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標題: | 醣基化修飾抗體與抗體受器複合體之結構研究 Structural studies of glyco-engineered IgG/Fc receptor complex |
作者: | Chia-Lin Chen 陳加林 |
指導教授: | 馬徹(Che Ma) |
共同指導教授: | 林俊宏 |
關鍵字: | 醣基化修飾,抗體Fc結構,免疫複合體,Fc受器,抗體依賴性細胞媒介毒殺作用, glyco-engineered antibody,Fc structure,immune complex,Fc receptor,antibody dependent cellular cytotoxicity, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | N-醣基化會影響抗體免疫球蛋白G中,Fc部位的結構和效用功能。若免疫球蛋白G經由人為的酵素優化後,使其帶有人類唾液酸複合型醣 (hSCT) 修飾,會增強此種抗體對於Fc第三A型受器的親和力並使修飾後的抗體具有更強的抗體依賴性細胞媒介毒殺作用。hSCT在結構上具有雙延伸支鏈,兩條支鏈的尾端都是alpha-2,6連結的唾液酸,且沒有岩藻醣基化。hSCT修飾後的抗體不僅能在抗原結合的狀況下增強對Fc受器的親和力,更能使某些本來不具抗體依賴性細胞媒介毒殺作用的抗體獲得此新功能。我們利用酵素優化大量的免疫球蛋白G抗體使其帶有hSCT修飾,並利用x光繞射結晶學方法解析其Fc部位的立體解構到1.85 Å的解析度。在此結構中我們發現同時存在一個閉鎖型和一個開放型的構形:在閉鎖型中, hSCT醣鏈利用醣分子間的作用形成兩處結合點,以穩定此閉鎖型的結構;在開放型中,hSCT醣鏈尾端的唾液酸會利用水分子間接結合免疫球蛋白上的D249-L251位置,並藉此調控抗體CH2和CH3部位的相對位置。這是第一個Fc部位帶有能增進抗體依賴性細胞媒介毒殺作用活性的醣基化修飾其立體結構被解析出來。此研究不僅解釋了免疫球蛋白G中Fc部位被hSCT修飾後,蛋白質結構彈性和效用功能間的關係,並為後續臨床抗體修飾工程提供重要的資訊。 N-glycosylation on IgG modulates Fc conformation and effector functions. An IgG contains a human sialo-complex type (hSCT) glycan of biantennary structure with two alpha-2,6-sialylations and without core-fucosylation is an optimized glycoform developed to targeting FcRIIIA and enhance the antibody dependent cellular cytotoxicity (ADCC). hSCT modification not only enhances the binding affinity to Fc receptors in the presence of antigen, but also in some cases provides gain-of-function effector activity. Binding kinetics analysis indicated the increased affinity to both alleles of FcRIIIA is mainly due to a 10-fold decrease of off rate. In addition, ADCC activity assay suggested IgGhSCT provides better killing effect no matter the targeted antigens are viral or tumor-related molecules. IgG-Fc with homogeneous hSCT attached to each CH2 domain was prepared by enzymatic glyco-engineering, and its crystal structure was solved in 1.85 Å resolution. A compact form and an open form were observed in the crystal. In the compact structure, the double glycan latches from the two hSCT chains stabilize the CH2 domains in a closed conformation. In the open structure, the terminal sialic acid residue interacts through water-mediated hydrogen bonds with the D249-L251 helix, to modulate the pivot region of CH2-CH3 interface. This is the first crystal structure of glyco-engineered Fc with enhanced effector activities. A structure-based engineering of IgG together with the hSCT modification were designed to provide even better ADCC and immune protecting effect. In addition, the structural studies of intact immune complex which includes IgGhSCT/antigen/FcRIIIA was conducted by x-ray crystallography and cryo-EM method. This work provides insights into the relationship between the structural stability and effector functions affected by hSCT modification and the development of better antibodies for therapeutic applications. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59597 |
DOI: | 10.6342/NTU201700712 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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