NEW DEVELOPMENT OF DC IMMUNOTHERAPY TO UNIDENTIFIED CANCER ANTIGEN

1)利用Flt3L基因进行树突状细胞的体内动员和成熟体外诱导外周血单核细胞来源的树突状细胞作为一种很有前途的肿瘤免疫治疗方法已开始在临床上应用。如果不需要体外操作就能获得相同类型的免疫刺激器，在临床环境中可能会非常方便。在这项研究中，我们利用体内电穿孔Flt3L质粒DNA(Flt3L-IVE)在胫前肌肉中进行了系统的Flt3L基因转移，以确定其对DC的影响。与对照组相比，Flt3L-IVE组小鼠脾和骨髓中DC数量显著增加，并显示出高水平的共刺激分子表达。免疫组织化学检测显示，与对照组相比，注射Flt3L-IVE 21d后，肿瘤局部不仅有树突状细胞，而且CD8和CD4阳性细胞也有明显的浸润，且仍在肿瘤内。然而，抗…在MCA205已建立的肿瘤中，Flt3L-IVE的更多肿瘤效应不明显。这些结果表明，体内电穿孔Flt3L基因转移可以动员DC进入肿瘤部位。通过IL-18联合Flt3L T细胞瘤内基因转移诱导强大的系统抗肿瘤免疫被认为是一种很好的肿瘤免疫治疗方法，IL-18可诱导干扰素-γ的产生并增强NK细胞的杀伤活性。然而，我们已经发现，仅用全身或局部给予IL-18治疗不足以诱导强大的全身抗肿瘤免疫。另一方面，我们也报道了树突状细胞(DC)可以从IL-18激活的NK细胞杀伤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。为了增强体内局部应用IL-18诱导的全身抗肿瘤反应，我们观察了IL-18和Flt3配体(Flt3L)联合治疗的效果。小鼠双侧皮内接种MCA205纤维肉瘤。在第5天和第12天，将携带人Flt3L或EGFP基因的DNA质粒体内电穿孔(IVE)到小鼠双侧后肢。作为联合治疗，部分小鼠还接受了携带IL-18基因(Ad.IL-18)或EGFP基因(Ad.EGFP)的腺病毒载体瘤内注射。在治疗的肿瘤中，单独使用IL-18以及Flt3L-IVE和Ad.IL-18联合治疗的小鼠与对照组相比有显著的抗肿瘤作用(p&lt；0.01)。与Flt3L-IVE单独治疗相比，联合应用Ad.IL-18产生了更强的抗肿瘤反应(P&lt；0.01)，与单独使用Ad.IL-18相比，联合治疗的小鼠完全根除的几率更高(100%比33%：P&lt；0.05)。在未注射的肿瘤中，只有联合治疗显示出显著的抗肿瘤作用。联合治疗组小鼠区域淋巴结中的淋巴样细胞对MCA205有明显的杀伤活性。此外，联合治疗对YAC-1(NK靶点)的杀伤活性明显高于单独应用Ad.IL-18(p&lt；0.05)。提示局部IL-18基因转移联合Flt3L原位动员DC可增强抗肿瘤效应，诱导强大的全身抗肿瘤免疫。较少

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