卵巢癌單株抗體的製備與功能之探討

Abstract

卵巢癌是致死率相當高的癌症。根據美國統計，在2011年裡有21,990位女性得到卵巢上皮細胞癌，其中高達15,460女性死於此癌症;是女性癌症死亡率第五位。根據台灣2012年國名健康局統計，卵巢癌發生率在女性癌症排名10位，而死亡率排名第八。卵巢癌可以分為上皮細胞癌，間質細胞癌，以及生殖細胞癌。其中上皮細胞癌占了約70%，是最為常見的種類，其又可細分為漿液性癌，子宮內膜癌，黏液性囊腺癌，及透明細胞膜癌等。卵巢癌的治療主要為經過診斷後以手術切除腫瘤，之後再搭配化療。卵巢癌在早期不易診斷，往往確診時已是晚期，即使治療過後仍有再度復發的機會。 標靶治療( targeted therapy) 是近年來治療癌症的新指標，透過專一性結合到癌細胞上以達到治療的目的。許多標靶藥物已廣泛的應用在癌症治療上。在治療卵巢癌的方面，最近有結合化療藥物與標靶藥物例如 Poly ADP-ribose polymerase (PARP) inhibitor以及bevacizumab的臨床試驗，除了比單一使用化療藥物有更好的療效之外，病人的Progression Free Survival (PFS) 也有延長的趨勢。近幾年因為實驗技術的創新，對於治療性抗體的研究與開發有相當大的進展。在本篇實驗中，我們希望得到具有專一性辨認到卵巢癌細胞的抗體。我們利用融合瘤技術挑選出了30株單株抗體，能夠專一性結合到SKOV-3卵巢癌細胞，而且不會與正常細胞如CCD-1112sk以及NNM反應。我們專注於研究幾株具有高結合力的抗體: OV-Ab 3-5，OV-Ab 7-12，OV-Ab 10-6，OV-Ab 13-2，OV-Ab 25-3，及OV-Ab 30-7。單株抗體OV-Ab 25-3與OV-Ab 30-7 能誘發癌細胞進入細胞凋亡。在臨床檢體的免疫染色實驗，我們也發現這些抗體可以辨識到卵巢癌病人的檢體。由以上實驗，得知我們所製備的單株抗體具有專一性辨認到癌細胞的能力。進一步去研究抗體的標的分子以及其功能是未來的發展目標。

Epithelial ovarian cancer is a lethal disease among women. Epithelial ovarian cancer, which accounted for 15,460 deaths in 2011, affects 21,990 women annually in the US. It is the fifth leading cancer cause of death among women. In Taiwan, ovarian cancer has been the 8th leading cause of cancer death among women and accounted for 3% of all cancer death (Department of Health of Taiwan, 2012). Almost 70% of ovarian cancers arise from the epithelial surface cells of the ovary. Epithelial ovarian cancer is the most common type of malignant ovarian tumor, including serous cystadenocarcinoma, endometrioid adenocarcinoma, mucinous cystadenocarcinoma, and clear cell adenocarcinoma. First line of treatment for ovarian cancer involves surgical resection and chemotherapy, and remission can often be attained in the majority of patients. The high mortality rate among women with ovarian cancer is largely due to late-stage diagnosis and its eventual development into chemotherapy resistance. In recent years, drugs such as liposomal doxorubicin and bevacizumab have been widely used in the case of recurrent disease. Targeted therapy is another choice for cancer treatments, with less side effects and toxicity. Combination of chemotherapy drugs and monoclonal antibodies are currently in clinical trials. Several recent studies combining Poly ADP-ribose polymerase (PARP) inhibitors with chemotherapy drug have shown improvement in the median Progression Free Survival (PFS) compared with historical data (9.5 versus 8.6 months). Therapeutic antibodies have established themselves as one of the most important and fast growing classes of drugs for cancer. In our study, we generated 30 mAbs, which showed specific binding to an ovarian cancer cell line: SKOV-3 without cross- reacted with normal cells, such as CCD-1112sk, HUVEC and NNM by cellular ELISA, flow cytometric and immunofluorescent analysis. We further focused on mAbs OV-Ab 3-5, OV-Ab 7-12,OV-Ab 10-6, OV-Ab 13-2, OV-Ab 25-3 and OV-Ab30-7 because their high binding affinity. By Western blot analysis, OV-Ab 7-12 recognizes a protein M.W. 170 kDa. OV-Ab 10-6 and OV-Ab 13-6 recognize a protein M.W. 38kDa. OV-Ab 3-5, OV-Ab 25-3, OV-Ab30-7 cannot recognize any protein by Western blotting even on non reducing gel. The expression of the target proteins was also identified in other cancers cell lines, including MDA-MB 231 and HCT 116. In addition, two of mAbs OV-Ab 25-3 and OV-Ab 30-7, can induce apoptosis of cancer cells, based on flow cytometric analysis. Our results suggest that these mAbs might be useful to identify tumor markers and develop therapeutic antibodies for treatment of ovarian cancer.