Natural killer (NK) cells rely on surface receptors to distinguish healthy cells from cancer cells. We designed a receptor termed NKG2D-DAP10-CD3z that is composed of the NK cell activating molecule NKG2D plus 2 key signaling molecules, DAP10 and CD3z, and evaluated its capacity to promote cancer cell killing. Retroviral transduction of NKG2D-DAP10-CD3z markedly increased NKG2D surface expression in NK cells, which became consistently more cytotoxic than mock-transduced cells against leukemia and solid tumor cell lines. In contrast, there was no increase in cytotoxicity against nontransformed blood and mesenchymal cells. NKG2D blockade abrogated gains in cytotoxicity to cancer cells. Receptor stimulation triggered signal transduction, secretion of IFN-g, GM-CSF, IL-13, MIP-1a, MIP-1b, CCL5, and TNF-a, and massive release of cytotoxic granules, which persisted after 48 hours of continuous stimulation. NKG2D-DAP10-CD3z-expressing NK cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas activated NK cells were ineffective. Thus, the cytotoxic potential of NK cells against a wide spectrum of tumor subtypes could be markedly enhanced by expression of NKG2D-DAP10-CD3z receptors. The development of an electroporation method that permits rapid expression of the receptor in a large number of human NK cells facilitates clinical translation of this NK-based strategy for a generalized cellular therapy that may be useful to treat a wide range of cancers. Cancer Res; 73(6);

Purpose To develop new therapies for children with solid tumors, we tested the cytotoxicity of natural killer (NK) cells expanded by coculture with K562-mb15-41BBL cells. We sought to identify the most sensitive tumor subtypes, clarify the molecular interactions regulating cytotoxicity, and determine NK anti-tumor potential in vivo. Experimental Design We tested in vitro cytotoxicity of expanded NK cells against cell lines representative of Ewing sarcoma (EWS) (n=5), rhabdomyosarcoma (n=4), neuroblastoma (n=3) and osteosarcoma (n=3), and correlated the results with expression of inhibitory and activating NK receptor ligands. We also compared expanded and primary NK cells, determined the effects of activating-receptor ligation and of chemotherapeutic drugs, and assessed the therapeutic effect of NK cell infusions in xenografts. Results In 45 experiments, EWS and rhabdomyosarcoma cell lines were remarkably sensitive to expanded NK cells, with median cytotoxicities at 1:1 effector:target ratio of 87.2% and 79.1%, respectively. Cytotoxicity was not related to levels of expression of NK receptor ligands, nor was it affected by pretreatment of target cells with daunorubicin or vincristine, but was markedly inhibited by preincubation of NK cells with a combination of antibodies against the NK activating receptors NKGD2 and DNAM-1. Expanded NK cells were considerably more cytototoxic than unstimulated NK cells, and eradicated EWS cells engrafted in NOD/scid IL2RGnull mice. Conclusions Among pediatric solid tumors, EWS and rhabdomyosarcoma are exquisitely sensitive to expanded NK cells. The NK expansion method described here has been adapted to large-scale conditions, and supports a Phase I clinical study including patients with these malignancies.

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