SSEA-4抗體的開發與應用於胰臟癌之研究

Abstract

異常的醣化是癌症的一個特徵。癌細胞其中一個的特點就是細胞表面具異常化的醣脂質，尤其是唾液酸化以及岩藻醣化。SSEA-4長期使用於胚胎幹細胞表面的標記，近來研究指出，在癌症研究中，SSEA-4和相似結構的Globo H和SSEA-3也被發現大量表現於癌細胞上。在這篇論文中，我們建立單株抗體來辨認SSEA-4 (MC45, MC46 and MC48)或同時辨認SSEA-4及Globo H和SSEA-3 (MC607)的抗體。我們並探討這些SSEA-4抗體之特性，並利用這些SSEA-4抗體來做癌細胞治療評估。首先，我們發現SSEA-4皆有表現在胰臟癌細胞。 MC48單株抗體對於SSEA-4表面解離常數約0.46 nM。在細胞實驗顯示，MC48抗體能夠結合於SSES-4抗原表現的胰臟癌細胞與組織，也可藉由細胞毒殺作用在殺死胰臟癌細胞。而在裸鼠動物實驗模式，顯示可抑制胰臟腫瘤的生長。將MC48轉換為嵌合單株抗體chimeric mAb (chi48)來做功能性的探討，chi48 antibody也可藉由補體的細胞毒殺實驗來殺死胰臟癌細胞株BxPC3。因此，本篇研究顯示SSEA-4的抗體具有發展於標靶治療的潛力，可應用於胰臟癌或其他帶有SSEA-4表現的腫瘤上。 另外針對Globo H的結合蛋白尚未報導，明確功能性也仍未被描述。利用蛋白質晶片、ELISA等方法，可初步發現一些Globo H 結合蛋白，利用醣晶片進一步發現Artemin蛋白可與Globo H有作用，而其Artemin/Globo H的相互作用也將探討於此篇論文中。

Aberrant glycolipids, especially the sialylated and fucosylated ones, give the most characteristic patterns of cancers. SSEA-4 (Stage-specific embryonic antigen-4), a sialyl-glycolipid, has long been used as a cell surface marker for pluripotent human embryonic stem cells. In cancer research, SSEA-4 and relative structure Globo H and SSEA-3 (Stage-specific embryonic antigen-3) also found in many cancer cells. In this thesis, we were developing antibodies against SSEA-4 for target validation study. We found SSEA-4 was expressed on pancreatic cancer cells and specimens. From mice immunized with a SSEA-4 synthetic conjugate, two IgG1 (MC45, MC48) and one IgM (MC46) monoclonal antibody were derived that recognizes the SSEA-4 structure. MC48 mAbs were calculated surface dissociation (KD) constant amount to 0.46 nM for SSEA-4 in glycan array assay. We have showed MC48 that is capable of binding to SSES-4 antigen expressed on pancreatic cancer cells and human pancreatic cancer tissue. The MC48 antibody also mediates complement-dependent cytotoxicity in pancreatic cancer cells and it also significantly inhibits pancreatic tumor growth in nude mice. Besides, MC48 convert to human IgG1, the chimeric antibody chi48 also showed that CDC in pancreatic cancer cells. The present studies indicated SSEA-4 mAbs could be explored as an immunotherapy in the treatment of pancreatic cancer patient. To investigate the glycan binding proteins for glycolipid, we used human protein microarray with biotinylated Globo H glycan epitope (Globo H-biotin). Then the glycan-binding protein candidates of human protein microarray were further analyzed by ELISA and glycan microarray. The Artemin/Globo H interaction was also investigated in this study.