血管新生是指由現存血管產生新血管的過程，是腫瘤生成的一個重要步驟，
新產生的血管主要功能是提供腫瘤養分和廢物的運送通道，並且可以作為癌症
轉移的路徑，因此，抑制血管新生也就成為了一個有潛力的治療標靶。在過去
的許多研究中證實了整合蛋白 α5β1 和血管內皮生長因子受體 2(VEGFR2)可共
同調控參與了血管新生的數個內皮細胞功能，其中包括細胞存活、遷移和管狀
形成。而在本研究中，我們利用纖維黏蛋白第三型區域作為蛋白鷹架，設計出
能夠拮抗 VEGFR2 以及整合蛋白 α5β1 的重組蛋白，並且利用 GGGGS 的重複
序列連接兩個拮抗蛋白，依照連接的順序以及連接序列重複次數的不同，設計
出 4 種能同時拮抗 α5β1 和 VEGFR2 的雙重專一性重組蛋白，並且成功的用
E.coli 表現系統取得重組蛋白。首先，進行蛋白穩定度的測試，在熱穩定度方
面，雙重專一性蛋白表現了比 VEGFR2 拮抗蛋白更佳的熱穩定度；另一方面，
溶解度方面比起 VEGFR2 拮抗蛋白也是有 2~5 倍不等的提昇，但和 α5β1 拮抗
蛋白相比則還有很大的進步空間。接下來進行細胞實驗，在 k562 細胞黏著實
驗中，我們發現 α5β1 拮抗蛋白區域在 N 端的組別(簡稱 I-n-V)保留了原本的專一性結合能力(IC 50 約 40nM)，而 α5β1 拮抗蛋白區域在 C 端的組別(簡稱 V-n-I)和單一專一性的 α5β1 拮抗蛋白相比(IC 50 約 50nM)，其專一性結合能力有上升(IC 50 約 20nM)。而在抑制血管新生實驗中，V-n-I 的組別在 HUVEC 的存活、遷移以及管狀形成的抑制能力上，都發現到比起單一專一性拮抗劑的抑制能力有顯著提昇，而 I-n-V 組別的抑制能力則是沒有顯著的提昇。基於以上實驗結果，我們選擇 V-n-I 組別中的 VEGFR2-(2)-α5β1 進行動物實驗，測試其抑制神經膠細胞瘤的腫瘤成長能力，結果發現確實對 U87 神經膠細胞瘤的生長有抑制能力，並且能夠提昇小鼠的存活時間，本次研究的結果是提出了一個能夠同時針對 VEGFR2 以及整合蛋白 α5β1 進行抑制的蛋白臨床藥物。

VEGFR2 and integrin α5β1 are involved in tumor angiogenesis, and the inhibition of these targets results in angiogenesis reduction and tumor repression. In this study we used fibronectin type III domain (Fn3) to develop dual VEGFR2- and integrin α5β1-specific Fn3 variants for cancer treatment. Dual-specific mutants were designed by linking VEGFR2-specific and integrin α5β1-specific 9,10Fn3 variants with the (G4S1)2 or (G4S1)3 repeat sequences. Endothelial cell proliferation analysis showed that integrin α5β1-specific, VEGFR2-specific, and dual-specific Fn3 variants inhibited VEGF-induced HUVEC proliferation with the IC 50 values of 504, 578, and 122 nM.
We also found that dual-specific proteins inhibited VEGF-induced HUVEC imgration with the IC 50 value of ~1μM, and VEGFR2-specific 10Fn3 mutant exhibited 2-fold less inhibitory activity with the IC 50 value of ~2μM. In contrast, integrin α5β1-specific 9,10Fn3 mutant only exhibited slight inhibitory activity in the same concentration. The analysis of endothelial cell tube-formation assay showed that dual-specific Fn3 variants exhibited higher inhibitory activity than mono-specific Fn3 variants. These results were consistent with our hypothesis that co-inhibition of integrin α5β1 and VEGFR2 could synergistically suppress the endothelial cell proliferation, migration and tube-formation in angiogenesis process. The result of xenograph animal model showed that bi-specific proteins can reduce the growth of U87 glioma tumor and increase its survival. This study indicated that dual VEGFR2- and integrin α5β1-specific Fn3 variant is a potential drug for cancer treatment.

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