Analysis of the function of Aminopeptidase N and neutral and peptidase and application of its cancer therapy.

我们已经建立了抑制血管生成的单克隆抗体（MAb）MH 8 -11，该抗体的克隆揭示了氨肽酶N（APN）/CD 13。APN具有潜在的肺转移能力。应用肺癌、结肠癌和胰腺癌的实际临床标本，我们发现APN的表达与血管生成有关，是预后不良的重要因素。然而，产生HAMA（人抗小鼠抗体）的小鼠单克隆抗体的临床应用存在问题。本研究建立了识别APN/CD 13的人源性单克隆抗体。我使用了麒麟啤酒公司开发的人型抗体生产小鼠“KM小鼠”。因此，我们采用与正常小鼠单克隆抗体相同的方法，通过免疫人纤维肉瘤细胞株HT 1080，建立了杂交瘤。建立人源性单克隆抗体后，用Transwell法筛选出抑制细胞运动的抗体。结果，我们建立了识别APN/CD 13的人源性单克隆抗体。在识别APN/CD 13的几种抗体中，MT 95 -4抗体对APN活性的抑制作用尤为显著。糖链的改变导致了这些变化，因此目前正在研究哪些糖链的改变可能与肿瘤转移有关。MT 95 -4抑制APN酶活性的能力强于小鼠单克隆抗体MH 8 -11。使用MT 95 -4，我们可能能够作为分子靶向治疗来治疗各种癌症。

We have established the monoclonal antibody(MAb) MH8-11 suppressing angiogenesis and the cloning of this antibody reveals Aminopeptidase N (APN) /CD13. The APN gained a potential ability of metastasis to the lung. Using the actual clinical specimens of lung cancer, colon cancer and pancreatic cancer, we found that the expression of APN had an association with angiogenesis, and was a significant factor of poor prognosis. However, there is a problem for the clinical application of mouse monoclonal antibody which produces HAMA (Human Anti-Mouse Antibody). We established the human type monoclonal antibody that recognized APN/CD13. I used the human type antibody production mouse "KM mouse" which Kirin Brewery developed. Thus, we established hybridoma by the same method as normal mouse monoclonal antibody by immunization by human fibrosarcoma cell line HT1080. After establishment in human type monoclonal antibody, we used assay of Transwell and sorted the antibody that inhibited cell motility. As the result, we established the human type monoclonal antibody that recognized APN/CD13. In particular, MT95-4 antibody significantly inhibited activity of APN among several kinds of antibody that recognized APN/CD13. The modification of the sugar chain resulted in such changes, thus currently it is studied which kinds of sugar chain changes might associate with the tumor metastasis. MT95-4 has a stronger ability suppressing APN enzyme activity than the mouse monoclonal antibody MH8-11. Using MT95-4 we might be able to treat the various kinds of cancers as a molecular targeting therapy.