研發應用於肝癌治療之人類抗體

Abstract

肝癌是全世界最常見的癌症之一，且肝癌在亞太地區有極高的盛行率。肝癌可以經由手術切除、化療、放射線治療、免疫療法或治療性抗體等方式治療。以手術切除的方式，常因癌細胞入侵到鄰近組織而效果有限；化療及放射線療法因會對正常組織造成傷害而產生副作用；標靶治療不但能提高腫瘤治療的專一性，也能降低藥物的副作用。因此，標靶治療及免疫療法為當前治療肝癌的發展趨勢。利用活體的噬菌體顯現法 (phage display) 技術可以篩選出與腫瘤組織專一性結合的人類單鏈抗體 (scFv) 以達到標的治療的目的。在本研究中，我們透過噬菌體顯現法 (phage display) 篩選出可以專一性地與肝癌細胞株結合的人類單鏈抗體，並成功獲得數個對SK-HEP-1細胞具有高度親和力的噬菌體株，經酵素免疫連結吸附反應及流式細胞儀的分析，發現其具有辨識大部分肝癌細胞株的能力，而不與正常組織結合。除此之外，藉由免疫組織染色也證實表現人類單鏈抗體的噬菌體能與肝癌病人檢體的腫瘤組織相結合，而正常組織則否。為了進一步了解這些對抗肝癌細胞的人類單鏈抗體，在活體內是否仍具有腫瘤的標的能力，故將噬菌體施打於移植人類腫瘤的免疫不全鼠，發現其在腫瘤組織相對於正常器官有較高量噬菌體的聚集。之後，再進一步建構表現人類全長IgG抗體的質體，於CHO-K1及FreeStyleTM 293-F細胞株進行生產，再以西方墨點法、酵素免疫連結吸附反應、免疫螢光染色法以及流式細胞分析等技術確認表現出的人類全長IgG抗體是否仍保有專一性結合至肝癌細胞株的能力。綜合以上之結果，我們所篩選出的人類抗體在未來應用於診斷及治療肝癌病人方面應有極大的潛力。

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Treatment of HCC through surgery is unsatisfactory, while systemic chemotherapy and radiotherapy are less efficacious. Targeted therapy and immunotherapy might become better alternatives for the treatment of HCC. Phage display technology is a powerful tool for selecting the specific monoclonal antibodies for development of targeted therapeutics. In this study, we utilized phage-displayed human single chain variable fragment (scFv) library to identify phage clones that specifically bind to HCC cells. After five rounds of in vitro biopanning, several phage clones with high binding affinity to SK-HEP-1 cells were identified. The phage clones were able to bind to HCC cell lines but not normal cells, as shown by ELISA and flow cytometry analysis. In addition, immunohistochemical staining also confirmed the binding of the selected phage clones to the tumor cells in surgical specimens of HCC, but not to their normal counterparts. The in vivo tumor homing assay was further confirmed the targeting ability of selected phage clones in xenograft model. Furthermore, the scFv genes of selected phage clones were successfully constructed and produced as recombinant fully human IgG in CHO-K1 cells or FreeStyleTM 293-F cells. Binding characteristics of recombinant IgGs from the respective phage clones were similar to original phage-displayed scFv clones. We conclude that recombinant fully human IgGs screening from phage display library have strong clinical potential as therapeutic antibodies for the treatment of HCC.