Natural killer (NK) cells are potent antitumor effectors that play an important role in innate and adaptive immunity. Despite recent clinical advances in the therapeutic use of NK cells, significant opportunities remain to harness their full potential in adoptive immunotherapy. For example, achieving consistent manufacturing of cancer immunotherapies using patient- and donor-sourced cells remains a significant challenge to delivering therapies to all patients who may benefit. There is also the need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote favorable patient outcomes. We have previously demonstrated that induced pluripotent stem cells (iPSCs) can be genetically edited at the single-cell level and expanded to create clonal master engineered pluripotent cell lines, representing a renewable and reliable starting material for the creation of off-the-shelf (OTS) adoptive NK cell therapy. Using this unique platform, we evaluated the combination of NK cell-specific chimeric antigen receptors (CARs) with an autonomous protein to create a highly effective, persistent, and targeted NK cell therapy. The NK cell optimized CAR (NK-CAR) backbone contains the NKG2D transmembrane domain and the 2B4 co-stimulatory and the CD3ζ signaling domains to mediate a strong increase in NK cell signaling. To provide directed antitumor activity, anti-mesothelin (meso) and anti-CD19 scFvs were added to the NK-CAR backbone, and then engineered into the iPSC and subsequently differentiated to NK cells expressing NK-CARmeso or NK-CAR19. Using an ovarian cancer xenograft model in the initial study, a single dose of NK-CARmeso NK cells markedly inhibited tumor growth and mediated enhanced survival (84 days) compared to various controls, including NK cells harboring a 3rd-generation T-cell CAR construct with CD28 and 4-1BB costimulatory domains (p Citation Format: Tom T. Lee, Ye E. Li, Ryan Bjordahl, Robert Blum, Sajid Mahmood, Huang Zhu, Gregory B. Bonello, Bahram Valamehr, Dan S. Kaufman. Cytokine-autonomous, CAR-directed, off-the-shelf natural killer cells derived from a clonal engineered master pluripotent cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3574.