Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92615
標題: 以重組岩藻醣水解酶修飾岩藻醣殘基化人類免疫球蛋白G之研究
Modification of fucosylated human IgG by recombinant alpha-L-Fucosidase
作者: 楊佩蓉
Pei-Jung Yang
關鍵字: 岩藻醣;抗體依賴型細胞毒殺作用;α-L-岩藻醣水解酶;細菌表面表現技術;core-fucose;ADCC;α-L-fucosidase;E. coli surface display technique
引用: 1. Aggarwal, S.R., What's fueling the biotech engine—2012 to 2013. Nature, 2014. 201: p. 4. 2. Chan, A.C. and P.J. Carter, Therapeutic antibodies for autoimmunity and inflammation. Nature Reviews Immunology, 2010. 10(5): p. 301-316. 3. Bruhns, P., et al., Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses. Blood, 2009. 113(16): p. 3716-3725. 4. Carter, P., Improving the efficacy of antibody-based cancer therapies. Nature Reviews Cancer, 2001. 1(2): p. 118-129. 5. Sondermann, P., et al., The 3.2-? crystal structure of the human IgG1 Fc fragment–FcγRIII complex. Nature, 2000. 406(6793): p. 267-273. 6. Shields, R.L., et al., Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity. Journal of Biological Chemistry, 2002. 277(30): p. 26733-26740. 7. Ferrara, C., et al., Modulation of therapeutic antibody effector functions by glycosylation engineering: Influence of Golgi enzyme localization domain and co-expression of heterologous β1, 4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Biotechnology and bioengineering, 2006. 93(5): p. 851-861. 8. Ferrara, C., et al., The Carbohydrate at FcγRIIIa Asn-162 an element required for high affinity binding to non-fucosylated IgG glycoforms. Journal of Biological Chemistry, 2006. 281(8): p. 5032-5036. 9. Junttila, T.T., et al., Superior in vivo efficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancer. Cancer research, 2010. 70(11): p. 4481-4489. 10. Schwab, I. and F. Nimmerjahn, Intravenous immunoglobulin therapy: how does IgG modulate the immune system? Nature Reviews Immunology, 2013. 13(3): p. 176-189. 11. Varki, A., et al., Biological roles of glycans. 2009. 12. Collin, M. and A. Ols?n, EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG. The EMBO journal, 2001. 20(12): p. 3046-3055. 13. Scanlan, C.N., D.R. Burton, and R.A. Dwek, Making autoantibodies safe. Proceedings of the National Academy of Sciences, 2008. 105(11): p. 4081-4082. 14. Yamane-Ohnuki, N. and M. Satoh. Production of therapeutic antibodies with controlled fucosylation. in MAbs. 2009. 15. Umekawa, M., et al., Efficient glycosynthase mutant derived from Mucor hiemalis endo-β-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan. Journal of Biological Chemistry, 2010. 285(1): p. 511-521. 16. Liu, S.-W., et al., Identification of Essential Residues of Human α-l-Fucosidase and Tests of Its Mechanism. Biochemistry, 2008. 48(1): p. 110-120. 17. Sela, D.A., et al., Bifidobacterium longum subsp. infantis ATCC 15697 α-fucosidases are active on fucosylated human milk oligosaccharides. Applied and environmental microbiology, 2012. 78(3): p. 795-803. 18. Rodriguez-Diaz, J., V. Monedero, and M.J. Yebra, Utilization of natural fucosylated oligosaccharides by three novel α-l-fucosidases from a probiotic Lactobacillus casei strain. Applied and environmental microbiology, 2011. 77(2): p. 703-705. 19. van Bloois, E., et al., Decorating microbes: surface display of proteins on Escherichia coli. Trends in biotechnology, 2011. 29(2): p. 79-86. 20. Ramesh, B., et al., Single-cell characterization of autotransporter-mediated Escherichia coli surface display of disulfide bond-containing proteins. Journal of Biological Chemistry, 2012. 287(46): p. 38580-38589. 21. Trastoy, B., et al., Crystal structure of Streptococcus pyogenes EndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies. Proceedings of the National Academy of Sciences, 2014. 111(18): p. 6714-6719. 22. Romano, P.R., et al., Development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycans. Biochemical and biophysical research communications, 2011. 414(1): p. 84-89. 23. Matsumura, K., et al., Carbohydrate Binding Specificity of a Fucose-specific Lectin from Aspergillus oryzae A NOVEL PROBE FOR CORE FUCOSE. Journal of biological chemistry, 2007. 282(21): p. 15700-15708.
