| Summary: | In contrast to T lymphocytes, natural killer (NK) cells do not require prior sensitization but are rapidly activated upon encountering virally infected or neoplastic cells. In addition, NK cells can be safely applied in an allogeneic setting, making them important effector cells for the development of off-the-shelf therapeutics for adoptive cancer immunotherapy. To further enhance their therapeutic potential, here, we engineered continuously expanding NK-92 cells as a clinically relevant model to express a humanized second-generation chimeric antigen receptor (CAR) with a composite CD28-CD3ζ signaling domain (hu14.18.28.z) that targets the disialoganglioside GD<sub>2</sub>, which is expressed at high levels by neuroblastoma cells and other tumors of neuroectodermal origin. In a separate approach, we fused an IL-15 superagonist (RD-IL15) to the GD<sub>2</sub>-CAR via a P2A processing site. Lentivirally transduced NK-92/hu14.18.28.z and NK-92/hu14.18.28.z_RD-IL15 cells both displayed high and stable CAR surface expression and specific cytotoxicity toward GD<sub>2</sub>-positive tumor cells. GD<sub>2</sub>-CAR NK cells carrying the RD-IL15 construct in addition expressed the IL-15 superagonist, resulting in self-enrichment and targeted cell killing in the absence of exogenous IL-2. Furthermore, co-culture with RD-IL15-secreting GD<sub>2</sub>-CAR NK cells markedly enhanced proliferation and cytotoxicity of bystander immune cells in a paracrine manner. Our results demonstrate that GD<sub>2</sub>-CAR NK cells co-expressing the IL-15 superagonist mediate potent direct and indirect antitumor effects, suggesting this strategy as a promising approach for the further development of functionally enhanced cellular therapeutics.
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