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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 翁啟惠(Chi-Huey Wong) | |
| dc.contributor.author | Chiu-Ping Liu | en |
| dc.contributor.author | 劉秋萍 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:11:16Z | - |
| dc.date.available | 2023-03-01 | |
| dc.date.copyright | 2018-03-01 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-01-17 | |
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Striebeck A, Robinson DA, Schuttelkopf AW, van Aalten DMF (2013) Yeast Mnn9 is both a priming glycosyltransferase and an allosteric activator of mannan biosynthesis. Open Biol 3(9). 44. Wang TY, et al. (2013) Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiae identifies mutants with improved heterologous exocellulase activity and host secretion. BMC Biotechnol. 13:71. 45. Wang LX, Lomino JV (2012) Emerging technologies for making glycan-defined glycoproteins. ACS Chem. Biol. 7(1):110-122. 46. Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23(9):1126-1136. 47. Holliger P, Winter G (1993) Engineering bispecific antibodies. Curr. Opin. Biotechnol. 4(4):446-449. 48. Werner RG (2004) Economic aspects of commercial manufacture of biopharmaceuticals. J. Biotechnol. 113(1-3):171-182. 49. Wang CC, et al. (2009) Glycans on influenza hemagglutinin affect receptor binding and immune response. Proc. Natl. Acad. Sci. U. S. A. 106(43):18137-18142. 50. Zhang N, et al. (2011) Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study. Mabs-Austin 3(3):289-298. 51. Ratner M (2014) Genentech's glyco-engineered antibody to succeed Rituxan. Nat. Biotechnol. 32(1):6-7. 52. Cartron G, et al. (2002) Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene. Blood 99(3):754-758. 53. Musolino A, et al. (2008) Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer. J. Clin. Oncol. 26(11):1789-1796. 54. Reusch D, Tejada ML (2015) Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 25(12):1325-1334. 55. Beck A, et al. (2008) Trends in Glycosylation, Glycoanalysis and Glycoengineering of Therapeutic Antibodies and Fc-Fusion Proteins. Curr. Pharm. Biotechnol. 9(6):482-501. 56. Monnet C, et al. (2014) Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody. Mabs-Austin 6(2):422-436. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68015 | - |
| dc.description.abstract | 近年來開發以單株抗體作為治療癌症，抗發炎及防禦病原體等之標靶藥物深受矚目; 然而，抗體Fc部位上的醣體組成對其與各種免疫細胞間的作用力具有相當大之影響。目前市面上以哺乳動物細胞作為宿主表達出來之抗體，除了其Fc部位上帶有非均相化的醣分子，且生產過程易受到病毒污染，純化費時，價格亦相對昂貴，因此，開發具大量生產均相化、且結構明確的抗體表達系統為亟待解決之課題。本研究利用醣化基因OCH1缺陷之甲醇誘導型畢赤酵母菌表現trastuzumab抗體，所產出之抗體Fc上的醣分子以五顆甘露糖鏈結 (Man5) 為主; 接著，我們從CAZymes資料庫中篩選並以大腸桿菌E. coli表達出十種新的內切糖苷酶 (Endoglycosidase)，作為後續抗體醣化修飾之酵素，且這些新發現的酵素可替代現有之內切糖苷酶EndoA與EndoH，及轉醣酶EndoS。此外，EndoE與EndoP兩種酵素不僅可利用雙觸鏈結寡糖鏈 (bi-antennary oligosaccharide chains)結構的醣分子作為抗體上Fc醣修飾的醣體來源，亦有催化三觸及四觸鏈結寡糖鏈 (tri- and tetra-antennary oligosaccharide chains) 結構的醣分子之潛力。本研究提供醣修飾及優化醣蛋白的系統性方法，並能提升IgG抗體之功效。 | zh_TW |
| dc.description.abstract | Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Since the glycosylation of mAbs in the Fc region influences their interaction with effector cells that kill antibody-targeted cells, and the current method of antibody expression in mammalian hosts exhibits heterogeneous glycosylation patterns and is relatively expensive, new efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAb with desirable glycoforms. The methylotrophic yeast Pichia pastoris that produces glycoproteins with high-mannose N-glycans has recently been engineered to express therapeutic glycoproteins. In this study, we demonstrate that