title: Growth and activation of natural killer cells ex vivo from children with neuroblastoma for adoptive cell therapy.
abstract: OBJECTIVE
  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).
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Propagation of large numbers of aNK cells that maintain potent antineuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18.
authors: Division of Hematology/Oncology and Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California, USA.
authors: Yin
authors: Y
authors: Liu
authors: Hong-Wei
authors: HW
authors: Wu
authors: Michael A
authors: MA
authors: Sheard
authors: Richard
authors: R
authors: Sposto
authors: Srinivas S
authors: SS
authors: Somanchi
authors: Laurence J N
authors: LJ
authors: Cooper
authors: Dean A
authors: DA
authors: Lee
authors: Robert C
authors: RC
authors: Seeger
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology; D019002:CD56 Antigen / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002648:Child; D018920:Coculture Techniques; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods; D020014:K562 Cells; D053208:Kaplan-Meier Estimate; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism / Q000637:transplantation; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008213:Lymphocyte Activation / Q000276:immunology*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009447:Neuroblastoma / Q000276:immunology* / Q000628:therapy; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D019002:CD56 Antigen; D016207:Cytokines; C112746:dinutuximab
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