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Nat Protoc:中科院上海药物所黄蔚课题组发展定点抗体药物偶联物新策略及抗体糖工程技术方案

摘要 : 2017年7月27日,国际学术权威刊物自然出版集团旗下子刊《Nature Protocols》在线发表了中国科学院上海药物研究所黄蔚课题组题为Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody-drug conjugates 的文章

2017年7月27日,国际学术权威刊物自然出版集团旗下子刊《Nature Protocols》在线发表了中国科学院上海药物研究所黄蔚课题组题为Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody-drug conjugates 的文章,研究论文报道研究组利用化学-酶催化方法制备糖工程抗体与基于糖链的定点抗体药物偶联物新策略。黄蔚课题组研究生唐峰为文章的第一作者,黄蔚为通讯作者。

抗体类生物药物在治疗肿瘤、自身免疫等疾病方面占据重要地位,其中抗体Fc区域N-糖链通过参与免疫细胞表面Fc受体识别,在抗体的免疫调节机制如抗体依赖细胞毒作用(ADCC)和补体依赖细胞毒作用(CDC)等方面发挥重要作用。黄蔚课题组在前期发展的抗体糖工程改造技术基础上(Org. Biomol. Chem. 2016, 9501; J. Am. Chem. Soc. 2012, 12308),利用一类糖苷酶及其突变酶的转糖基化活性,“一锅法”实现抗体的糖基化单一结构控制,优化抗体药物疗效。同时在抗体糖工程化基础上,在糖链底物上引入标记基团,实现小分子毒素药物的定点偶联,为新型抗体药物偶联物的设计提供了新的定点策略。

抗体药物偶联物(ADC)通过将小分子毒素共价连接到抗体上,利用抗体对肿瘤表面抗原的高特异性识别,靶向输送小分子药物。传统的ADC采用随机偶联方法将小分子偶联至抗体赖氨酸或半胱氨酸,在偶联小分子数量和位点等方面存在不均一性,为ADC药物的质量控制、PK/PD稳定性等带来困难。采用糖工程技术的糖链定点ADC药物,克服了随机偶联的不均一性,不仅在小分子偶联位点和数量上高度可控,而且将抗体多组分糖链结构转化为单一组分,实现了ADC分子的高度均一性,为ADC药物设计和成药性优化提供了结构优势。

同时,该方法在非治疗性抗体试剂的定点偶联和标记方面也具有广泛的应用价值,利用Fc糖链定点偶联技术可以避免标记修饰对抗体Fab区域(抗原识别区)的影响,克服随机偶联对抗体抗原识别造成的实验差异。黄蔚课题组长期从事蛋白糖基化调控、细胞表面糖链编辑、糖结构药物等方向的研究。

定点抗体药物偶联物新策略及抗体糖工程技术方案

原文链接:

Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody–drug conjugates

原文摘要:

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 remainingN-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.

来源: Nature Protocols 浏览次数:0

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