摘要: 
近年來抗體藥物的研發越趨重要,如何讓抗體藥物更具療效成為一個重要的課題。目前已知,若抗體缺乏核心岩藻醣殘基將會造成強烈的抗體依賴型細胞毒殺作用 (ADCC),另外同時可以藉由與作用細胞 (effector cell)上受體 (Fc gamma recepter IIIa, FcγRIIIa)高度結合來抑制血清中免疫球蛋白G (IgG)與同個受體結合的稀釋作用。因此,我們將策略定在找到有效切除人類抗體上岩藻醣基的岩藻醣水解酶,及做基因重組的改良以達到目的。
我們從乳酸桿菌 (Lactobacillus)、比菲德氏菌 (Bifidobacterium)及擬桿菌(Bacteroides)中尋找、利用不同的標記 (tag)修飾來做基因重組表現,發現來自凱氏乳酸桿菌BL23菌株的岩藻醣水解酶重組蛋白搭配內切醣酵素 (endoglycosidase)具有切除人類抗體及其他蛋白上岩藻醣基的活性。為了鑑定岩藻醣水解酶活性我們建立了幾種方法:(1)凝集素墨點法 ?使用對岩藻醣基有專一性的凝集素 (Aleuria aurantia Lectin, AAL);(2)胞外酵素活性分析 使用可釋放可見光的岩藻醣基化合物4-nitrophenyl fucopyranoside;(3)抗體的N聚醣譜型分析。結果顯示,來自凱氏乳酸桿菌BL21菌株基因體的岩藻醣水解酶組胺酸重組蛋白AlfC與醣苷內切酶共同作用時可以水解人類IgG及其他醣蛋白上的岩藻醣修飾。
另外,我們將大腸桿菌 (E. coli)表面主動運輸蛋白 (autotransporter)與岩藻醣水解酶重組,將酵素表現在細菌表面,如此則不需要經由蛋白質純化過程即可測得具切除岩藻醣基化合物活性。

Glycoengineered therapeutic antibodies lacking core fucose residues exhibit strong ADCC, in the same time, the high binding with the Fc gamma recepter IIIa on the effector cell surface evade the serum IgG inhibitory effect. We aimed to use enzymatic modification of N-glycans to produce non-fucosylated forms on human antibodies.
In this study, we searched for α-L-fucosidase candidates from Lactobacillus, Bifidobacterium and Bacteroides species. To confirm fucosidase activities, several methods was established: (1) Lectin blot with the use of Aleuria aurantia Lectin; (2) in vitro enzymatic analysis with 4-nitrophenyl fucopyranoside; (3) N-glycan profiling of antibodies. The results showed that (His)6-tagged recombinant α-L-fucosidase AlfC from the Lactobacillus casei strain BL23, combined with endoglycosidases, has the activity to release fucose residues on human IgG and other glycoproteins.
Further, we use surface display techniques to express AlfC on the surface of E. coli cells. In the end, we identified a suitable system 183 for surface display of AlfC.
URI: http://hdl.handle.net/11455/92615
其他識別: U0005-2811201416182881
Rights: 同意授權瀏覽/列印電子全文服務,2016-08-31起公開。
Appears in Collections:生命科學院碩士在職專班

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