NEW DEVELOPMENT OF DC IMMUNOTHERAPY TO UNIDENTIFIED CANCER ANTIGEN

1)利用FLT3L基因治疗树突状细胞的体内动员和成熟外周单核细胞诱导树突状细胞作为一种很有前景的抗癌免疫治疗方法，已开始在体外临床应用。如果相同类型的免疫刺激器可以在没有体外操作的情况下实现，它可能在临床环境中非常方便。在本研究中，我们通过在胫骨前肌肉中电穿孔Flt3L质粒DNA （Flt3L- ive）进行了Flt3L的全身基因转移，以确定其对原位DCs的影响。与对照组相比，经Flt3L-IVE治疗后，dc数量显著增加，脾脏和骨髓中均出现高度共刺激分子表达。免疫组化评价显示，与对照组相比，不仅dc， CD8和CD4阳性细胞也明显浸润到肿瘤局部，并且在单次Flt3L-IVE后21天仍在肿瘤中存活。而Flt3L-IVE在MCA205建立的肿瘤中抗肿瘤作用不明显。这些结果表明，利用体内电穿孔技术转移Flt3L基因可以将dc动员到肿瘤部位。诱导这些dc成熟的其他方法可能对该策略的抗肿瘤效果产生积极影响。2)利用肿瘤内IL-18基因转移联合FLT3L治疗诱导强效和全身性抗肿瘤免疫白介素-18 （IL-18）可诱导IFN-γ产生并增强NK细胞的细胞溶解活性，被认为是用于肿瘤免疫治疗的良好候选者。然而，我们已经发现仅全身或局部给药IL-18不足以诱导有效的全身抗肿瘤免疫。另一方面，我们也报道了树突状细胞（dc）可以从被IL-18激活的NK细胞杀死的肿瘤细胞中捕获肿瘤抗原，并在体外有效地诱导肿瘤特异性CTL。为了增强体内局部给药IL-18诱导的全身抗肿瘤反应，我们研究了IL-18与Flt3配体（Flt3L）联合治疗的效果。小鼠双侧皮肤内接种MCA205纤维肉瘤。在第5天和第12天，用携带人Flt3L或EGFP cDNA的DNA质粒对小鼠双侧后腿进行活体电穿孔（IVE）处理。作为联合治疗，部分小鼠还在瘤内注射携带IL-18基因（Ad.IL-18）或EGFP基因（Ad.EGFP）的腺病毒载体。在治疗的肿瘤中，Ad对小鼠有明显的抗肿瘤作用。IL-18单用及Flt3L-IVE与Ad联合治疗。IL-18与对照组比较，差异有统计学意义（p<0.01）。与Ad联合治疗。与单独使用Flt3L-IVE治疗相比，IL-18产生了更有效的抗肿瘤反应（p<0.01），并且与Ad相比，联合治疗小鼠的完全根除频率更高（100% vs 33%: p<0.05）。单独IL-18治疗。在未注射的肿瘤中，只有联合治疗有明显的抗肿瘤作用。联合治疗小鼠局部淋巴结淋巴样细胞对MCA205表现出明显的细胞溶解活性。此外，联合治疗对YAC-1 （NK靶点）的细胞溶解活性显著高于Ad。单纯IL-18治疗组差异有统计学意义（p<0.05）。这些结果表明，IL-18的局部基因转移联合dc原位动员Flt3L可增强抗肿瘤作用，并诱导有效的全身抗肿瘤免疫。少

1)IN VIVO MOBILIZATION AND MATURATION OF DENDRITIC CELLS USING FLT3L GENE THERAPYThe clinical application of DCs induced from peripheral monocytes in vitro has been initiated as a promising immunological therapy against cancer. If the same type of immuno-stimulator could be achieved without in vitro manipulation, it might be very convenient in clinical settings. In this study, we performed systemic gene transfer of Flt3L using in vivo electroporation of Flt3L plasmid DNA (Flt3L-IVE) in pretibial muscles in order to determine the effects on DCs in situ. The number of DCs was significantly increased and showed highly co-stimulatory molecules expressions both in spleen and bone marrow after Flt3L-IVE compared to those of control groups. Immunohistochemical evaluation revealed that not only DCs but also CD8 and CD4 positive cells were significantly infiltrated into the local tumor site compared with those of control and remained in the tumor 21 days after a single Flt3L-IVE. However, anti- … More tumor effects of Flt3L-IVE were not significant in MCA205 established tumor. These results suggest that Flt3L gene transfer using in vivo electroporation could mobilize DCs into tumor site. Additional means to induce maturation of these DCs could have positive impact on anti-tumor effects of this strategy.2)INDUCTION OF POTENT AND SYSTEMIC ANTITUMOR IMMUNITY USING INTRA-TUMORAL GENE TRANSFER OF IL-18 IN COMBINATION WITH FLT3L THERAPYInterleukin-18 (IL-18), which induces IFN-γ production and enhances the cytolytic activity of NK cells, is considered to be a good candidate to be used for cancer immunotherapy. However, we have already found that treatment with systemic or local administration of IL-18 alone was not enough to induce a potent systemic anti-tumor immunity. On the other hand, we also have reported that dendritic cells (DCs) can capture tumor antigens from tumor cells killed by NK cells activated with IL-18 and efficiently induce tumor-specific CTL in vitro. In order to enhance the systemic anti-tumor response induced by local administration of IL-18 in vivo, we examined the effects o」 the combination therapy with IL-18 and Flt3 ligand (Flt3L). The mice received intra-dermal inoculation of MCA205 fibrosarcoma in the bilateral flanks. On day 5 and 12, mice were treated with in vivo electroporation (IVE) with DNA plasmids carrying cDNA of human Flt3L or EGFP to bilateral hind legs. As the combination therapy, some of the mice were also treated with intra-tumoral injection of adenoviral vector carrying IL-18 gene (Ad.IL-18) or EGFP gene (Ad.EGFP). In the treated tumors, significant anti-tumor effect was observed in mice treated with Ad.IL-18 alone and the ones treated with combination therapy of Flt3L-IVE and Ad.IL-18 when compared to those mice with control (p<0.01). The combination treatment with Ad.IL-18 resulted in the more potent anti-tumor response when compared to Flt3L-IVE treatment alone (p<0.01), and the complete eradication was observed more frequently (100% vs 33% : p<0.05) in mice treated with the combination therapy when compared to ones with Ad.IL-18 treatment alone. In the un-injected tumors, only the combination therapy showed significant anti-tumor. Lymphoid cells in regional lymph nodes of the mice treated with the combination therapy showed a significant cytolytic activity against MCA205. Moreover, cytolytic activity of the combination therapy against YAC-1 (NK target) was significantly higher than that of Ad.IL-18 treatment alone (p<0.05). These results suggested that local gene transfer of IL-18 combined with DCs mobilization in situ with Flt3L may enhance the anti-tumor effect and induce a potent systemic anti-tumor immunity. Less