Development of cellular cancer immunotherapy by using humaniPS-cell-derived dendritic cells and ideal cancer-associated antigens

我们建立了一种方法，可以通过简单的程序从人 iPS 细胞中获得大量功能性树突状细胞 (DC)。我们用与增殖或抗衰老作用相关的 cMYC 和 BMI1 基因转导 iPS 细胞衍生的 CD11b 骨髓细胞。这使得细胞能够以 M-CSF 依赖性方式增殖超过 4 个月，同时保留分化为 DC 的能力。我们将iPS细胞衍生的增殖性骨髓细胞命名为“iPS-ML”，将iPS-ML衍生的树突状细胞命名为“ML-DC”。此外，我们通过锌指核酸酶辅助的靶向基因破坏产生了 T AP2 缺陷的 iPS 细胞克隆。 T AP2 缺陷的 iPS-ML 避免了预激活的同种异体反应性 CD8 T 细胞的识别。 T AP2 缺陷型 ML-DC 表达外源引入的 HLA-A2 基因，刺激从 HLA-A2 阳性同种异体供体获得的 HLA-A2 限制性肿瘤抗原特异性 CD8 T 细胞，从而产生肿瘤抗原特异性 CTL 系。引入各种 HLA I 类基因的 T AP 缺陷 iPS-ML 可以作为疫苗接种治疗的 DC 的无限来源。基于目前的研究，我们提出了一种 DC 产生系统，该系统简单、安全，适用于任何患者，无论其 HLA 类型如何。该技术将为治疗用途 DC 的大规模生产铺平道路。我们还通过腹腔注射经基因改造可分泌干扰素-β的人 iPS-巨噬细胞，成功抑制了免疫缺陷小鼠腹腔内播散的人类癌细胞的肿瘤生长。

We established a method by which we can obtain a large number of functional dendritic cells (DC)with a simple procedure from human iPS cells. We transduced iPS cell-derived CD11b+myeloid cellswith cMYC and BMI1 genes associated with proliferative or anti-senescence effects. This made the cellscapable of propagating for more than 4 months in an M-CSF-dependent manner while retaining thecapacity to differentiate into DC. We named the iPS cell-derived proliferating myeloid cells “iPS-ML”,and the iPS-ML-derived dendritic cells “ML-DC”. In addition, we generated T AP2-deficient iPS cellclones by zinc finger nuclease-aided targeted gene disruption. T AP2-deficient iPS-ML avoidedrecognition by pre-activated allo-reactive CD8+T cells. The T AP2-deficient ML-DC expressingexogenously introduced HLA-A2 genes stimulated HLA-A2-restricted tumor antigen-specific CD8+Tcells obtained from HLA-A2-positive allogeneic donors, resulting in generation of tumorantigen-specific CTL lines. T AP-deficient iPS-ML introduced with various HLA class I genes may serveas an unlimited source of DC for vaccination therapy . Based on the present study , we propose aDC-producing system, which is simple, safe, and applicable to any patients irrespective of their HLAtypes. This technology will pave the way for the mass production of DC for therapeutic use. We havealso succeeded in inhibition of tumor growth of intraperitoneal dissemination of human cancer cells inimmune-deficient mice by intraperitoneal injection of human iPS-macrophage genetically modified tosecrete interferon-beta.