利用噬菌體顯現法識別活化內皮細胞的結合配體

惡性腫瘤已成為世界及國人重要死因之一。在腫瘤傳統治療上，由於腫瘤細胞的基因不穩定性（genetic instability）高，容易產生抗藥性（drug resistant）的形成。腫瘤生長和轉移仰賴血管生成作用（angiogenesis）的觀念提出後，加上血管內皮細胞的基因變化遠比腫瘤細胞的變化小，使得血管內皮細胞成為重要的癌症治療標的，而血管生成作用的抑制也成為熱門的研究。在本研究中，我們利用噬菌體顯現法（phage display）找尋腫瘤血管內皮細胞的特有結合配位標記。血管靜止蛋白（angiostatin）是內生性的腫瘤血管生成抑制蛋白，具專一性和腫瘤血管內皮細胞結合的能力。利用噬菌體顯現胜肽庫，我們從四個對抗血管靜止蛋白功能性的單株抗體上找到了B細胞抗原決定位。運用這些B-細胞抗原決定位所合成的胜肽配體將有助於尋找出血管靜止蛋白上的功能性區域及發展抗血管生成治療（anti-angiogenesis therapy）。我們也利用噬菌體顯現法和血管內皮生長因子（VEGF）活化處理的人類臍靜脈內皮細胞（HUVEC）作用，可以尋找和增生內皮細胞結合的專一性胜肽序列。我們找出了數個可以和增生內皮細胞具較高結合性的胜肽，其中部份胜肽序列已見於其他發表文章，部份尚未發表仍屬新穎，由此可證明此方法的可行性。在活體動物實驗，我們也證明PCH118 噬菌體株可以專一性的標定到口腔癌細胞的異體移植腫瘤組織。本研究所找尋到具有專一性和活化人類臍靜脈內皮細胞結合的胜肽，未來可以運用於增生人類臍靜脈內皮細胞抗原的純化及確認，並可以發展標的性治療。

關鍵字

噬菌體顯現法；抗血管新生；標的性治療；血管內皮細胞標記

並列摘要

Malignant tumor has become the most important cause of death around the world and in our country. Drug resistance formation due to high genetic instability of tumor cells were reported in conventional tumor therapies. Tumor progress and metastases depend on angiogenesis imply tumor vascular endothelial cells is a potent target of cancer therapy. Endothelial cells are stable in genetic mutations and inhibition of angiogenesis becomes hot research area. In this study, phage display was used to identify tumor vascular endothelial cells specific binding ligands. Angiostatin is a potent endogenous inhibitor of angiogenesis that specific bind to tumor vessels. Using phage-displayed peptide library, we have identified B-cell epitopes from several functional MAbs against angiostatin. These peptide ligands will be useful to identify the functional domain of angiostatin and develop anti-angiogenesis therapy. To identify peptide ligands specific binding to proliferated endothelial cells, phage display biopanning using VEGF stimulated HUVEC was performed. We have identified several peptide ligands with higher capability to bind stimulated-HUVEC. Some motifs of these peptide ligands have been published and some peptide ligands are novel. In vivo homing test showed that phage clone PCH118 targeting specifically to tumor tissue vasculature of oral cancer xenograft. Stimulated HUVEC-binding ligands identify in this study will be valuable to identify proliferated endothelial cell markers as well as develop targeted therapy.

並列關鍵字

phage display ； anti-angiogenesis ； targeting therapy ； vascular endothelial cell marker

參考文獻

An, P., Lei, H., Zhang, J., Song, S., He, L., Jin, G., Liu, X., Wu, J., Meng, L., Liu, M., and Shou, C. (2004). Suppression of tumor growth and metastasis by a VEGFR-1 antagonizing peptide identified from a phage display library. Int J Cancer 111, 165-173.

Arap, W., Pasqualini, R., and Ruoslahti, E. (1998). Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279, 377-380.

Bergers, G., and Benjamin, L. E. (2003). Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3, 401-410.

Bindra, R. S., and Glazer, P. M. (2005). Genetic instability and the tumor microenvironment: towards the concept of microenvironment-induced mutagenesis. Mutat Res 569, 75-85.

Borsum, T., Hagen, I., Henriksen, T., and Carlander, B. (1982). Alterations in the protein composition and surface structure of human endothelial cells during growth in primary culture. Atherosclerosis 44, 367-378.

國際替代計量

利用噬菌體顯現法識別活化內皮細胞的結合配體

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