研發對抗EpCAM單株抗體運用於癌症之診斷與治療

Abstract

單株抗體在過去這20幾年來一直是癌症診斷或治療上非常重要的發展方向。癌症抗體療法概念最初源自1960年代藉由血清學技術觀察到腫瘤細胞膜上各種抗原的存在，這些癌症抗原多半都具有過度表現或是突變的特性。上皮細胞黏附因子 (EpCAM)即是在抗體療法中良好的標的蛋白。目前發現許多上皮細胞癌都有EpCAM的高度表現，包括大腸直腸癌、乳癌、卵巢癌、胰臟癌與肺癌等。過去研究顯示EpCAM 的過度表達與腫瘤增生、轉移、侵襲，造成化療的抗性以及病患存活率降低有相當密切的關係。因此，發展專一性辨認上皮細胞黏附因子，並用於診斷或治療功能之單株抗體成為目前之趨勢。因此在本篇研究中，我們製備出十株單株抗體，其中八株EpAb21-1、EpAb23-1、 EpAb28-3、EpAb34-4、EpAb40-8、 EpAb41-9與EpAb42-1能夠專一性辨識細胞的上皮細胞貼附因子之膜外構造，另兩株EpAb19-24、 EpAb32-16與EpAb43-19能夠辨認上皮細胞貼附因子之膜內構造。除此，利用細胞早期凋亡試驗找出其中兩株EpAb23-1與EpAb34-4能夠誘發細胞凋亡，並利用流式細胞儀分析其凋亡機制，再以噬菌體呈現技術 (Phage display) 經由親和性篩選方法，從大量不同蛋白胜肽序列噬菌體中選取到專一結合EpAb34-4噬菌體株，用以了解抗體之抗原決定位。這些具高度專一與親和力之上皮細胞黏附因子之單株抗體，日後將可用於癌症之診斷或治療。

In the past two decades, monoclonal antibody therapy for cancer has been a pivotal component in the diagnosis and treatment of cancer. The fundamental basis of antibody-based therapy for cancer was originally derived from the observation of the presence of various antigens on tumor cell membrane by serological techniques in the 1960s, and these cancer antigens are mostly characterized by overexpression or mutation. Epithelial cell adhesion molecule (EpCAM) is an emerging target in antibody-based therapy. EpCAM is overexpressed in a wide array of carcinomas such as breast cancers, ovarian cancers, pancreatic cancers and colon cancers. Numerous studies have shown that EpCAM overexpression enhances untoward malignant phenotypes of cancer cells, and reduce the survival of cancer patients. Therefore, it is rational and accretive to develop monoclonal antibodies against EpCAM for cancer theranostics. In this study, we generated ten monoclonal antibodies with high specificity to EpCAM via hybridoma. Among the 10 clones, seven of them bind to extracellular domain of EpCAM (EpEX), whereas the least of them target to the cytoplasmic tail of EpCAM (EpICD). Cell apoptosis assay was conducted and EpAb23-1 and EpAb34-4 were shown being capable to induce cancer cell apoptosis. Mechanisms of apoptosis was further elucidated through flow cytometry. Phage display technology was used to select the matching phage clones specific to EpAb34-4 from a large number of peptide library. In conclusion, we acquired apoptosis-inducing anti-EpCAM antibody with high affinity and specificity in this study, and it paves the way to further evolution into EpCAM-targeting cancer theranostics in the future.