纖維化的慢性組織損傷導致組織結構破壞、器官功能障礙和最終的器官衰竭。整合蛋白是穿膜的異源二聚體 (ab) 受體，介導細胞與細胞和細胞與細胞外基質的相互作用。av整合素家族成員的調節對多種器官和疾病狀態的纖維化表現出深遠的影響。特別是抑制整合蛋白avb6 (肺中TGF-b的關鍵激活劑) 是一種有潛力的治療策略。血管生成在生長發育中起重要作用。整合蛋白avb3和血管內皮生長因子受體2（VEGFR2）可以共同調節許多與血管生成有關的細胞活動，包括內皮細胞遷移、血管生成和血管內造血細胞功能。在我們之前的研究中成功地使用了纖維黏蛋白第三型結構域的第十個模組 (10Fn3) 包含1491CPRGDMPDC序列來設計整合蛋白avb3拮抗劑。在這項研究中，將使用10Fn3作為支架來設計抗纖維化和血管生成劑。我已經表達了十種Fn3蛋白，包含六個抗纖維化突變蛋白和四個抗血管生成突變蛋白，並將它們純化至同質。 M1496R突變體抑制整合蛋白avb6的能力有11.9倍的增加，IC50值為590.2 nM。另一方面，先前研究中發現C9KW (Fn3和Disintegrin的融合蛋白)存在降解問題。然而，靶向VEGFR2和整合蛋白avb3的雙特異性Fn3融合蛋白沒有明顯降解問題。此外，雙特異性Fn3融合蛋白表現出抗內皮細胞增殖能力，IC50值約為20 nM。 另一方面，三特異性融合蛋白在抗遷移試驗中顯示出抑制作用，IC50值為約為10 nM。這項研究的結果將用於設計使用Fn3的抗纖維化和血管生成藥物。

Chronic tissue injury with fibrosis results in the disruption of tissue architecture, organ dysfunction and eventual organ failure. Integrins are membrane-spanning heterodimeric (ab) receptors that mediate cell-cell and cell-extracellular matrix interactions. Modulation of members of the αv integrin family has exhibited profound effects on fibrosis in multiple organs and disease states. In particular, inhibition of integrin avb6, a key activator of TGF-b in lung, is an attractive therapeutic strategy. Angiogenesis plays an important role in growth and development. Integrin avb3 and vascular endothelial growth factor receptor 2 (VEGFR2) could co-regulate many cellular activities associated with angiogenesis, including endothelial cell migration, tube formation, and hematopoietic cell functions within vasculature. In our previous study we have successfully used the tenth module of fibronectin type III domain (Fn3) to design integrin avb3 antagonist containing a 1491CPRGDMPDC motif. In this study we aim to use Fn3 as a scaffold to design anti-fibrotic and -angiogenic agents. I have expressed ten Fn3 proteins, including six mutants for anti-fibrotic agent and four mutants for anti-angiogenic agent, and purified them to homogeneity. I found that M1496R mutant exhibited 11.9-fold increase in inhibiting avb6 with the IC50 value of 590.2 nM. In our previous study we found C9KW, a fusion protein of Fn3 and disintegrin, with degradation problem. I found that bi-specific Fn3 fusion protein targeting VEGFR2 and integrin avb3 did not have degradation problem. In addition, bi-specific Fn3 fusion proteins exhibited anti-proliferation ability with the IC50 value of ~20 nM. Furthermore, a tri-specific fusion protein showed inhibitory effects on anti-migration assay with the IC50 value of ~10 nM. The results of this study will be used to design anti-fibrotic and -angiogenic agents using Fn3.

Abhinand, C. S., Raju, R., Soumya, S. J., Arya, P. S., & Sudhakaran, P. R. (2016). VEGF-A/VEGFR2 signaling network in endothelial cells relevant to angiogenesis. J Cell Commun Signal, 10(4), 347-354.

Bloom, L., & Calabro, V. (2009). FN3: a new protein scaffold reaches the clinic. Drug Discov Today, 14(19-20), 949-955.

