Please use this identifier to cite or link to this item:
標題: 重組抗-吲哚胺2,3雙加氧酶單鏈抗體之製備與分析
Preparation and characterization of recombinant anti- Indoleamine 2,3-dioxygenase single-chain variable fragment (scFv)
作者: 蘇彥兆
Yan-Zhao Su
關鍵字: 吲哚胺2,3雙加氧酶;單鏈抗體;IDO,single-chain variable fragment;scFv
引用: [1] Leistikow, B., Lung cancer rates as an index of tobacco smoke exposures: validation against black male approximate non-lung cancer death rates, 1969-2000. Prev Med, 2004; 38(5): 511-515. [2] Warnes, A.M., UK and western European late-age mortality: trends in cause-specific death rates, 1960-1990. Health Place, 1999; 5(1): 111-118. [3] Dai W, Gupta SL. Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA. Biochem. Biophys. Res. Commun. 1990; 168 (1): 1–8 [4] Yamamoto, S., & O. Hayaishi. Tryptophan Pyrrolase of Rabbit Intestine D- and L-Tryptophan-cleaving enzyme or enzymes. J. Biol. Chem. 1967; 242:5260–5266. [5] Knox, W. E., & A. H. Mehler. The relation of liver kynureninase to tryptophan metabolism in pyridoxine deficiency. J. Biol. Chem. 1950; 187:419–430. [6] van der Bruggen, P., & B. J. van den Eynde. Processing and presentation of tumor antigens and vaccination strategies. Curr. Opin. Immunol. 2006; 18:98-104. [7] Taylor, M. W., & G. Feng. Relationship between interferon-γ, indoleamine 2,3-dioxygenase, and tryptophan catabolism, FASEB J. 1991; 5:2516-2522. [8] Munn, D.H., M. Zhou, J.T. Attwood, I. Bondarev, S. J. Conway, B. Marshall, C. Brown, & A. L. Mellor. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science. 1998; 281:1191-1193. [9] Hayashi, T., S. P. Rao, K. Takabayashi, J. H. Van Uden, R. S. Kornbluth, S. M. Baird, M. W. Taylor, D. A. Carson, A. Catanzaro, & E. Raz. Enhancement of Innate Immunity against Mycobacterium avium Infection by Immunostimulatory DNA Is Mediated by Indoleamine 2,3-Dioxygenase. Infect Immun. 2001; 69: 6156-6164. [10] Nelson, D. E., D. P. Virok, H. Wood, C. Roshick, R. M. Johnson, W. M. Whitmire, D. D. Crane, O. Steele-Mortimer, L. Kari, G. McClarty, & H. D. Caldwell. Chlamydial IFN-γ immune evasion is linked to host infection tropism. Proc Natl Acad Sci. 2005; 102:10658-10663. [11] Hwu, P., M. X. Du, R. Lapointe, M. Do, M. W. Taylor, & H. A. Young. Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. J. Immunol. 2000; 164:3596-3599. [12] Terness, P., T. M. Bauer, L. Rose, C. Dufter, A. Watzlik, H. Simon, & G. Opelz. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J Exp Med. 2002; 196:447-457. [13] Fallarino, F., C. Vacca, C. Orabona, M. L. Belladonna, R. Bianchi, B. Marshall, D. B. Keskin, A. L. Mellor, M. C. Fioretti, U. Grohmann, & P. Puccetti. Functional expression of indoleamine 2,3-dioxygenase by murine CD8α+ dendritic cells. Int. Immunol. 2002; 14:65-68. [14] von Bubnoff, D., H. Matz, C. Frahnert, M. L. Rau, D. Hanau, H. de la Salle, & T. Bieber. FcεRI induces the tryptophan degradation pathway involved in regulating T cell responses. J. Immunol. 2002; 169:1810-1816. [15] Grohmann, U., C. Orabona, F. Fallarino, C. Vacca, F. Calcinaro, A. Falorni, P. Candeloro, M. L. Belladonna, R. Bianchi, M. C. Fioretti, & P. Puccetti. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 2002; 3:1097-1101. [16] Munn, D. H., J. Pressey, A. C. Beall, R. Hudes, & M. R. Alderson. Selective activation-induced apoptosis of peripheral T cells imposed by macrophages: a potential mechanism of antigen-specific peripheral lymphocyte deletion. J. Immunol. 1996; 156:523-532. [17] Alexander A., Crawford M, Bertera S, Rudert WA, Takikawa O, Robbins PD, Trucco M. Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes. Diabetes. 