Tang Feng, Shi Wei, 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, Pudong, Shanghai, China.
University of Chinese Academy of Sciences, Beijing, People's Republic of China.
Methods Mol Biol. 2019;2033:221-238. doi: 10.1007/978-1-4939-9654-4_15.
Conventional antibody-drug conjugates (ADCs) randomly assemble small-molecule drugs onto Lys or Cys residues of a tumor-targeting antibody, featured with heterogeneity in payload numbers and conjugation positions. Glycosite-specific ADCs (gsADCs) link payload drugs onto IgG Fc N-glycans with high homogeneity that facilitates structural optimization and quality control for ADC drug development. In this protocol, we report two strategies for preparation of homogeneous ADCs via chemoenzymatic glycoengineering. First, an azido-tagged unnatural N-glycan substrate is transferred onto Fc glycosites of a therapeutic antibody through Endo-S-catalyzed glycoremodeling, followed by click reaction with an alkyne-tagged payload drug to give a well-defined gsADC. In an alternative way, glycoengineering of antibody with a natural sialylated N-glycan and successive selective oxidation of sialic acid moieties using sodium periodate provided an aldehyde handle on the glycans for conjugation with an aminooxy-assembled payload. These two strategies both enable gsADCs with high homogeneity in their conjugation sites, payload numbers, and glycoforms, which are characterized of a single mass under mass-spectral detection.
传统的抗体药物偶联物（ADC）将小分子药物随机组装到肿瘤靶向抗体的赖氨酸或半胱氨酸残基上，其特点是载药量和偶联位置存在异质性。糖基位点特异性ADC（gsADC）将载药以高度均一性连接到IgG Fc N-聚糖上，这有利于ADC药物开发的结构优化和质量控制。在本方案中，我们报告了两种通过化学酶促糖基工程制备均一ADC的策略。首先，通过内切糖苷酶S催化的糖基重塑，将叠氮标记的非天然N-聚糖底物转移到治疗性抗体的Fc糖基位点上，然后与炔烃标记的载药进行点击反应，得到结构明确的gsADC。另一种方法是，用天然唾液酸化N-聚糖对抗体进行糖基工程改造，并使用高碘酸钠对唾液酸部分进行连续选择性氧化，在聚糖上提供一个醛基用于与氨氧基组装的载药偶联。这两种策略都能使gsADC在偶联位点、载药量和糖型方面具有高度均一性，在质谱检测下表现为单一质量特征。
