肺癌專一性單株抗體的製備與功能之探討

Generation and Characterization of Monoclonal Antibodies Against Lung cancer

陳慧宇(Hui-Yu Chen)

指導教授 ： 吳漢忠

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.10354

摘要

根據世界衛生組織的統計，最近二十年來全世界的癌症死亡率正快速上升，其中肺癌目前是全世界癌症死因的第一名。肺癌的細胞種類通常分成兩大類型：小細胞肺癌（約佔肺癌發生率的15~20%）與非小細胞肺癌（約佔肺癌發生率的80~85%）。其中非小細胞肺癌又細分為三小項: 肺腺癌(Lung adenocarcinoma)、鱗狀細胞癌 (Squamous cell carcinoma)及大細胞肺癌 (Large cell carcinoma)。以往肺癌的治療方式有手術切除、放射線治療及化學治療，而標的治療始於二十世紀末，用於治療末期肺癌。通常大部分病人治療後的五年存活率都小於15％。由於一般療法常會帶給病人極大的副作用，再加上近年來治療性抗體與生物技術的進步，因而使得抗體在臨床癌症治療上越趨重要。在本篇研究中，我們以一株人類高度轉移肺腺癌CL1-5細胞株免疫BALB/cJ老鼠後，進行融合瘤技術，生產十二株專一性對抗肺癌的單株抗體。接著進行酵素免疫連結吸附反應，流式細胞分析，西方墨點法及免疫組織染色等研究。結果發現其中有四株單株抗體皆能專一性的辨認到肺癌細胞，而和正常上皮細胞、周邊血球細胞及人類正常組織都沒有反應，而且這四株單株抗體對於肺癌的細胞膜蛋白都有很好的結合能力。除此之外，這些單株抗體也可辨認到其他癌細胞的蛋白質，例如：口腔癌及乳癌。另外在細胞凋亡實驗中，也可觀察到LC-Ab 2-37、LC-Ab 8-5、LC-Ab 9-5可以造成肺癌細胞有細胞凋亡的現象產生。另外，我們也使用這些抗體去對肺癌病人組織切片做免疫染色,結果發現這四株單株抗體皆有反應產生。經過以上結果顯示，我們所製備對抗肺癌之單株抗體，的確能夠專一性對抗到癌細胞，因而未來可以運用於腫瘤抗原之基礎研究；也可運用於臨床上，來偵測肺癌抗原的表現及有效的發展肺癌的治療。

關鍵字

肺癌 ； 融合瘤 ； 治療性抗體 ； 細胞凋亡

並列摘要

Lung cancer is the leading cause of cancer deaths, accounting for one third of all deaths from cancer worldwide. In the United States, accounting for about 29% of all cancer deaths, are expected to happen in 2008. In Taiwan, lung cancer is the first most common cancer and the death rate is the highest among all cancers, accounting for approximately 19.7% of all cancer deaths in 2006. Lung cancer is classified clinically as small (SCLC) (15-20%) or non-small cell lung cancer (NSCLC) (80-85%) for the purposes of treatment. Current treatment options include surgical resection, platinum-based doublet chemotherapy, and radiation therapy alone or in combination. However, there are many side effects and drug resistance of these treatments. Despite these therapies, the disease is rarely curable and the prognosis is poor, with an overall 5-year survival rate of only 15%. Because monoclonal antibodies (mAbs) have the ability to target tumours, and hence enables them to improve the selectivity of other types of anticancer agent. Therapeutic antibodies have established themselves as one of the most important and fastest growing classes of drugs for cancer. In this study, we have generated 12 mAbs which were specifically against CL1-5 and did not cross-react to normal cells including NNM cells, HUVEC, PBMC and normal human tissues. Four mAbs LC-Ab 1-7, LC-Ab 2-37, LC-Ab 8-5 and LC-Ab 9-5 exhibited high specificities against CL1-5. Therefore, we focused on these 4 mAbs to further characterize their biological properties. In Western blot analysis, LC-Ab 1-7, LC-Ab 2-37 and LC-Ab 4-12 recognize a protein with M.W. 60 kDa, 120 kDa and 58 kDa, respectively. These target proteins were also identified in other cancer cell line, including SAS and MDA-MB 231 by flow cytometric analysis. Furthermore, LC-Ab 2-37, LC-Ab 8-5 and LC-Ab 9-5 can induce apoptosis of cancer cells using flow cytometric analysis. Results from immunohistochemical staining of human surgical specimen sections by LC-Ab 1-7, LC-Ab 2-37, LC-Ab 8-5 and LC-Ab 9-5 indicated that these mAbs might be promising to be applied in the diagnosis and treatment for NSCLC. According to these data, the target proteins of these mAbs and their possible usage as tumor markers and development of Ab-targeted chemotherapy is warranted.

並列關鍵字

lung cancer ； hybridoma ； therapeutic antibody ； apoptosis

參考文獻

Adams, G.P., and Weiner, L.M. (2005). Monoclonal antibody therapy of cancer. Nat Biotechnol 23, 1147-1157.

Alberg, A.J., Brock, M.V., and Samet, J.M. (2005). Epidemiology of lung cancer: looking to the future. J Clin Oncol 23, 3175-3185.

Bailey-Wilson, J.E., Amos, C.I., Pinney, S.M., Petersen, G.M., de Andrade, M., Wiest, J.S., Fain, P., Schwartz, A.G., You, M., Franklin, W., et al. (2004). A major lung cancer susceptibility locus maps to chromosome 6q23-25. Am J Hum Genet 75, 460-474.

Belinsky, S.A., Liechty, K.C., Gentry, F.D., Wolf, H.J., Rogers, J., Vu, K., Haney, J., Kennedy, T.C., Hirsch, F.R., Miller, Y., et al. (2006). Promoter hypermethylation of multiple genes in sputum precedes lung cancer incidence in a high-risk cohort. Cancer Res 66, 3338-3344.

Blackhall, F., Ranson, M., and Thatcher, N. (2006). Where next for gefitinib in patients with lung cancer? Lancet Oncol 7, 499-507.