Shi Wei, Li Wanzhen, Zhang Jianxin, Li Tiehai, Song Yakai, Zeng Yue, Dong Qian, Lin Zeng, Gong Likun, Fan Shuquan, Tang Feng, Huang Wei
CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Center for Biotherapeutics Discovery Research, Shanghai Institute of Materia Medica Chinese Academy of Sciences, Shanghai 201203, China.
School of Pharmaceutical Science and Technology, Hangzhou Institute of Advanced Study, Hangzhou 310024, China.
Acta Pharm Sin B. 2022 May;12(5):2417-2428. doi: 10.1016/j.apsb.2021.12.013. Epub 2021 Dec 24.
Glycosite-specific antibody‒drug conjugatess (gsADCs), harnessing Asn297 -glycan of IgG Fc as the conjugation site for drug payloads, usually require multi-step glycoengineering with two or more enzymes, which limits the substrate diversification and complicates the preparation process. Herein, we report a series of novel disaccharide-based substrates, which reprogram the IgG glycoengineering to one-step synthesis of gsADCs, catalyzed by an --acetylglucosaminidase (ENGase) of Endo-S2. IgG glycoengineering ENGases usually has two steps: deglycosylation by wild-type (WT) ENGases and transglycosylation by mutated ENGases. But in the current method, we have found that disaccharide LacNAc oxazoline can be efficiently assembled onto IgG by WT Endo-S2 without hydrolysis of the product, which enables the one-step glycoengineering directly from native antibodies. Further studies on substrate specificity revealed that this approach has excellent tolerance on various modification of 6-Gal motif of LacNAc. Within 1 h, one-step synthesis of gsADC was achieved using the LacNAc-toxin substrates including structures free of bioorthogonal groups. These gsADCs demonstrated good homogeneity, buffer stability, and anti-tumor activity. This work presents a novel strategy using LacNAc-based substrates to reprogram the multi-step IgG glycoengineering to a one-step manner for highly efficient synthesis of gsADCs.
糖基位点特异性抗体-药物偶联物（gsADCs）利用IgG Fc的Asn297聚糖作为药物有效载荷的偶联位点，通常需要用两种或更多种酶进行多步糖工程改造，这限制了底物的多样性并使制备过程复杂化。在此，我们报道了一系列基于二糖的新型底物，它们将IgG糖工程改造重新编程为一步合成gsADCs，由Endo-S2的N-乙酰葡糖胺酶（ENGase）催化。IgG糖工程改造的ENGases通常有两个步骤：由野生型（WT）ENGases进行去糖基化和由突变的ENGases进行转糖基化。但在目前的方法中，我们发现二糖乳糖-N-乙酰葡糖胺恶唑啉可以由WT Endo-S2有效地组装到IgG上而不水解产物，这使得能够直接从天然抗体进行一步糖工程改造。对底物特异性的进一步研究表明，这种方法对LacNAc的6-Gal基序的各种修饰具有极好的耐受性。在1小时内，使用包括不含生物正交基团结构的LacNAc-毒素底物实现了gsADC的一步合成。这些gsADCs表现出良好的均一性、缓冲液稳定性和抗肿瘤活性。这项工作提出了一种使用基于LacNAc的底物将多步IgG糖工程改造重新编程为一步方式以高效合成gsADCs的新策略。