Borges, E., Jan, Y., & Ruoslahti, E. (2000). Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain. J Biol Chem, 275(51), 39867-39873.

Bredel, M., Bredel, C., Juric, D., Harsh, G. R., Vogel, H., Recht, L. D., & Sikic, B. I. (2005). Functional network analysis reveals extended gliomagenesis pathway maps and three novel MYC-interacting genes in human gliomas. Cancer Res, 65(19), 8679-8689.

Brooks, P. C. (1996). Role of integrins in angiogenesis. Eur J Cancer, 32A (14), 2423-2429.

Brooks, P. C., Montgomery, A. M., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G., & Cheresh, D. A. (1994). Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell, 79(7), 1157-1164.

Byzova, T. V., Goldman, C. K., Pampori, N., Thomas, K. A., Bett, A., Shattil, S. J., & Plow, E. F. (2000). A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol Cell, 851-860.

Chandler, P. G., & Buckle, A. M. (2020). Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain. Cells, 9(3).

Chen, Y., Lee, H., Tong, H., Schwartz, M., & Zhu, C. (2017). Force regulated conformational change of integrin alphaVbeta3. Matrix Biol, 60-61, 70-85.

Connolly, D. T., Heuvelman, D. M., Nelson, R., Olander, J. V., Eppley, B. L., Delfino, J. J., Siegel, N. R., Leimgruber, R. M., & Feder, J. (1989). Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. The Journal of clinical investigation, 84(5), 1470–1478.
Desgrosellier, J. S., & Cheresh, D. A. (2010). Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer, 10(1), 9-22.

Djudjaj, S., & Boor, P. (2019). Cellular and molecular mechanisms of kidney fibrosis. Mol Aspects Med, 65, 16-36.

Durowoju, I. B., Bhandal, K. S., Hu, J., Carpick, B., & Kirkitadze, M. (2017). Differential Scanning Calorimetry - A Method for Assessing the Thermal Stability and Conformation of Protein Antigen. J Vis Exp(121).

Erratum: Hayward IP, Bridle KR, Campbell GR, Underwood PA, Campbell JH (1995) Effect of Extracellular Matrix Proteins on Vascular Smooth Muscle Cell Phenotype. Cell Biology International 19: 839-846.

Ferrara, N., & Henzel, W. J. (2012). Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. 1989. Biochem Biophys Res Commun, 425(3), 540-547.

Foubert, P., & Varner, J. A. (2012). Integrins in tumor angiogenesis and lymphangiogenesis. Methods Mol Biol, 757, 471-486.

Gill, P., Moghadam, T. T., & Ranjbar, B. (2010). Differential scanning calorimetry techniques: applications in biology and nanoscience. J Biomol Tech, 21(4), 167-193.

Ginsberg, M. H., Du, X., & Plow, E. F. (1992). Inside-out integrin signalling. Curr Opin Cell Biol, 4(5), 766-771.

Gupta, P., Sata, T. N., Yadav, A. K., Mishra, A., Vats, N., Hossain, M. M., . . . Venugopal, S. K. (2019). TGF-beta induces liver fibrosis via miRNA-181a-mediated down regulation of augmenter of liver regeneration in hepatic stellate cells. PLoS One, 14(6), e0214534.

Hsu, A. R., Veeravagu, A., Cai, W., Hou, L. C., Tse, V., & Chen, X. (2007). Integrin alpha v beta 3 antagonists for anti-angiogenic cancer treatment. Recent Pat Anticancer Drug Discov, 2(2), 143-158.

Janouskova H, Maglott A, Leger DY, Bossert C, Noulet F, Guerin E, Guenot D, Pinel S, Chastagner P, Plenat F, Entz-Werle N, Lehmann-Che J, Godet J, Martin S, Teisinger J, Dontenwill M. (2012) Integrin α5β1 plays a critical role in resistance to temozolomide by interfering with the p53 pathway in high-grade glioma. Cancer Res. Jul 15;72(14):3463-70.

