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標題: | 免疫球蛋白G的Fc醣體改造與其相關活性研究 Engineering of IgG Fc Glycosylation and the Relevant Activity Studies |
作者: | Chin-Wei Lin 林津瑋 |
指導教授: | 吳宗益(Chung-Yi Wu) |
關鍵字: | 抗體醣化修飾,醣均質化,抗體依賴細胞介導的細胞毒殺作用,唾液酸醣化,噁唑咻醣, Fc glycosylation,glycoengineered antibodies,homogeneous antibodies,endoglycosidase,sugar oxazoline,antibody-dependent cell-mediated cytotoxicity, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | Antibodies are crucial glycoproteins that consist of two antigen binding fragments (Fab) and one crystalizable fragment (Fc). At the Fc region, there are asparagine-linked glycosylations which function to mediate and modulate its recruitments or interactions with various effector cells or receptors. Through such modulations, one antibody can display different strength in functions, including cancer killing, pathogen defending, and anti-inflammation, etc. Pharmaceutically, many FDA-approved immunoglobulin G monoclonal antibodies have been successfully administrated to patients with leukemia, solid tumors, or autoimmune diseases. However, these antibodies are all heterogeneous in glycosylation and may contain few or none of the optimal glycan form for best efficacy of treatment. Therefore, high dosage of antibody is often indispensable and it follows high cost and high frequency of injection. In addition, the inaccessibility of pure antibody with homogeneous glycosylation further troubles researchers in defining and comparing the impact of each specific Fc glycan on activity, half life and side effects.
To overcome the aforementioned problems, various well-defined homogeneous glycosylated antibodies were prepared using one-pot enzymatic reaction to remove the whole bunch of glycans, followed by re-attaching the defined glycans back to antibodies for activity assessment. Significantly, it was found that antibodies with the 2,6-sialylated Fc glycosylation, a known potent composition of intravenous immunoglobulin for exhibiting anti-inflammatory activity, display superior binding affinity towards FcγRIIIa/FcγRI, remarkable antibody-dependent cell-mediated cytotoxicity(ADCC)against both breast cancer cells, and good anti-H1N1 influenza activity, implying multifaceted functions derived from single Fc glycan form of 2,6-sialylation. Furthermore, both the pairing and the different modifications of Fc glycans, including the core-fucose, the branch, the length, and the sialylation linkage were investigated to supply a clear picture about the interactions with the FcγRIIIa/FcγRI and the corresponding effectors functions, including the ADCC and oxidative burst. The results indicate that the 1,6’-branch is more potent to increase the affinity towards FcγRIIIa/FcγRI than that of the 1,3’-branch and such interactions intensify as the length of Fc glycans increase. Meanwhile, it was also illustrated that the 2,3-sialylation disrupts the aforementioned interaction and shows elevated dissociation rate constants whereas the 2,6-sialylation maintains the affinity compared to the non-sialylated counterpart. Among the various antibodies with different Fc glycan modifications, the adverse effect of core-fucosylation on the affinity exceeds the others in the case of anti-FcγRIIIa but lessens for FcγRI. Moreover, not only the affinity but also the threshold for activating FcγRIIIa was found to be mediated by various Fc glycans. Interestingly, the Fab/Fc interdomain interaction and the minor adjustments of Fab binding by the Fc glycans were observed as well. The affinity and threshold for FcγRs activation might relate to the the efficiency of antigen uptake whereas the fine-tuning of the Fab recognition could lead to the distinct footprint for presenting to T cells after the proteolytic processing. Based on the results in this study and previous reports, an alternative affinity maturation mediated by the Fc glycosylation was implied. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59763 |
DOI: | 10.6342/NTU201700515 |
全文授權: | 有償授權 |
顯示於系所單位: | 化學系 |
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