success has primarily been limited to hematological cancers. Challenges of CAR-T cell therapy include tumor immune evasion through loss of target antigen expression by tumor cells and inhibition of CAR-T cell function by tumor expressed inhibitory molecules. Natural killer (NK) cells present an alternative to T cells that could be more effective due to their ability to perform both antigen dependent and independent killing. NK cells have demonstrated antigen specific killing when engineered to express T cell CARs and NK cells also mediate the direct killing of transformed cells with reduced or absent MHC expression. In fact, NK cells carry out antibody dependent cell mediated cytotoxicity (ACDD) of cells that bind antibodies via the NK cell CD16A receptor. Due to the multiple modalities for cancer cell killing, there is an increased interest in NK cells for cancer immunotherapy. As NK cells are not associated with graft versus host disease, neurotoxicity, long-term autoimmunity, nor cytokine release syndrome, they are more suited for use in allogeneic settings than T cells and have significant clinical potential for use as off-the-shelf products. However, previous publications and clinical trials have demonstrated that the use of unmanipulated NK cells to treat cancer is minimally effective, likely due to limited engraftment, little in vivo expansion, and suppression by the tumor microenvironment. NK cells activated and expanded with feeder cells expressing membrane bound interleukin-21 (mbIL-21) have shown promising results clinically with high-risk myeloid malignancies and preclinically in several solid tumor models. Therefore, we hypothesize that activated/expanded NK cells that have be genetically edited can be used to successfully treat osteosarcoma, a disease for which patient outcome has not improved in over thirty years. Our proposed objectives are to evaluate the baseline response of rested- and activated-NK cells against various osteosarcoma cell lines, knockout negative regulators of NK cell function (specifically, c-CBL, IL-1R8, and SMAD3), and implement a specific CAR that optimally activates NK cell antigen-specific killing. Genetically engineered NK cells will be evaluated for enhanced therapeutic efficacy and safety in osteosarcoma models. Our preliminary data strongly supports the hypothesis that NK cell-based cancer immunotherapy can be fully realized using activated, genome engineered NK cells.