Kemperman, H., Wijnands, Y. M., & Roos, E. (1997). alphaV Integrins on HT-29 colon carcinoma cells: adhesion to fibronectin is mediated solely by small amounts of alphaVbeta6, and alphaVbeta5 is codistributed with actin fibers. Exp Cell Res, 234(1), 156-164.

Kim, S., Bell, K., Mousa, S. A., & Varner, J. A. (2000). Regulation of angiogenesis in vivo by ligation of integrin alpha5beta1 with the central cell-binding domain of fibronectin. Am J Pathol, 156(4), 1345-1362.

Koide, A., Bailey, C. W., Huang, X., & Koide, S. (1998). The fibronectin type III domain as a scaffold for novel binding proteins. J Mol Biol, 284(4), 1141-1151.

Krayem, N., Abdelkefi-Koubaa, Z., Gargouri, Y., & Luis, J. (2018). Integrin-mediated human glioblastoma cells adhesion, migration and invasion by native and recombinant phospholipases of Scorpio maurus venom glands. Arch Biochem Biophys, 645, 19-25.

Leask, A., & Abraham, D. J. (2004). TGF-beta signaling and the fibrotic response. FASEB J, 18(7), 816-827.

Lee, J. O., Bankston, L. A., Arnaout, M. A., & Liddington, R. C. (1995). Two conformations of the integrin A-domain (I-domain): a pathway for activation? Structure, 3(12), 1333-1340.

Li, C., Wen, A., Shen, B., Lu, J., Huang, Y., & Chang, Y. (2011). FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnol, 11, 92.

Lipovsek, D. (2011). Adnectins: engineered target-binding protein therapeutics. Protein Eng Des Sel, 24(1-2), 3-9.

Lowell, C. A., & Mayadas, T. N. (2012). Overview: studying integrins in vivo. Methods Mol Biol, 757, 369-397.

Lu, R. M., Hwang, Y. C., Liu, I. J., Lee, C. C., Tsai, H. Z., Li, H. J., & Wu, H. C. (2020). Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci, 27(1), 1.
Mahabeleshwar, G. H., Chen, J., Feng, W., Somanath, P. R., Razorenova, O. V., & Byzova, T. V. (2008). Integrin affinity modulation in angiogenesis. Cell Cycle, 7(3), 335-347.

Mahabeleshwar, G. H., Feng, W., Phillips, D. R., & Byzova, T. V. (2006). Integrin signaling is critical for pathological angiogenesis. J Exp Med, 203(11), 2495-2507.

Mamluk, R., Carvajal, I. M., Morse, B. A., Wong, H., Abramowitz, J., Aslanian, S., Lim, A. C., Gokemeijer, J., Storek, M. J., Lee, J., Gosselin, M., Wright, M. C., Camphausen, R. T., Wang, J., Chen, Y., Miller, K., Sanders, K., Short, S., Sperinde, J., Prasad, G., … Furfine, E. S. (2010). Anti-tumor effect of CT-322 as an adnectin inhibitor of vascular endothelial growth factor receptor-2. mAbs, 2(2), 199–208.

Mao, Y., & Schwarzbauer, J. E. (2005). Fibronectin fibrillogenesis, a cell-mediated matrix assembly process. Matrix Biol, 24(6), 389-399.

Mattern, R. H., Read, S. B., Pierschbacher, M. D., Sze, C. I., Eliceiri, B. P., & Kruse, C. A. (2005). Glioma cell integrin expression and their interactions with integrin antagonists: Research Article. Cancer Ther, 3A, 325-340.

Meng, X. M., Nikolic-Paterson, D. J., & Lan, H. Y. (2016). TGF-beta: the master regulator of fibrosis. Nat Rev Nephrol, 12(6), 325-338.

