The results of this study indicate that, among pediatric solid tumors, EWS cells are exquisitely sensitive to the cytotoxicity exerted by expanded, activated NK cells. Expanded NK cells could kill nearly all EWS cells within 4 hours at a 1 : 1 ratio, and still be considerably cytotoxic at a 1 : 10 or lower ratios if cultures were prolonged for 24 hours. Expanded NK cells had much more powerful anti-EWS capacity than primary, non-expanded, NK cells. The anti-EWS effect of NK cells was also seen in experiments with immunodeficient mice bearing EWS tumors, where NK cell infusions produced durable remissions. The interaction between NKG2D and DNAM-1 on the surface of NK cells with their ligands on tumors cells appears to be critical for NK cell cytotoxicity against EWS cells, as interference with these molecules considerably reduced cell killing. However, the key ligands remain to be identified.
The results with EWS cells recall those that we previously obtained with expanded NK cells against acute myeloid leukemia (AML) cells [32
], suggesting that EWS and AML are the main targets for NK cell therapy of cancer in children. Most cell lines derived from other cancer cell types were also sensitive to expanded NK cell cytotoxicity. For example, rhabdomyosarcoma cells were as sensitive as EWS at 1 : 1 E :T ratio but less sensitive when the proportion of NK cells was reduced. The sensitivity of osteosarcoma cells varied widely. The molecular mechanisms underlying these differences remain to be elucidated but cannot be attributed to the levels of HLA Class I expression in the tumor cells alone. Neuroblastoma cell lines were significantly less sensitive than EWS cells to expanded NK cells, suggesting that neuroblastoma should have a lower priority among pediatric solid tumors eligible for NK cell therapy. NK cell cytotoxicity against neuroblastoma cells might be considerably enhanced by directing NK cells with antibodies reacting with neuroblastoma cell markers [46
]. Moreover, Altvater et al. [47
] using the method described here to expand NK cells demonstrated that cytotoxicity could be considerably enhanced by transducing NK cells with a chimeric signaling receptor recognizing the neuroblastoma cell marker GD2.
Expanded NK cells resulted in a substantial anti-tumor effect in mice engrafted with EWS cells, apparently achieving disease eradication in some animals. In our experiments, we also administered IL-2, as we previously observed that this cytokine significantly prolongs the survival of NK cells in immunodeficient mice [32
]. Moreover, administration of IL-2 is often included in clinical NK cell infusions [17
]. We also administered multiple infusions of NK cells, as in preliminary experiments, single infusions of NK cells had anti-tumor activity but did not eradicate the disease. As a caveat, the xenografts that we included in our experiments are not orthotopic models of EWS, and NK cells might not migrate effectively to tissues were EWS tumor cells typically reside, resulting in suboptimal E : T ratios at the tumor site. However, we suggest that NK cell infusions might be more effective against tumor cells metastasizing to the bone marrow and lymphoid organs (tissues where NK cells thrive), rather than against primary tumor masses. Thus, infusions of allogeneic expanded NK cells should enhance the effects of chemotherapy followed by autologous hematopoietic cell transplantation, while reducing the risk of disease recurrence from carry over tumor cells infused with the autograft. Alternatively, NK cells could be directly injected into the tumor site after surgical removal, in efforts to deplete the margins of residual tumor cells.
To be effective, NK cell infusions must achieve E : T ratios estimated to exert substantial tumor cytoreduction. In this regard, the capacity of our method to specifically expand NK cells many fold, in addition to activate them, is particularly useful. Seven-day cultures, such as those used in this study, should yield sufficient NK cells for single infusions. Additional stimulation with K562-mb15-41BBL cells and extended cultures can dramatically increase the number of NK cells obtained, which should be adequate for multiple infusions. The NK expansion method described here has now been adapted to large scale, clinical grade, conditions, and a clinical protocol testing the feasibility and toxicity of this approach is now open. Because of the results of this study, eligibility includes patients with EWS and rhabdomyosarcoma.