肿瘤细胞治疗研究
英文原题：Development of anti-GD2 Antibody-producing Mesenchymal Stem Cells as Cellular Immunotherapy.
新开发出的抗GD2-MSCs能产生功能性抗体，这些抗体对NB细胞上的GD2抗原具有亲和力，并能诱导ADCC介导的细胞毒性。基于抗GD2-MSCs的细胞免疫疗法有潜力成为难治性NB的一种新型治疗选择。
利用酪氨酸羟化酶（TH）-MYCN小鼠神经母细胞瘤（NB）模型，我们此前已报道了小鼠间充质干细胞（mMSCs）在体内肿瘤上的聚集，以及转染小分子（IFN-β）表达基因的mMSCs的抗肿瘤效果。在本研究中，我们开发了分泌抗双唾液酸神经节苷脂GD2抗体的新型MSCs（anti-GD2-MSCs），并在体外评估了其抗肿瘤效果。
我们构建了一种抗GD2抗体构建体（14.G2a-Fcx2-GFP），其包含针对GD2的FLAG标签单链可变区片段，融合至连接序列、人IgG1恒定区片段以及GFP蛋白。该构建体经慢病毒转导至mMSCs中，并通过GFP表达评估转导效率。使用抗FLAG抗体通过Western blotting检测FLAG标签抗GD2抗体的分泌。通过流式细胞术确认抗体结合能力。使用人NB细胞和人自然杀伤（NK）细胞评估抗体依赖性细胞介导的细胞毒性（ADCC），以评估在所产生的抗体存在下抗肿瘤活性是否增强。
抗GD2-MSCs的转导效率超过90%。抗GD2-MSCs在细胞外分泌抗体，这些抗体对表达GD2的人NB细胞具有高亲和力。ADCC实验显示，加入抗GD2-MSCs分泌的抗体显著增强了NK细胞对NB细胞的细胞毒活性。
BACKGROUND/AIM: Using the tyrosine hydroxylase (TH)-MYCN mouse neuroblastoma (NB) model, we have previously reported the accumulation of mouse mesenchymal stem cells (mMSCs) on tumors in vivo and the antitumor effect of mMSCs transfected with a small molecule (IFN-β) expression gene. In this study, we have developed novel MSCs secreting anti-disialoganglioside GD2 antibody (anti-GD2-MSCs) and evaluated their antitumor effects in vitro. MATERIALS AND METHODS: We generated an anti-GD2 antibody construct (14.G2a-Fcx2-GFP) incorporating FLAG-tagged single-chain fragment variable against GD2 fused to a linker sequence, a fragment of the constant portion of human IgG1, and GFP protein. The construct was lentivirally transduced into mMSCs and the transduction efficiency was assessed by GFP expression. The secretion of FLAG-tagged anti-GD2 antibody was detected by Western blotting using anti-FLAG antibody. Antibody binding capacity was confirmed by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using human NB cells and human natural killer (NK) cells to assess whether the antitumor activity was enhanced in the presence of the produced antibodies. RESULTS: The transduction efficiency of anti-GD2-MSCs was more than 90%. anti-GD2-MSCs secreted antibodies extracellularly and these antibodies had high affinity to GD2-expressing human NB cells. ADCC assays showed that the addition of antibodies secreted from anti-GD2-MSCs significantly increased the cytotoxic activity of NK cells against NB cells. CONCLUSION: Newly developed anti-GD2-MSCs produced functional antibodies that have affinity to the GD2 antigen on NB cells and can induce ADCC-mediated cytotoxicity. Anti-GD2-MSCs based cellular immunotherapy has the potential to be a novel therapeutic option for intractable NB.
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