Miotto M, Olimpieri PP, Di Rienzo L, Ambrosetti F, Corsi P, Lepore R, Tartaglia GG, Milanetti E. (2019) Insights on protein thermal stability: a graph representation of molecular interactions. Bioinformatics. 1;35(15):2569-2577.

Mulgrew K, Kinneer K, Yao XT, Ward BK, Damschroder MM, Walsh B, Mao SY, Gao C, Kiener PA, Coats S, Kinch MS, Tice DA. (2006) Direct targeting of alphavbeta3 integrin on tumor cells with a monoclonal antibody, Abegrin. Mol Cancer Ther ;5(12):3122-9.

Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, Rifkin DB, Sheppard D. (1999) The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell. 5;96(3):319-28.

Napione, L., Pavan, S., Veglio, A., Picco, A., Boffetta, G., Celani, A., . . . Bussolino, F. (2012). Unraveling the influence of endothelial cell density on VEGF-A signaling. Blood, 119(23), 5599-5607.

Niu, G., & Chen, X. (2011). Why integrin as a primary target for imaging and therapy. Theranostics, 1, 30-47.

Park, H. J., Zhang, Y., Georgescu, S. P., Johnson, K. L., Kong, D., & Galper, J. B. (2006). Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis. Stem Cell Rev, 2(2), 93-102.

Patsenker, E., Popov, Y., Stickel, F., Jonczyk, A., Goodman, S. L., & Schuppan, D. (2008). Inhibition of integrin alphavbeta6 on cholangiocytes blocks transforming growth factor-beta activation and retards biliary fibrosis progression. Gastroenterology, 135(2), 660-670.

Piao, Y., Liang, J., Holmes, L., Zurita, A. J., Henry, V., Heymach, J. V., & de Groot, J. F. (2012). Glioblastoma resistance to anti-VEGF therapy is associated with myeloid cell infiltration, stem cell accumulation, and a mesenchymal phenotype. Neuro Oncol, 14(11), 1379-1392.

Puthawala K, Hadjiangelis N, Jacoby SC, Bayongan E, Zhao Z, Yang Z, Devitt ML, Horan GS, Weinreb PH, Lukashev ME, Violette SM, Grant KS, Colarossi C, Formenti SC, Munger JS. (2008) Inhibition of integrin alpha(v)beta6, an activator of latent transforming growth factor-beta, prevents radiation-induced lung fibrosis. Am J Respir Crit Care Med. 1;177(1):82-90.

Redick, S. D., Settles, D. L., Briscoe, G., & Erickson, H. P. (2000). Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis. J Cell Biol, 149(2), 521-527.

Sarabipour, S., Ballmer-Hofer, K., & Hristova, K. (2016). VEGFR-2 conformational switch in response to ligand binding. Elife, 5, e13876.
Seguin, J., Nicolazzi, C., Mignet, N., Scherman, D., & Chabot, G. G. (2012). Vascular density and endothelial cell expression of integrin alpha v beta 3 and E-selectin in murine tumours. Tumour Biol, 33(5), 1709-1717.

Senger, D. R., Galli, S. J., Dvorak, A. M., Perruzzi, C. A., Harvey, V. S., & Dvorak, H. F. (1983). Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science, 219(4587), 983-985.

Shen, B., Delaney, M. K., & Du, X. (2012). Inside-out, outside-in, and inside-outside-in: G protein signaling in integrin-mediated cell adhesion, spreading, and retraction. Curr Opin Cell Biol, 24(5), 600-606.

Shimaoka M, Xiao T, Liu JH, Yang Y, Dong Y, Jun CD, McCormack A, Zhang R, Joachimiak A, Takagi J, Wang JH, Springer TA. (2003) Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell. 10;112(1):99-111.

Singh, P., Carraher, C., & Schwarzbauer, J. E. (2010). Assembly of fibronectin extracellular matrix. Annu Rev Cell Dev Biol, 26, 397-419.

Soldi, R., Mitola, S., Strasly, M., Defilippi, P., Tarone, G., & Bussolino, F. (1999). Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2. EMBO J, 18(4), 882-892.