2002; 51(2):356-365. [18] Miki, T., H. Sun, Y. Lee, A. Tandin, A. M. Kovscek, V. Subbotin, J. J. Fung, & L. A. Valdivia. Blockade of tryptophan catabolism prevents spontaneous tolerogenicity of liver allografts. Transplant. Proc. 2001; 33:129-130. [19] Hwang SL, Chung NP, Chan JK, Lin CL. Indoleamine 2, 3-dioxygenase (IDO) is essential for dendritic cell activation and chemotactic responsiveness to chemokines. Cell Res. 2005; 15(3):167-175. [20] Boon, T., & P. van der Bruggen. Human tumor antigens recognized by T lymphocytes. J. Exp. Med. 1996; 183:725-729. [21] Lennerz, V., M. Fatho, C. Gentilini, R. A. Frye, A. Lifke, D. Ferel, C. Wolfel, C. Huber, & T. Wolfel. The response of autologous T cells to a human melanoma is dominated by mutated neoantigens. Proc. Natl. Acad. Sci. U. S. A. 2005; 102:16013-16018. [22] Ercolini, A. M., B. H. Ladle, E. A. Manning, L. W. Pfannenstiel, T. D. Armstrong, J.P. H. Machiels, J. G. Bieler, L. A. Emens, R. T. Reilly, & E. M. Jaffee. Recruitment of latent pools of high-avidity CD8(+) T cells to the antitumor immune response. J. Exp. Med. 2005; 201: 1591-1602. [23] Nishikawa, H., T. Kato, I. Tawara, T. Takemitsu, K. Saito, L. Wang, Y. Ikarashi, H. Wakasugi, T. Nakayama, M. Taniguchi, K. Kuribayashi, L. J. Old, & H. Shiku. Accelerated chemically induced tumor development mediated by CD4+CD25+ regulatory T cells in wild-type hosts. Proc. Natl. Acad. Sci. U. S. A. 2005; 102:9253-9257. [24] Yu, P., Y. Lee, W. Liu, T. Krausz, A. Chong, & H. Schreiber. Intratumor depletion of CD4+ cells unmasks tumor immunogenicity leading to the rejection of late-stage tumors. J. Exp. Med. 2005; 201:779-791. [25] Fallarino, F., U. Grohmann, K. W. Hwang, C. Orabona, C. Vacca1, R. Bianchi1, M. L. Belladonna1, M. C. Fioretti1, M. L. Alegre, & P. Puccetti1. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 2003; 4:1206-1212. [26] Witkiewicz, A., T. K. Williams, J. Cozzitorto, B. Durkan, S. L. Showalter, C. J. Yeo, J. R. Brody, R. Schulick, J. E. Lee, & J. G. Chandler. Expression of Indoleamine 2,3-Dioxygenase in Metastatic Pancreatic Ductal Adenocarcinoma Recruits Regulatory T Cells to Avoid Immune Detection. J. AM. Coll. Surg. 2008; 206:849-856. [27] Grohmann U, Fallarino F, Puccetti P. Tolerance, DCs and tryptophan: much ado about IDO. Trends Immunol. 2003; 24(5):242-248. [28] Woof JM, Burton DR. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat Rev Immunol. 2004; 4(2):89-99. [29] Nemazee D. Receptor editing in lymphocyte development and central tolerance. Nat Rev Immunol. 2006; 6(10):728-740. [30] Natsume A1, Niwa R, Satoh M. Improving effector functions of antibodies for cancer treatment: Enhancing ADCC and CDC. Drug Des Devel Ther. 2009; 21:(3)7-16. [31] Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975; 256(5517):495-497. [32] Steinitz M. Three decades of human monoclonal antibodies: past, present and future developments. Hum Antibodies; 2009;18(1-2):1-10. [33] Molina MA, Codony-Servat J, Albanell J, Rojo F, Arribas J, Baselga. Trastuzumab (herceptin), a humanized anti-Her2 receptor monoclonal antibody, inhibits basal and activated Her2 ectodomain cleavage in breast cancer cells. J.Cancer Res. 2001; 61(12):4744-4749. [34] Maloney DG, Grillo-Lopez AJ, White CA, Bodkin D, Schilder RJ, Neidhart JA, Janakiraman N, Foon KA, Liles TM, Dallaire BK, Wey K, Royston I, Davis T, Levy R. IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma. Blood. 1997; 90(6):2188-2195. [35] Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988; 85(16):5879-5883. [36] Coloma MJ, Hastings A, Wims LA, Morrison SL. Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. Immunol Methods. 1992; 152(1):89-104. [37] Li J, Wang Y, Li QX, Wang YM, Xu JJ, Dong ZW. Cloning of 3H11 mAb variable region gene and expression of 3H11 human-mouse chimeric light Chain. World J Gastroenterol. 1998; 4(1):41-44.. [38] Miller BR Demarest SJ, Lugovskoy A, Huang F, Wu X, Snyder WB, Croner LJ, Wang N, Amatucci A, Michaelson JS, Glaser SM. tability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel. 2010; 23(7):549-557. S [39] Mathew JP, Shernan SK, White WD, Fitch JC, Chen JC, Bell L, Newman MF. Preliminary report of the effects of complement suppression with pexelizumab on neurocognitive decline after coronary artery bypass graft surgery. Stroke. 