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Title: | 研發具有治療卵巢癌潛力之新型單株抗體對抗α3整合素 Novel Monoclonal Antibody Against Integrin α3 Shows Therapeutic Potential for Ovarian Cancer |
Authors: | Feng-Yi Ke 柯鋒翌 |
Advisor: | 郭冠廷(Kuan-Ting Kuo) |
Co-Advisor: | 吳漢忠(Han-Chung Wu) |
Keyword: | 卵巢癌,單株抗體,整合素α3,focal adhesion kinase,層連結蛋白,細胞凋亡, ovarian cancer,monoclonal antibody,integrin α3,focal adhesion kinase,laminin,cell apoptosis, |
Publication Year : | 2020 |
Degree: | 博士 |
Abstract: | 卵巢癌是現今最致命的婦科(gynecology)惡性腫瘤,在美國每年造成大約一萬五千名婦女死亡;在台灣卵巢癌的發生率每年節節上升,根據統計2016年因卵巢癌死亡的人數首次超越了子宮頸癌成為國人女性十大癌症死因的第七位。而造成高死亡率的原因主要有兩個,第一個是目前沒有有效篩檢早期卵巢癌的方法,導致確診的病人多為晚期;另一個原因則是現行治療,晚期的患者在經過手術及化學藥物治療後,仍有很高的機率復發且具有抗藥性。因此,目前急需發展新型藥物來更有效治療卵巢癌病患。 抗體藥物與小分子化療藥物相比,抗體藥具有較高的專一性以及較低的副作用,此外目前卵巢癌也缺乏有效的標靶藥物,因此尋找卵巢癌的分子標記以及治療性抗體是相當重要的。因此在本研究中我們利用SKOV-3這株卵巢癌細胞當作抗原打入小鼠腹腔,待小鼠產生免疫反應後之後取出脾臟,利用融合瘤技術生產2800多個融合瘤細胞株,再利用酵素免疫法篩選可專一性辨認SKOV-3細胞而不會辨認正常細胞如CCD-1112sk及NNM,最終得到30株單株抗體。我們進一步以Annexin V-FITC及PI染色並用流式細胞儀偵測凋亡細胞,發現其中一株抗體OV-Ab 30-7具有使SKOV-3以及ES-2兩株卵巢細胞株走向細胞凋亡的能力。因此我們好奇OV-Ab 30-7所對抗的抗原為何種分子,利用抗原抗體結合特性,將SKOV-3細胞溶解產物與OV-Ab 30-7以免疫沉澱法純化出抗原,再利用液相層析串聯質譜儀得知抗原為整合素α3 (integrin α3),並以shRNA減弱SKOV-3細胞本身整合素α3的表現,可阻止OV-Ab 30-7所造成的細胞凋亡現象。此外我們也發現OV-Ab 30-7可阻止整合素α3與其配體層連結蛋白(laminin)結合,並阻止曾連結蛋白所導致的下游分子focal adhesion kinase (FAK)的磷酸化現象。在小鼠的治療實驗中,同時注射SKOV-3細胞及OV-Ab 30-7至老鼠腹腔中可顯著延長小鼠的存活時間,此外OV-Ab 30-7單獨治療小鼠可顯著抑制腹腔中ES-2與SKOV-3細胞生長並延長小鼠存活時間,我們也發現OV-Ab 30-7搭配小分子化療藥物卡鉑及紫杉醇藥物在小鼠實驗中具有顯著的加成療效。用免疫組織化學法分析臨床病人組織切片當中整聯蛋白α3的表現量,發現癌化的組織相較於正常的卵巢組織表現高量的整聯蛋白α3,並且利用線上資料庫分析發現高表現整聯蛋白α3的病人有較差的預後。 綜合以上結果我們認為OV-Ab 30-7是一株具有作為治療卵巢癌潛力的單株抗體。 Ovarian cancer has the highest mortality rate among gynecological cancers due to low rates of early detection and high recurrence after platinum-based chemotherapy. Because therapeutic antibodies are often efficacious with low toxicity, they are considered a promising option for novel ovarian cancer therapies. In order to identify ovarian cancer-specific antibodies, we immunized mice with the human ovarian carcinoma cell line, SKOV-3, and generated hybridoma clones. Several rounds of screening yielded 30 monoclonal antibodies (mAbs) with no cross-reactivity to normal cells. Among these mAbs, OV-Ab 30-7 was found to target integrin α3 and upregulate p53 and p21, while stimulating apoptosis of cancer cells. Integrin α3 (ITGA3)-knockdown in SKOV-3 cells with shRNA suppressed OV-Ab 30-7-induced cancer cell apoptosis. We further found that binding of integrin α3 by OV-Ab 30-7 impaired laminin-induced focal adhesion kinase (FAK) proteins phosphorylation. The mAb also inhibited tumor progression and prolonged the survival rate of tumor-bearing mice. In an intraperitoneal xenograft mouse model for ovarian cancer, the combination of OV-Ab 30-7 with carboplatin and paclitaxel significantly extended the survival compared to the carboplatin and paclitaxel combination without mAb. Moreover, immunohistochemical staining of integrin α3 in ovarian patient specimens revealed higher levels in cancer cells compared with normal cells. By querying online clinical databases, we found that elevated ITGA3 expression in ovarian cancer is associated with poor prognosis. Taken together, our data suggest that the novel mAb, OV-Ab 30-7, displays potential as a therapeutic for ovarian cancer. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19843 |
DOI: | 10.6342/NTU202003432 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 病理學科所 |
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