研發對抗VEGFR2之人類抗體運用於癌症之治療

Abstract

在腫瘤生長過程當中，腫瘤血管新生 (angiogenesis) 是必經的過程之一，癌細胞藉由新生血管獲得養分及釋放廢物得以進一步擴大惡化。根據以往研究發現，腫瘤新生血管之型態結構以及表現於血管細胞膜上之受體相較於正常血管有很大的不同，故可利用這些受體做為癌症診斷及治療的標的物質；其中，VEGFR2是調控血管新生重要的受體，血管細胞藉由VEGF/VEGFR2訊息傳遞路徑可促進細胞之增生、存活力、轉移以及血管通透性。本篇研究中，透過噬菌體顯現法 (phage display) 篩選出專一性結合VEGFR2的人類單鏈抗體 (scFv)。進一步利用酵素免疫連結吸附反應、西方點墨法、免疫螢光染色法以及流式細胞分析等技術，確認四株表現不同單鏈抗體之噬菌體分別對於VEGFR2蛋白質、經VEGFR2轉染後的293T細胞、以及人類臍帶靜脈內皮細胞 (HUVEC) 有專一性辨認的能力，並且亦可競爭VEGFA結合到VEGFR2。另外，純化後的這四株單鏈抗體蛋白對癌症病患腫瘤血管也具有高度辨識能力。為了更進一步了解這些對抗VEGFR2單鏈抗體在活體之功能，將這幾株噬菌體施打於移植人類腫瘤的免疫不全鼠之後，發現它們對於腫瘤有高度的辨認之特性。綜合以上之結果，我們所篩選出對抗VEGFR2之單鏈抗體的確可辨識細胞及活體腫瘤所表現之VEGFR2，因此，這些對抗VEGFR2之抗體具有應用於發展配體藥引之藥物傳輸系統 (ligand-mediated drug delivery system) 以及建構治療性抗體之潛力，進而改善目前腫瘤治療的困境，亦可結合癌症造影之技術，針對癌症患者做更精確的診斷與追蹤。

Angiogenesis is an essential pathological process during tumor development and progression. The growth of tumor cells need to recruit oxygen and nutrition supplied through new blood vessels. According to the previous study, the structural and molecular diversity of tumor-associated vasculature which very differently from normal vasculature provides a basis for the development of targeted diagnostics and therapeutics. Among these tumor vasculature-associated molecules, VEGFR2 is a crucial receptor which plays an important role in regulation of angiogenesis. Activation of VEGF/VEGFR2 signaling pathway results in the promotion of cell proliferation, migration, survival, and vascular permeability. In this study, several anti-VEGFR2 scFvs were selected from a large human naïve scFv library displayed by phage. After ELISA, Western blot analysis, immunofluorescence staining, and FACS analysis, we identified four anti-VEGFR2 scFvs with high binding affinity against VEGFR2 proteins, VEGFR2 transfected 293T cells, HUVEC, and tumor vessels of cancer patients. Besides, these scFvs were able to inhibit the binding of VEGF to VEGFR2. For further investigation of the binding ability of these scFvs in vivo, we injected these anti-VEGFR2 scFv displayed phages to tumor bearing mice. The tumor homing ability of these four targeting phages were remarkable compared with other normal organs. Our results showed that several anti-VEGFR2 scFv antibodies with the specific recognition ability to cell-expressed VEGFR2 and tumor mass were isolated successfully. Therefore, these anti-VEGFR2 scFvs are promising agents for development of ligand-mediated drug delivery system or for further generation of therapeutic antibodies to overcome the obstacles of cancer treatment. In addition, combination of the scFvs to imaging agents is also attractive for cancer diagnosis.