glycosylation remodeling of the monoclonal antibody trastuzumab expressed in glycoengineered P. pastoris, through de-glycosylation by endoglycosidases identified from the CAZymes database, and transglycosylation using glycans with stable leaving group will generate a homogeneous antibody designed to optimize the effector functions. The ten newly identified recombinant bacterial endoglycosidases are complementary to existing endoglycosidases (EndoA, EndoH, EndoS); and two of which can even accept sialylated tri- and tetra-antennary glycans as substrates. This study provides a new platform for use to modulate and optimize the functions of glycoproteins, including the effector functions of IgG antibodies. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:11:16Z (GMT). No. of bitstreams: 1 ntu-107-D98642009-1.pdf: 59050343 bytes, checksum: 66c6dafeaa6db43d0f8aeb5ed8b70ae4 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 摘要…………………………………………………………………………………… i
ABSTRACT……………………………………………………………...…………… ii Significant………………………………………………………………..…………… iv Abbreviations…………………………………………………………….……...…… v Table of Contents………………………………………………………..…………… viii Chapter 1. Introduction 1.1 Immunoglobulin G antibodies.………………………………………...…..……… 1 1.2 Trastuzumab (Herceptin®).…………………………………………....……...…… 2 1.3 Fc glycosylation and interaction towards FcγRs……………………………..…… 3 1.4 Alternative mAb expression system: Pichia pastoris…………………………...… 4 1.5 Fc glycosylation engineering and pertinent glycosynthases…………………..….. 5 Chapter 2. Results and Discussion 2.1 Construction of plasmids in yeast strains………………….…………………….... 9 2.2 Expression, purification and characterization of recombinant trastuzumab expressed in P. pastoris………………….………………….………………..…… 15 2.3 Expression of a library of bacterial endoglycosudases and characterization of their de-glycosylation activity on glycoproteins……………... 21 2.4 ENGases catalyzed hydrolysis of glycans with C5-amine linker………………..... 30 2.5 EndoE and EndoP catalyzed hydrolysis of glycans with 2-aminobenzamide (2-AB) tag……………….………………….………………..………………….… 35 2.6 EndoE and EndoP catalyzed hydrolysis of glycans with peptide……………….… 43 2.7 De-glycosylation activity of EndoE for cleavage of high-mannose glycans on yeast-produced recombinant trastuzumab….………………..………………….… 45 2.8 Synthesis of homogeneous trastuzumab…….………………..…………………… 47 2.9 Transglycosylation test of selected ENGases mutants…………………………….. 53 2.10 Glycoengineered trastuzumab with strong FcγRIIIA binding affinity……..……. 56 Chapter 3. Conclusion…….………………….………………..………………….…. 58 Chapter 4. Discussion and Future Work………….………………..………………. 60 Chapter 5. Materials and Methods………….………………..………………….…. 62 5.1 Materials…….………………….………………..………………….….…………. 62 5.2 Construction of antibody expression vectors……..………………….….………… 62 5.3 Site-directed mutagenesis……..………………….….………………..…………... 63 5.4 Expression of enzymes……..………………….….………………..……………... 63 5.5 Transformation of antibody expression plasmids into P. pastoris…………….. 64 5.6 Transformation of antibody expression plasmids into GlycoSwitch® strains... 64 5.7 Fermentation conditions……..………………….….………………..………….. 65 5.8 Antibody purification……..………………….….………………..…………….... 65 5.9 SDS-PAGE detection of E. coli expressed ENGases and glycoengineered Herceptin antibodies……..………………….….………………..……………..... 65 5.10 Preparation of mono-GlcNAc-trastuzumab……..………………….….………. 66 5.11 Transglycosylation of mono-GlcNAc trastuzumab (rHer-G) with SGP.……….. 67 5.12 Western blot……..………………….….………………..……………………….. 67 5.13 Analysis of glycopeptides using LC-MS/MS…………..