Song, N., Liu, B., Wu, J., Zhang, R., Duan, L., He, W., & Zhang, C. (2014). Vascular endothelial growth factor (VEGF) -2578C/A and -460C/T gene polymorphisms and lung cancer risk: a meta-analysis involving 11 case-control studies. Tumour Biol, 35(1), 859-870.

Tamkun, J. W., & Hynes, R. O. (1983). Plasma fibronectin is synthesized and secreted by hepatocytes. J Biol Chem, 258(7), 4641-4647.

Techasen, A., Loilome, W., Namwat, N., Khuntikeo, N., Puapairoj, A., Jearanaikoon, P., . . . Yongvanit, P. (2014). Loss of E-cadherin promotes migration and invasion of cholangiocarcinoma cells and serves as a potential marker of metastasis. Tumour Biol, 35(9), 8645-8652.

Teleanu, R. I., Chircov, C., Grumezescu, A. M., & Teleanu, D. M. (2019). Tumor Angiogenesis and Anti-Angiogenic Strategies for Cancer Treatment. J Clin Med, 9(1).
To, W. S., & Midwood, K. S. (2011). Plasma and cellular fibronectin: distinct and independent functions during tissue repair. Fibrogenesis Tissue Repair, 4, 21.

Traister, A., Li, M., Aafaqi, S. et al. (2014) Integrin-linked kinase mediates force transduction in cardiomyocytes by modulating SERCA2a/PLN function. Nat Commun 5, 4533.

Trevino, S. R., Scholtz, J. M., & Pace, C. N. (2007). Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa. J Mol Biol, 366(2), 449-460.

Tzakas, P., Wong, B. Y., Logan, A. G., Rubin, L. A., & Cole, D. E. (2005). Transforming growth factor beta-1 (TGFB1) and peak bone mass: association between intragenic polymorphisms and quantitative ultrasound of the heel. BMC Musculoskelet Disord, 6, 29.

Wang, Y., Fei, D., Vanderlaan, M., & Song, A. (2004). Biological activity of bevacizumab, a humanized anti-VEGF antibody in vitro. Angiogenesis, 7(4), 335-345.

Weekes CD, Rosen LS, Capasso A, Wong KM, Ye W, Anderson M, McCall B, Fredrickson J, Wakshull E, Eppler S, Shon-Nguyen Q, Desai R, Huseni M, Hegde PS, Pourmohamad T, Rhee I, Bessudo A. (2018) Phase I study of the anti-α5β1 monoclonal antibody MINT1526A with or without bevacizumab in patients with advanced solid tumors. Cancer Chemother Pharmacol. ;82(2):339-351.

Bessudo, A. (2018). Phase I study of the anti-alpha5beta1 monoclonal antibody MINT1526A with or without bevacizumab in patients with advanced solid tumors. Cancer Chemother Pharmacol, 82(2), 339-351.

Wynn, T. A., & Ramalingam, T. R. (2012). Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med, 18(7), 1028-1040.

Zhijun, X., Shulan, Z., & Zhuo, Z. (2007). Expression and significance of the protein and mRNA of metastasis suppressor gene ME491/CD63 and integrin alpha5 in ovarian cancer tissues. Eur J Gynaecol Oncol, 28(3), 179-183.

歐陽睿. (2020). 使用不同類型和長度的鏈接器設計抗VEGFR2及抗整合蛋白αvβ3雙特異性融合蛋白. 國立成功大學生物化學暨分子生物學研究所碩士論文

蔡景皓. (2018). 利用去整合突變蛋白設計具有專一性辨認整合蛋白αvβ6或αIIbβ3之拮抗劑. 國立成功大學生物化學暨分子生物學研究所碩士論文

林舉征. (2017). 蛋白質工程以改善利用第三型纖維黏蛋白片段之VEGFR2專一性拮抗劑的熱穩定性與溶解度. 國立成功大學生物化學暨分子生物學研究所碩士論文