研發Fab抗體片段嵌合微脂體藥物運用於抗癌標靶藥物傳輸

Abstract

HER2蛋白質是EGFR酪氨酸激酶受體家族之一，其在細胞度過度表現在腫瘤細胞增殖和轉移中扮演重要作用。許多卵巢癌患者都具有極高HER2蛋白質的表達，同時伴隨不良的預後。儘管臨床上對於卵巢癌患者的治療多為傳統手術切除或合併化療，但大多數的患者在五年內會因為產生抗藥性而復發，於是目前的藥物開發研究主要著重在如何利用標靶治療的方式有效解決抗藥性。 上皮細胞粘附分子（EpCAM）是一種穿膜醣蛋白，其在結腸癌患者中具有高表達現象，目前上皮細胞粘附分子的細胞外結構（EpEX）已知會促進結腸癌細胞增殖。透過標靶治療的方式，可以避免化療藥物對正常細胞的損害並提升化療藥物的治療效率。 在本研究中，我們運用先前由噬菌體展示技術挑選出與HER2或EpEX專一性結合的抗原呈現片段（Fab），接著透過酵素連結免疫吸附分析法（ELISA）和免疫螢光染色證實它們各自的專一性與結合強度。我們進而將HER2或EpEX抗體結合區片段接於微脂體藥物之外膜，發現HER2或EpEX抗體結合區片段皆可提升微脂體藥物對細胞的結合能力與吞噬作用。最後，我們用接上抗原呈現片段的微脂體藥物運用於治療異體移植免疫缺陷小鼠，並比較帶有標靶的微脂體藥物與一般微脂體藥物兩者之療效與存活率，發現帶有抗原呈現片段的微脂體藥物具有較佳的存活率，且結合不同種類的微脂體藥物有更好的療效。綜合上述結果，HER2與EpEX抗原呈現片段可應用於標靶藥物傳輸系統，透過其對腫瘤細胞的高專一性與對生物體的低毒性之兩大優勢，可望提升癌症患者的整體存活率。

HER2 belongs to EGFR tyrosine kinase receptor family, the overexpression of HER2 plays an important role in tumor cell proliferation and metastasis. Many ovarian cancer patients have a high expression level of HER2 and are associated with poor prognosis. Although the combination of traditional surgery and chemotherapy is commonly used for the treatment of ovarian cancer patients, most of the patients would relapse within five years because of drug resistance. Therefore, many targeted therapies have been developed to solve drug resistance and improve the patient’s outcome. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein which is frequently overexpressed in colon cancer patients, and the extracellular domain of EpCAM (EpEX) is associated with colon cancer cell proliferation. Through treating targeted therapies to patients with cancer, the damage caused by chemo drugs can be avoided, and the therapeutic efficacy of chemo drugs can be promoted as well. In this study, we synthesized anti-HER2 Fab and anti-EpEX Fab which was selected by phage display technology. The specificity and binding affinity of anti-HER2 or anti-EpEX Fab were confirmed by ELISA and immunofluorescence. Next, we found that the conjugation of anti-HER2 or anti-EpEX Fab onto liposomal drugs could effectively improve the binding and targeting ability of liposomal drugs to cancer cell line. Eventually, in NOD/SCID tumor-bearing mice model, targeting liposomal drugs and non-targeting liposomal drugs were used to evaluate the therapeutic efficacy and the survival rate. The results revealed that Fab-conjugated liposomal drugs could prolong the survival rate. Furthermore, the combination of different liposomal drugs can harvested the better therapeutic efficacy. In summary, both anti-HER2 Fab and anti-EpEX Fab can be applied to targeting drug delivery system. With the advantages of high specificity and low cytotoxicity, anti-HER2 Fab and anti-EpEX Fab have the potential to prolong the survival of patients.