Immune system cells have been used for cancer treatment in recent years. T-cells genetically modified with a chimeric antigen receptor (CAR) has shown promising clinical results for leukemias and lymphomas. However, some adverse effects associated to CAR-T cells have been described and many efforts have been made to improve alternative strategies for cancer treatment. Studies with natural killer cells (NK) expressing CAR (CAR-NK) have shown exciting results with reduced adverse effects associated. Nevertheless, limitations still exist, such as decreased cell proliferation in vitro and in vivo and low expression of CAR vector. Strategies focusing on the development of specific CAR molecules for NK cells may generate CAR-NK cells with greater capacity for proliferation, activation, cytokine secretion and cytolytic activity, resulting in greater therapeutic efficacy. The aim of this study is to develop a new combinations of CAR intracellular domains and to improve CAR-NK signaling and cytotoxicity functions. Methods: Lentiviral vectors containing CARs composed of an extracellular single-chain variable fragment (scFv) anti-CD19 followed by different combination of co-receptors and cytokines involved to activation of NK cells were designed to generate CAR-NK cells. Conformational protein analysis was performed to confirm the potential functionality of CARs. Results and discussions: Although CAR-T cells strategies in NK cells are effective, initiation of signal transduction in NK cells can involve different pathways and diverge significantly from T cells. Therefore, the systematic application of CAR-T-cell strategies in NK cells might not be an optimized strategy for NK cells. For the improvement of CAR-NK cells we developed three new vectors containing CD3z or DAP12 as stimulatory domain and 4-1BB or 2B4 as co-stimulatory domain. In addition, all vectors contain the interleukin 15: 1) CD19.CAR-41BB-DAP12-IL15; 2) CD19.CAR-2B4-CD3z-IL15; 3) CD19.CAR-2B4-DAP12-IL15. Conclusion: The results of this work may help to establish a good condition for CAR-NK transduction and expansion, as well the development of strategies to improve the persistence of CAR-NK in vivo, what is extremely important for improvement of protocols for cancer therapy in the future. The next steps include the transduction of primary NK and NK cell line with different vectors and the expansion analysis of each CAR-NK cell in vitro. Lastly, the therapeutic effectiveness of these cells will be evaluated in vitro and in vivo. Financially supported by the FAPESP 2019/25309-0, CTC Center for Cell-based Therapies (FAPESP 2013/08135-2) and National Institute of Science and Technology in Stem Cell and Cell Therapy (CNPq 573754-2008-0 and FAPESP 2008/578773).