Development of the next generation cancer therapy using "Exosomes" by molecular tumor engineering technique

沿着研究计划，我们在此报告研究期间的研究结果。1.高外泌体分泌细胞的制备：单核细胞来源的树突状细胞（DC）分泌大量的外泌体并且其分泌是稳定的。2.外泌体的分离和纯化：DC衍生的外泌体和肿瘤衍生的外泌体分别通过抗HLA DR抗体和针对肿瘤抗原的抗体有效纯化。3.改变外泌体的功能囊泡：我们使用腺病毒载体将EGFR或CEA基因导入DC。这些DC分泌分别表达EGFR或CEA的外泌体。也就是说，通过基因转染的方法，可以在外泌体上产生任意的蛋白质。4.外泌体的分子生物学分析：已证实肿瘤来源的外泌体分泌多种蛋白质，这些蛋白质可用于癌症疫苗。因此，确认了肿瘤来源的外泌体作为用于提供癌相关分子的纳米颗粒的效用。5.外来体的免疫功能分析：DC衍生的外来体：1）自然杀伤细胞活性的增强，2）癌症患者衍生的DC的功能改善，和3）调节性T细胞的存活延长。癌细胞来源的外泌体：诱导CTL样细胞。如上所述，我们阐明了外泌体的几个独特的生物学和免疫学特性，并提出了外泌体在肿瘤免疫治疗中的临床应用。另一方面，最初计划在外泌体上表达Patched，但没有成功。然而，在本研究期间，我们成功地制备了几种具有中和活性的抗Patched抗体。

Along a study plan, we report here results of research during the study period. 1. Making of high exosome-secretion cells : Monocyte-derived dendritic cells (DCs) secrete a large amount of exosomes and their secretion is stable. 2. Separation and purification of Exosomes : DCs-derived exosomes and tumor-derived exosomes were efficiently purified by anti-HLA DR antibodies and by the antibodies against tumor antigens, respectively. 3. Change to functional vesicles of exosomes : We introduced EGFR or CEA gene using adenovirus vectors to DCs. These DCs secreted exosomes expressing EGFR or CEA, respectively. In other words, the possibility that could produce arbitrary protein on exosome by gene transfection was shown. 4. Molecular biologic analysis of exosomes : It was confirmed that tumor-derived exosomes secrete various kinds of protein which are useful for cancer vaccine. Thus, a utility of tumor-derived exosomes as nanoparticle for supplying carcinoma-related molecules was confirmed. 5. Immunological functional analysis of exosomes : DCs-derived exosomes : 1) augmentation of natural killer cells activity, 2) functional improvement of cancer patients-derived DCs, and 3) survival prolongation of regulatory T cells. Cancer cell-derived exosomes : Induction of CTL-like cells. As described above, we clarified several unique biological and immunological characteristics of exosomes and suggested a clinical application of exosomes to tumor immunotherapy. On the other hand, it was a plan to express Patched on exosomes at first, but did not succeed. However, during this study period, we succeeded in making several anti-Patched antibodies that have neutralization activity.