鼻咽癌專一性單株抗體的製備與功能之探討

Abstract

鼻咽癌為中國人特有的癌症。在台灣鼻咽癌佔國內癌症的第十四位。目前鼻咽癌的治療方式有手術、放射線治療及化學治療，但以放射治療為主。而這種癌症對放射線極為敏感，故療效很好。經治療之結果，大部分病人五年之存活率超過90%，但轉移嚴重的病人五年存活率低於50%，其中局部復發及遠端轉移是治療的主要失敗原因，加上一般療法常會帶給病人相當大的副作用，因此我們嘗試找出其他更好的治療方式。近年來，因治療性抗體發展與生物技術的進步，使抗體在癌症治療上逐漸扮演重要的角色。目前已有不少治療性抗體已通過美國食品藥物管理局認可，在臨床治療上使用。本篇研究，我們以NPC TW01免疫BALB/c老鼠後，利用單株抗體融合的技術，產生十四株專一性對抗到鼻咽癌的單株抗體，進行一連串的功能測試。我們選擇其中五株單株抗體來進行ELISA、流式細胞儀、西方墨漬法、免疫化學染色等研究，結果發現這五株抗體皆能專一對抗鼻咽癌細胞，卻和正常鼻咽細胞沒有反應。在西方墨漬法中觀察到NPC-Ab 1-1、 NPC-Ab 6-1、NPC-Ab 9-2 和NPC-Ab 14-1可偵測到NPC 細胞的蛋白質，分子量分別為 47 kDa、170 kDa、47 kDa、220 kDa。除此之外，這些抗體也可辨認到其他癌細胞的蛋白質，例如 胰臟癌、口腔癌、大腸癌、肺癌等，對於鼻黏膜細胞、血球細胞和人類內皮細胞則無反應。除此，從細胞流式儀的實驗中，發現NPC-Ab 4-2 和 NPC-Ab 6-1對於鼻咽癌的細胞膜蛋白有較佳的結合能力。在細胞凋亡實驗中，更證明其中一株抗體NPC-Ab 4-2 可以造成鼻咽癌細胞進行細胞凋亡，並且也進一步證明具有抑制鼻咽癌細胞增生的能力。最後我們也研究鼻咽癌的標的治療，以NPC-Ab 6-1 的抗體接上Lipo-Dox來治療鼻咽癌腫瘤的SCID mice，結果發現含有此抗體的標的微脂體對老鼠腫瘤生長的抑制，明確比其他控制組更具療效。經過以上結果顯示，我們所製備的單株抗體，有其專一性對抗癌症的特性，可用來偵測鼻咽癌抗原表現及運用在鼻咽癌治療上。

Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in southern China, Taiwan, and Singapore. The 5-year survival rate has been improved for localized NPC cases to > 90% in some hospital, but for advanced cases, the survival rate remains below the 50% margin. Radiotherapy, surgical removal, and chemotherapy have been used for decades with varying degrees of success. However, the side-effects of radiotherapy are substantial, including mucositis, central nervous system toxicity, alterations in taste, and problems with saliva and trismus. Furthermore, chemotherapy can easily induce chemoresistant tumor cells. There is a great need for developing new modalities of therapy such as targeted therapy. Over the last few years, monoclonal antibodies (mAbs) have repeatedly made the successful transition from the bench to the bedside by the US Food and Drug Adminstration (FDA) for use in various clinical settings, including cancer therapy. In this study, we generated 14 mAbs specific against NPC cells. Five mAbs NPC-Ab 1-1, NPC-Ab 4-2, NPC-Ab 6-1, NPC-Ab 9-2 and NPC-Ab 14-1 revealed different binding pattern to nine cancer and two normal cells were further characterized. The specificity of these antibodies was further confirmed by the ELISA, Western blot, immunohistochemistry stain and flow cytometric assays. In Western blot analysis, NPC-Ab14-1 can recognize a protein of 220 kDa; NPC-Ab 9-2, a protein of 47 kDa; NPC-Ab 1-1, protein of 47 kDa; NPC-Ab 6-1 can bind a protein of 170 kDa. These target proteins were also identified in other cancer cell lines, such as PaCa-2, SAS, HCT116 and H460. Results of FACS analysis further demonstrated that the NPC-Ab 4-2 and NPC-Ab 6-1 can detect the antigens expressed on the cell surface of NPC but not NNM cells. Furthermore, the NPC-Ab 4-2 can even inhibit NPC cell proliferation and induce apoptosis by MTT and flow cytometry assays. Finally, to develop of ligand-targeted therapy using diagnostic animal experiments, we found that NPC-Ab 6-1 antibody-conjugated targeting liposome can significantly enhanced the therapeutic efficacy of the drug against NPC. These data suggest that these mAbs might be useful to identify tumor antigens, and developing ligand-targeted therapy in the treatment of nasopharyngeal carcinoma.