Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody-drug conjugates.
Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody-drug conjugates.
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糖工程化 IgG 抗体和糖位点特异性抗体-药物缀合物的化学酶合成
DOI：
10.1038/nprot.2017.058
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发表时间：
2017-08
期刊：
影响因子：
14.8
通讯作者：
Huang W
中科院分区：
文献类型：
--
作者：
Tang F;Wang LX;Huang W
糖工程化治疗性抗体和糖位点特异性抗体-药物缀合物（gsADC）由于其治疗潜力而引起了研究人员的极大兴趣。内切糖基化酶催化的体外糖工程技术是IgG Fc（片段cystallizable）N-糖基化重塑的有力工具。在该方案中，首先用野生型内切糖苷酶将天然非均匀糖基化IgG N-聚糖去糖基化。接下来，使用缺乏水解活性但具有糖工程化的转糖基化活性的突变体内切糖苷酶（也称为内切糖合酶），将如本文所述预合成的均质N-聚糖底物连接至IgG的剩余N-乙酰葡糖胺（GlcNAc）。与体内糖基工程技术和糖基转移酶激活的体外工程方法相比，目前的方法是稳健的，并且具有定量产率、产生的抗体和ADC的均一糖型、与多种天然和非天然聚糖结构的相容性、方便地利用天然IgG作为起始材料以及用于抗体修饰的明确的缀合位点的特征。该方法的潜在应用涵盖了广泛的抗体相关研究，包括开发具有增强功效的新型糖工程化治疗性抗体、位点特异性抗体-药物缀合以及用于荧光标记、PEG化、蛋白质交联、免疫脂质体形成等的抗体的位点特异性修饰，而不损失抗原结合亲和力。制备天然或修饰的N-聚糖底物需要5-8 d，工程化IgG N-糖基化需要3-4 d，合成小分子毒素和制备gsADC需要2-5 d。
Glycoengineered therapeutic antibodies and glycosite-specific antibody–drug conjugates (gsADCs) have generated great interest among researchers because of their therapeutic potential. Endoglycosidase-catalyzed in vitro glycoengineering technology is a powerful tool for IgG Fc (fragment cystallizable) N-glycosylation remodeling. In this protocol, native heterogeneously glycosylated IgG N-glycans are first deglycosylated with a wild-type endoglycosidase. Next, a homogeneous N-glycan substrate, presynthesized as described here, is attached to the remaining N-acetylglucosamine (GlcNAc) of IgG, using a mutant endoglycosidase (also called endoglycosynthase) that lacks hydrolytic activity but possesses transglycosylation activity for glycoengineering. Compared with in vivo glycoengineering technologies and the glycosyltransferase-enabled in vitro engineering method, the current approach is robust and features quantitative yield, homogeneous glycoforms of produced antibodies and ADCs, compatibility with diverse natural and non-natural glycan structures, convenient exploitation of native IgG as the starting material, and a well-defined conjugation site for antibody modifications. Potential applications of this method cover a broad scope of antibody-related research, including the development of novel glycoengineered therapeutic antibodies with enhanced efficacy, site-specific antibody–drug conjugation, and site-specific modification of antibodies for fluorescent labeling, PEGylation, protein cross-linking, immunoliposome formation, and so on, without loss of antigen-binding affinity. It takes 5–8 d to prepare the natural or modified N-glycan substrates, 3–4 d to engineer the IgG N-glycosylation, and 2–5 d to synthesize the small-molecule toxins and prepare the gsADCs.
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影响因子：
46.9
作者：
Li, HJ;Sethuraman, N;Gerngross, TU
通讯作者：
Gerngross, TU
影响因子：
16.6
作者：
Ning, Xinghai;Guo, Jun;Boons, Geert-Jan
通讯作者：
Boons, Geert-Jan
DOI：
10.1073/pnas.0810163105
发表时间：
2008-12-16
影响因子：
11.1
作者：
Anthony, Robert M.;Wermeling, Fredrik;Ravetch, Jeffrey V.
通讯作者：
Ravetch, Jeffrey V.
影响因子：
3.8
作者：
Gramer, Michael J.;Eckblad, Jackie J.;van Berkel, Patrick H. C.
通讯作者：
van Berkel, Patrick H. C.
影响因子：
4.3
作者：
Goetze, Andrew M.;Liu, Y. Diana;Flynn, Gregory C.
通讯作者：
Flynn, Gregory C.