Growth and activation of natural killer cells ex vivo from children with neuroblastoma for adoptive cell therapy.
Growth and activation of natural killer cells ex vivo from children with neuroblastoma for adoptive cell therapy.
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DOI：
10.1158/1078-0432.ccr-12-1243
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发表时间：
2013-04-15
期刊：
影响因子：
--
通讯作者：
Seeger RC
中科院分区：
文献类型：
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作者：
Liu Y;Wu HW;Sheard MA;Sposto R;Somanchi SS;Cooper LJ;Lee DA;Seeger RC
自然杀伤（NK）细胞的过继转移与肿瘤特异性单克隆抗体（mAb）相结合具有治疗恶性肿瘤的潜力。我们确定是否可以使用人工抗原呈递细胞 (aAPC) 从高危神经母细胞瘤儿童的外周血单核细胞 (PBMC) 中离体培养大量活化的 NK (aNK) 细胞。将经辐射的 K562 衍生克隆 9.mbIL21 aAPC 与 PBMC 共培养，并通过流式细胞术、细胞毒性测定、Luminex® 多细胞因子测定和播散性神经母细胞瘤 NOD/SCID 小鼠模型对增殖的 NK 细胞进行表征。将患者 PBMC 与 aAPC 共培养 14 天，诱导 CD56+CD3−CD14− NK 细胞扩增 2,363±443 倍，纯度为 83±4% (n=10)。结果与正常捐献者的 PBMC 相似 (n=5)。与第0天相比，第14天DNAM-1、NKG2D、FcγRIII/CD16和CD56的表达分别增加6±3、10±2、21±20和18±3倍，表明NK细胞被激活。在体外，aNK 细胞对神经母细胞瘤细胞系具有高度细胞毒性，GD2 特异性单克隆抗体 ch14.18 可增强杀伤作用。当使用 ch14.18 介导细胞毒性时，aNK 细胞释放的 TNFα、GM-CSF、IFNγ、sCD40L、CCL2/MCP-1、CXCL9/MIG 和 CXCL11/I-TAC 增加了 4、5-6、15、265、917 和 363 倍（151 至 9,121 pg/mL），分别与单独的 aNK 细胞相比。与未治疗的小鼠相比，当用解冻并立即静脉输注冷冻保存的 aNK 细胞治疗时，携带播散性神经母细胞瘤的 NOD/SCID 小鼠的存活率得到改善，并且当添加 ch14.18 时，存活率进一步改善。冷冻保存时保持有效抗神经母细胞瘤活性的大量 aNK 细胞的增殖支持了 ch14.18 过继细胞疗法的临床测试。
Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAbs) 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 co-cultured with PBMC, and propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex® multi-cytokine assays, and a NOD/SCID mouse model of disseminated neuroblastoma. Co-culturing patient PBMC with aAPC for 14 days induced 2,363±443-fold expansion of CD56+CD3−CD14− NK cells with 83±4% purity (n=10). Results were similar with 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 to day 0, demonstrating activation of NK cells. In vitro, aNK cells were highly cytotoxic against neuroblastoma cell lines, and killing was enhanced with GD2-specific monoclonal antibody ch14.18. When mediating cytotoxicity with ch14.18, release of TNFα, GM-CSF, IFNγ, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cells increased 4-, 5- 6-, 15-, 265-, 917- and 363-fold (151 to 9,121 pg/mL), respectively, compared to aNK cells alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved aNK cells compared to un-treated mice and was further improved when ch14.18 was added. Propagation of large numbers of aNK cells that maintain potent anti-neuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18.