2004; 35(10):2335-2339. [40] Peipp M, Saul D, Barbin K, Bruenke J, Zunino SJ, Niederweis M, Fey GH. Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications. J Immunol Methods. 2004; 285(2):265-280. [41] Yan J, Gu GJ, Jost C, Hammond M, Pluckthun A, Landegren U, Kamali-Moghaddam M. A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis. PLoS One. 2014; 9(9) e108061 [42] Schweitzer B, Wiltshire S, Lambert J, O'Malley S, Kukanskis K, Zhu Z, Kingsmore SF, Lizardi PM, Ward DC. Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection. Proc Natl Acad Sci U S A. 2000; 97(18): 10113–10119 [43] Yuan Y. A survey and evaluation of population-based screening for gastric cancer. Cancer Biol Med. 2013; 10(2): 72–80. [44] Pourhassan-Moghaddam M, Rahmati-Yamchi M, Akbarzadeh A, Daraee H, Nejati-Koshki K, Hanifehpour Y, Joo SW. Protein detection through different platforms of immuno-loop-mediated isothermal amplification. Nanoscale Res Lett. 2013; 8(1): 485. [45] Brandacher, G., A. Perathoner, R. Ladurner, S. Schneeberger, P. Obrist, C. Winkler, E. R. Werner, G. Werner-Felmayer, H. G. Weiss, G. Gobel, R. Margreiter, A. Konigsrainer, D. Fuchs, & A. Amberger. Prognostic value of indoleamine 2,3-dioxygenase expression in colorectal cancer: effect on tumor-infiltrating T cells. Clin. Cancer. Res. 2006. 12: 1144-1151.
本研究成功地自表現IDO抗體之融合瘤細胞中取得其可變區基因並進一步建構成融合His-tag之單鏈抗體之表現質體,在篩選最佳之大腸桿菌表現宿主後,以BL21(DE3)pLysS大量表現並使用Ni-resin親和性管柱純化獲得IDO的單鏈抗體。藉由抗體競爭性試驗同時證實了IDO單鏈抗體對IDO的親和力並證實了單鏈抗體與其單株抗體競爭相同區域。未來更可分析IDO單鏈抗體在細胞內抑制IDO活性的能力,並進一步地利用易錯聚合酶鏈反應(error prone PCR)來增加親和力或融合蛋白質轉導區(PTD domain)使單鏈抗體更準確投遞到癌細胞以提升抑制癌細胞內IDO活性的能力。

Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme which plays an important role in tryptophan catabolism. In case of highly IDO-expressed tumor cells, the micro-environmental tryptophan was metabolized into proapoptotic kynurenines. The decline of tryprophan level and accumulation of kynurenines results in suppression of the surrounding T cell's proliferation. Hence, this phenomenon has made the cancer cell flee from the tracking and killing of the T cells thus contributed to the gain-of-function immune-escape of the tumor cells.
It is an innovative anti-cancer strategy to reboot T cell's tumor cells recognition by inactivating IDO using a specific monoclonal antibody. Due to the fact that the antibody is too large to enter the cytosol, employing recombinant DNA technology to generate a small size single-chain variable fragment (scFv) other than large antibody might help breakthrough this bottleneck.
In this project, we have successfully cloned IDO-scFv gene from hybridoma mRNA and fused the gene with a His-tag gene onto an expression vector. The optimized expression of this protein in E. coli BL21(DE3) pLysS was utilized to produce the recombinant IDO-scFv protein. Additionally, the IDO-scFv protein was purified with Ni-IMAC. Using the competition assay with IDO monoclonal antibody and its derived scFv has demonstrated that IDO-scFv could contend with its source monoclonal antibody in the same antigen site. In the future, the inhibition of IDO activity can be monitored by metabolic methods. Furthermore, to enhance scFv's binding affinity by error-prone PCR approach or to improve scFv's cancer-cell-targeting specificity by fusing the tumor specific protein transduction domain (PTD) flank to the scFv could promisingly inactivate IDO in the cancer cell.
Rights: 同意授權瀏覽/列印電子全文服務,2015-02-02起公開。
Appears in Collections:生物化學研究所

Files in This Item:
File Description SizeFormat Existing users please Login
nchu-104-7101058020-1.pdf5.62 MBAdobe PDFThis file is only available in the university internal network    Request a copy
Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.