………………………... 68 5.14 FcγRIIIA analyte biotinylation…………..……………………….…………….... 69 5.15 Biolayer interferometry (BLI) analysis…..……………………….…………….... 69 5.16 Glycan microarray on NHS-activated glass slides……………….…………….... 70 5.17 Quantitative analysis of the substrate specificity by UPLC……….…………….. 71 References……..………………….….………………..……………………………… 72 Appendices……..………………….….………………..……………………………... 77 Appendix 1. SDS-PAGE of ENGases and their mutations expressed in E. coli BL21.. 78 Appendix 2. EndoE and EndoP catalyzed hydrolysis of A1 glycan with 2-AB tag evaluated by UPLC…….….………………..……………………………. 79 Appendix 3. EndoE and EndoP catalyzed hydrolysis of A1F glycan with 2-AB tag evaluated by UPLC…….….………………..……………………………. 80 Appendix 4. EndoE and EndoP catalyzed hydrolysis of A2 glycan with 2-AB tag evaluated by UPLC…….….………………..……………………………. 81 Appendix 5. EndoE and EndoP catalyzed hydrolysis of A2F glycan with 2-AB tag evaluated by UPLC…….….………………..……………………………. 82 Appendix 6. EndoE and EndoP catalyzed hydrolysis of NA2 glycan with 2-AB tag evaluated by UPLC…….….………………..……………………………. 83 Appendix 7. EndoE and EndoP catalyzed hydrolysis of NA2F glycan with 2-AB tag evaluated by UPLC…….….………………..……………………………. 84 Appendix 8. EndoE and EndoP catalyzed hydrolysis of NGA2 glycan with 2-AB tag evaluated by UPLC…….….………………..……………………………. 85 Appendix 9. EndoE and EndoP catalyzed hydrolysis of NGA2F glycan with 2-AB tag evaluated by UPLC…….….………………..………………………... 86 Appendix 10. EndoE and EndoP catalyzed hydrolysis of NA3 glycan with 2-AB tag evaluated by UPLC…….….………………..………………………... 87 Appendix 11. EndoE and EndoP catalyzed hydrolysis of NA3 glycan with 2-AB tag evaluated by UPLC…….….………………..………………………... 88 Appendix 12. EndoE and EndoP catalyzed hydrolysis of NGA3 (2,4,2) glycan with 2-AB tag evaluated by UPLC…….….………………..………………..... 89 Appendix 13. EndoE and EndoP catalyzed hydrolysis of NGA3F (2,4,2) glycan with 2-AB tag evaluated by UPLC…….….………………..………………..... 90 Appendix 14. EndoE and EndoP catalyzed hydrolysis of NGA3 (2,2,6) glycan with 2-AB tag evaluated by UPLC…….….………………..………………..... 91 Appendix 15. EndoE and EndoP catalyzed hydrolysis of NGA3F (2,4,2) glycan with 2-AB tag evaluated by UPLC…….….………………..………………..... 92 Appendix 16. EndoE and EndoP catalyzed hydrolysis of NGA4 (2,4,2,6) glycan with 2-AB tag evaluated by UPLC…….….……………………………... 93 Appendix 17. Cell growth rate of P. pastoris X33 and ΔOCH1 strains by OD600…... 94 | |
| dc.language.iso | en | |
| dc.subject | 均相化 | zh_TW |
| dc.subject | Fc醣體改造 | zh_TW |
| dc.subject | 畢赤酵母菌 | zh_TW |
| dc.subject | 內切醣?? | zh_TW |
| dc.subject | 賀癌平 | zh_TW |
| dc.subject | 醣修飾抗體 | zh_TW |
| dc.subject | Glycoengineered antibodies | en |
| dc.subject | Pichia | en |
| dc.subject | Trastuzumab | en |
| dc.subject | Endoglycosidase | en |
| dc.subject | Fc-glycosylation | en |
| dc.subject | Homogeneous | en |
| dc.title | 利用酵素法修飾酵母菌表現之重組蛋白上之醣分子以製備均相化抗體 | zh_TW |
| dc.title | Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 吳漢忠(Han-Chung Wu),吳宗益(Chung-Yi Wu),馬徹(Che Alex Ma),劉?睿(Je-Ruei Liu) | |
| dc.subject.keyword | 醣修飾抗體,畢赤酵母菌,賀癌平,內切醣??,Fc醣體改造,均相化, | zh_TW |
| dc.subject.keyword | Glycoengineered antibodies,Pichia,Trastuzumab,Endoglycosidase,Fc-glycosylation,Homogeneous, | en |
| dc.relation.page | 94 | |
| dc.identifier.doi | 10.6342/NTU201800085 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-01-18 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 生物科技研究所 | zh_TW |
| 顯示於系所單位： | 生物科技研究所 | |
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