| 摘要: | 促紅血球生成素產生的肝細胞受體 (Erythropoietin-producing hepatocellular receptor, Eph) 與Ephrin結合，開啟雙向訊號，調控細胞形態，黏附、增生，並影響腫瘤的生成。EphA4 屬於Eph家族之一，在神經系統中的作用已有許多研究。然而，EphA4與癌症的相關性，目前還未能證明。近期研究指出，在胃癌病人的組織切片中偵測到EphA4的過度表現，並且可能與不良的臨床病理特徵相關。因此，EphA4被認為是除了HER2/neu外，具有潛力的胃癌治療藥物作用標的。本研究以過度表現於胃癌細胞表面的EphA4為標的，來亨雞 (Gallus domesticus) 為免疫動物，建構單鏈抗體基因庫，進而運用噬菌體展現技術，製備並篩選具有專一性的單鏈抗體 (scFv)。其中，單鏈抗體S3對胃癌細胞具有良好結合力和特異性。經由定性實驗測試，單鏈抗體S3對胃癌細胞增生和遷移都具有顯著抑制效果。同時，單鏈抗體S3作用下，也觀察到胃癌細胞的EphA4降解，LAMP1和LAMP2訊號上升，暗示溶?體 (lysosome) 可能參與其中反應；pAKT、pERK、和pSTAT3訊號下調，顯示單鏈抗體S3影響癌細胞增生和遷移的調控路徑。實驗動物模型中，發現單鏈抗體S3能抑制HER2/neu低表現胃癌細胞 (SNU-16) 生長達71.6%，相較於臨床用藥Trastuzumab 之單鏈抗體，具有更顯著的抑制效果。在腫瘤組織染色也發現在單鏈抗體S3作用下，Ki-67上升和 cleaved caspase 3 下降，暗示抗體可能誘發細胞凋亡機制，進而影響腫瘤細胞的生長。最後，進行抗原決定位分析 (epitope mapping)，建立抗體-抗原結合體 (antibody-antigen complex) 結構模型。經過分析，單鏈抗體S3與EphA4 的關鍵位置R162特異性結合，進而影響EphA4在癌細胞中的功能。綜合以上結果，證明利用抗體結合EphA4能有效抑制胃癌細胞的生長，未來有潛力進一步發展為胃癌的治療藥物，為EphA4過度表現或訊號異常活化的癌症患者提供更有效且更低副作用的醫療。
Erythropoietin-producing hepatocellular receptor (Eph) binds to Ephrins to turn on a two-way signal. The interactions extensively regulate cell morphology, adhesion, proliferation, and affect tumorigenesis. EphA4 is a member of Eph family. The roles of EphA4 in the nervous system have been well studied in past studies. However, correlation between EphA4 and cancer is unclear. Recent studies have indicated that high expression of EphA4 in gastric cancer tissues and may be correlated with unfavorable clinical pathological characteristics. Therefore, EphA4 is considered a potential target for clinical therapy of gastric cancer in addition to HER2/neu. In this study, we isolated single chain variable fragment (scFv) that can target EphA4, which is overexpressed in gastric cancer by using phage display technique. Leghorn chicken (Gallus domesticus) was used as an alternative animal model for immunization, and the scFv cDNA library was constructed from the spleen. After bio-panning, characterization tests were carried out to screen and generate the potential antibodies. Among the antibodies, scFv S3 can bind endogenous EphA4 of gastric cancer cells and has notable membrane staining in immunocytochemistry and immunofluorescence. Additionally, scFv S3 binding to EphA4 inhibits the growth and metastasis of cancer cells directly and the growth induction that Ephrin A1 generates in gastric cancer cell SNU-16. Moreover, we found that EphA4 molecules may degrade through antibody treatment of cells. The increase in LAMP1 and LAMP2 signaling indicates that lysosome is involved in cell reactions. The molecular signals pAKT, pERK, and pSTAT3 were also decrease in cancer cells. Next, the xenograft model established using HER2/neu low expressing gastric cancer cell SNU-16 exhibits better therapeutic effects by scFv S3 than Trastuzumab scFv. By targeting EphA4, scFv S3 can effectively inhibit the growth of SNU-16 gastric tumors in mice showing a tumor growth inhibition rate of 71.6%. The scFv S3 treatment in vivo can degrade EphA4 molecules in tumor tissues, decreasing Ki67 and increasing cleaved caspase 3 molecule expression. Furthermore, through molecular docking of the interaction between scFv S3 and EphA4, we identified and validated that scFv S3 generates essential ionic bonding with R162 on EphA4. Therefore, based on the findings, the bonding between antibodies and EphA4 may reduce potential side effects. The antibody may provide effective treatment for patients with cancer and abnormal activation or overexpression of EphA4 signaling. |
