- 學位論文
探討新型MET抗體抑制MET受體活化之療效與機轉
A novel MET antibody antagonizes MET receptor activation
摘要
肝細胞生長因子(Hepatocyte growth factor, HGF)為細胞表面上的酪胺酸激酶受體MET之受質。HGF/MET 的訊息傳遞路徑調節了許多細胞生理過程，包括了胚胎發育及組織修復等。然而不正常的MET訊號活化透過促進癌細胞增生、增加癌細胞移動與侵犯的能力及產生癌細胞對藥物的抗性等，增進了癌症進程的惡化。因此以抑制MET訊息路徑為標的，是對於多種癌症的治療之良好方針。在我們實驗室先前的研究當中發現肝臟分泌的白血球趨化因子2 (Leukocyte cell-derived chemotoxin 2, LECT2) 能內生性和MET受體結合，降低MET訊息路徑活化並抑制肝癌的血管侵犯。因此我們以MET對LECT2結合的片段作為抗原，生產了新型的MET單株抗體#5-9，評估其對於MET專一性的結合及抑制MET訊息路徑活化之效益。我們評估MET抗體#5-9的結合型態，發現此抗體能在不與HGF競爭下和MET受體結合。接著我們評估MET抗體#5-9 抑制MET訊號活化的效果，無論在是否經由HGF所誘導的MET活化模式當中，MET 5-9抗體皆能抑制癌細胞增生、移動與侵犯的能力。此外MET 抗體#5-9 抑制腦癌細胞球體形成 (sphere formation)，可能與MET抗體降低癌細胞產生對藥物的抗性有關。接著進一步去探討MET抗體#5-9 抑制MET訊息路徑的分子機制，我們發現MET 抗體#5-9 能防止MET形成活化的二聚體(dimerization)、促進MET受體內吞作用(internalization)而降低表面MET的表現，進而在無論是否由HGF所誘導的模式中都能降低MET磷酸化及達到下游訊息的抑制。最後在癌症異種移植(xenograft)的動物模式中驗證了MET抗#5-9拮抗MET訊息傳遞及抑制腫瘤生長的效果。總結來說，新型的MET單株抗體 #5-9能以抑制癌症MET訊息路徑為標的，未來若能應用於臨床可能是對於多種癌症的治療之良好方針。
並列摘要
The MET tyrosine kinase is a cell surface receptor for hepatocyte growth factor (HGF). The HGF/MET signaling regulates many cellular processes, such as embryogenesis and tissue regeneration. However, aberrant activation of MET pathway contributes to cancer malignancy by promoting proliferation, migration, and invasion and resistance. Thus, targeting the MET oncogenic pathway is considered to be a promising therapeutic strategy in multiple cancers. In our previous study, we have identified Leukocyte cell-derived chemotaxin 2 (LECT2) as a novel endogenous MET antagonist to suppress vascular invasion without competing HGF binding in hepatocellular carcinoma (HCC). By using the LECT2 binding site as an antigen, we have generated a monoclonal antibody, anti-MET mAb #5-9 against the specific binding site of MET protein. The in vitro binding assay and competitive ELISA analysis demonstrates that this neutralizing antibody specifically binds to extracellular domain of MET without competing HGF binding. We investigate the effect of the anti-MET mAb #5-9 to inhibit both HGF-dependent and HGF-independent MET activation. The antagonistic effect of anti-MET mAb #5-9 was evaluated by the suppression of MET phosphorylation, cell proliferation, cell migration, sphere formation, and the tumor growth in xenograft model. Moreover, we characterized the molecular mechanism of antibody-mediated MET inactivation, including phosphatases recruitment, receptor internalization and degradation, as well as the ability to prevent MET receptor from dimerization. Collectively, this novel MET antibody may provide a new therapeutic strategy for blocking MET-dependent signaling and eliminating the cancer progression.