Tang Feng, Wang Lai-Xi, Huang Wei
CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
University of Chinese Academy of Sciences, Beijing, China.
Nat Protoc. 2017 Aug;12(8):1702-1721. doi: 10.1038/nprot.2017.058. Epub 2017 Jul 27.
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.
糖基工程改造的治疗性抗体和糖基位点特异性抗体-药物偶联物（gsADC）因其治疗潜力而引起了研究人员的极大兴趣。内切糖苷酶催化的体外糖基工程技术是用于IgG Fc（可结晶片段）N-糖基化重塑的有力工具。在本方案中，首先用野生型内切糖苷酶将天然异质性糖基化的IgG N-聚糖去糖基化。接下来，使用一种缺乏水解活性但具有用于糖基工程的转糖基化活性的突变型内切糖苷酶（也称为内切糖基合酶），将如本文所述预先合成的均匀N-聚糖底物连接到IgG剩余的N-乙酰葡糖胺（GlcNAc）上。与体内糖基工程技术和糖基转移酶介导的体外工程方法相比，当前方法稳健，具有定量产率、所产生抗体和ADC的糖型均匀、与多种天然和非天然聚糖结构兼容、便于以天然IgG为起始材料进行开发以及抗体修饰的共轭位点明确等特点。该方法的潜在应用涵盖了广泛的抗体相关研究，包括开发具有增强疗效的新型糖基工程改造治疗性抗体、位点特异性抗体-药物偶联以及用于荧光标记、聚乙二醇化、蛋白质交联、免疫脂质体形成等的抗体位点特异性修饰，且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天，改造IgG N-糖基化需要3-4天，合成小分子毒素并制备gsADC需要2-5天。
