Division of Hematology/Oncology and Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California, USA.
Clin Cancer Res. 2013 Apr 15;19(8):2132-43. doi: 10.1158/1078-0432.CCR-12-1243. Epub 2013 Feb 1.
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.
过继性转移自然杀伤 (NK) 细胞联合肿瘤特异性单克隆抗体 (mAb) 对恶性肿瘤具有治疗潜力。我们通过人工抗原呈递细胞 (aAPC) 确定能否从高危神经母细胞瘤患儿的外周血单个核细胞 (PBMC) 中体外大量扩增激活的 NK (aNK) 细胞。
用辐照的 K562 衍生的 Clone 9.mbIL21 aAPC 与 PBMC 共培养，并通过流式细胞术、细胞毒性测定、Luminex 多细胞因子测定和非肥胖型糖尿病/严重联合免疫缺陷 (NOD/SCID) 小鼠模型评估传播性神经母细胞瘤来鉴定扩增的 NK 细胞。
用 aAPC 共培养患者 PBMC 14 天可诱导 CD56(+)CD3(-)CD14(-) NK 细胞扩增 2363±443 倍，纯度为 83%±3%（n=10）。结果与正常供者 PBMC 相似（n=5）。与第 0 天相比，第 14 天 NK 细胞上的 DNAM-1、NKG2D、FcγRIII/CD16 和 CD56 的表达分别增加了 6-±3-、10-±2-、21-±20-和 18-±3-倍，表明 NK 细胞被激活。体外，aNK 细胞对神经母细胞瘤细胞系具有高度细胞毒性，与 GD2 特异性 mAb ch14.18 联合使用可增强杀伤作用。当用 ch14.18 介导细胞毒性时，与 aNK 细胞单独作用相比，aNK 细胞释放的 TNF-α、粒细胞巨噬细胞集落刺激因子、IFN-γ、sCD40L、CCL2/MCP-1、CXCL9/MIG 和 CXCL11/I-TAC 分别增加了 4-、5-、6-、15-、265-、917-和 363-倍（151-9121 pg/mL）。与未治疗的小鼠相比，患有播散性神经母细胞瘤的 NOD/SCID 小鼠在接受解冻并立即静脉输注冷冻保存的 aNK 细胞治疗后存活时间得到改善，当添加 ch14.18 时存活时间进一步改善。
大量扩增并冷冻保存的 aNK 细胞能保持强大的抗神经母细胞瘤活性，支持用 ch14.18 进行过继细胞治疗的临床研究。
