Overcome to avoidance from the anti-leukemia immunity by using combination therapy with HSP and dendritic cells targeted to both tumor and tumor angiogenesis

本研究尝试利用热休克蛋白（HSP）同时靶向肿瘤和肿瘤血管生成诱导抗白血病免疫。我们通过内皮细胞（EC）标记物CD 31免疫染色证明了A20荷瘤小鼠中肿瘤血管生成的存在。用抗CD 31抗体（Ab）的MACS法难以获得足够量的EC。因此，我们产生GFP转导的A20细胞，然后从A20肿瘤组织中分选GFP阴性部分。目前，我们试图通过使用分选的GFP阴性组分来分离A20-肿瘤相关EC（A20-EC）。为了增加EC的数量，构建了含有永生化基因teromerase reverse transcriptase（TERT）的载体。A20-EC分离成功后，将TERT基因导入A20-ES细胞并扩增，然后计划纯化A20-EC来源的HSP。为了获得肿瘤内皮抗原在B细胞肿瘤中的有用信息，我们对POEMS综合征的孤立性浆细胞瘤（被称为血管生成性B细胞肿瘤之一）标本进行了血管生成因子免疫组化染色，结果表明VEGF和IGF在肿瘤中的表达水平很高。此外，我们还发现，抗独特型抗体的小鼠B细胞肿瘤可能会抑制血管生成的微环境中的B细胞肿瘤通过体液免疫的CDC。这些发现使得能够通过针对具有独特基序的肿瘤EC的新型免疫疗法的方法成为可能，例如来自B细胞肿瘤相关EC或抗独特型Ab的HSP。

In this study, we tried induction of the anti-leukemia immunity by using heat shock protein (HSP) targeted to both tumor and tumor angiogenesis. We demonstrated the presence of tumor angiogenesis in the A20-bearing mouse by endothelium cell (EC)-marker CD31 immunostaining. It was difficult to obtain the enough amount of EC by MACS with anti-CD31 antibody (Ab). We therefore originated GFP transduced A20-cells, and then GFP-negative fraction was sorted from A20-tumor tissue. Now we tried to separate the A20-tumor-associated EC (A20-EC) by using sorted GFP-negative fraction. To increase the number of EC, a vector containing immortalizing gene teromerase reverse transcriptase (TERT) was constructed. After successful A20-EC separation, transduction of TERT gene to A20-ESs and expansion of them will be performed, and then purification of A20-EC-derived HSP will be planed. To obtain the useful information of tumor endotherial antigen in the B-cell neoplasms, the specimen of solitary plasmacytoma in POEMS syndrome, which is known as one of the angiogenic B-cell neoplasms, was immunostained with angiogenic factors, and we demonstrated that the expression levels of VEGF and IGF were very high in the tumor. In addition, we showed the possibility that anti-idiotype Ab of mouse B-cell neoplasm may inhibit the angiogenesis in the microenvironment of the B-cell tumor through humoral immunity by the CDC. These findings enable the approach by novel immunotherapy against tumor ECs with unique motifs, such as HSPs derived from B-cell neoplasm associated ECs or anti-idiotype Ab.