Abstract
Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC). Irradiated K562-derived Clone 9.mbIL21 aAPC were cocultured with PBMC, and propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex multicytokine assays, and a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of disseminated neuroblastoma. Coculturing patient PBMC with aAPC for 14 days induced 2,363- ± 443-fold expansion of CD56(+)CD3(-)CD14(-) NK cells with 83% ± 3% purity (n = 10). Results were similar to PBMC from normal donors (n = 5). Expression of DNAM-1, NKG2D, FcγRIII/CD16, and CD56 increased 6- ± 3-, 10- ± 2-, 21- ± 20-, and 18- ± 3-fold, respectively, on day 14 compared with day 0, showing activation of NK cells. In vitro, aNK cells were highly cytotoxic against neuroblastoma cell lines and killing was enhanced with GD2-specific mAb ch14.18. When mediating cytotoxicity with ch14.18, release of TNF-α, granulocyte macrophage colony-stimulating factor, IFN-γ, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cells increased 4-, 5-, 6-, 15-, 265-, 917-, and 363-fold (151-9,121 pg/mL), respectively, compared with aNK cells alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved aNK cells compared with untreated mice and was further improved when ch14.18 was added. Propagation of large numbers of aNK cells that maintain potent antineuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18.
Highlights
Outcome has steadily improved over the past 20 years for patients with high-risk, metastatic neuroblastoma (NBL), long-term event-free survival (EFS) is still only 45% [1,2,3]
ANK cells were highly cytotoxic against neuroblastoma cell lines, and killing was enhanced with GD2-specific monoclonal antibody ch14.18
Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved activated NK (aNK) cells compared to un-treated mice and was further improved when ch14.18 was added
Summary
Outcome has steadily improved over the past 20 years for patients with high-risk, metastatic neuroblastoma (NBL), long-term event-free survival (EFS) is still only 45% [1,2,3]. Strategies include modifying monoclonal antibodies to have high affinity interaction with NK cell FcγRIII/CD16 or to deliver cytokines via mAb-cytokine fusion proteins to the tumor microenvironment [4], enhancing activation of NK cells in vivo using immune modulating drugs such as lenalidomide [5, 6], or growing and activating NK cells ex vivo for adoptive cell therapy. Some of these approaches may be combined with cytotoxic chemotherapy or targeted therapy for more effective treatment of measurable disease
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