The gsADC concept was originally developed using glycosyltransferase to introduce azido-tagged monosaccharides on external residues of IgG N-glycans, followed by small-molecule drug conjugation 30– 32. Recently, we reported a one-pot chemoenzymatic method using azido-tagged N-glycopeptide precursors, and successive ‘click’ conjugation with alkyne-tagged small molecules for the synthesis of gsADCs 26. pyogenes serotype M49, Endo-S2, and mutants thereof exhibit even more relaxed substrate specificity and are capable of acting on high-mannose, hybrid, and complex-type Fc N-glycans 25, 29. In addition, another endo-β- N-acetylglucosaminidase from S. These unique features of Endo-S make it a perfect enzyme for in vitro IgG glycoengineering that is specific for the Fc domain and compatible with a diverse range of glycan substrates 22. The glycan substrate specificity of Endo-S covers a wide range of natural and modified N-glycan structures. Endo-β- N-acetylglucosaminidase from Streptococcus pyogenes (Endo-S), as one of these endoglycosidases, specifically recognizes the IgG Fc domain and has been used for efficient glyco-remodeling of therapeutic antibodies 21– 26. Next, endoglycosidase mutants 28, also called glycosynthases-which lack hydrolytic activity but possess trans-glycosylation activity through the use of a homogeneous synthetic N-glycan oxazoline as the activated substrate-are used to assemble this intact N-glycan substrate on the GlcNAc-bearing glycoprotein generated by the aforementioned deglycosylation by the WT enzyme to accomplish the glycoengineering. This chemoenzymatic method exploits the hydrolytic activity of wild-type (WT) endoglycosidases to cleave heterogeneous N-glycans and leave the innermost GlcNAc motifs on the glycoproteins. In addition to the approaches to in vivo N-glycoengineering through glycosyltransferase knockout/knock-in expression systems 2, 15, 16 and in vitro treatment of antibodies with glycosyltransferases 17, 18, endoglycosidase-catalyzed N-glycosylation remodeling has become a powerful tool for the synthesis of homogeneous N-glycoproteins 19– 27. Various glycoengineering technologies have emerged, aiming at enhancing the efficacy of therapeutic antibodies through N-glycosylation 10. These interactions are regulated by N-glycosylation of the IgG Fc domains 11– 14. The interaction between the Fc domain of IgG antibodies and Fc receptors has an important role in antibody-dependent cell-mediated cytotoxicity (ADCC) 1– 3, complement-dependent cytotoxicity (CDC) 4, anti-inflammatory activity 5– 7, pharmacokinetic half-life 8, 9, and immunogenicity 10. 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. 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. 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. 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. In this protocol, native heterogeneously glycosylated IgG N-glycans are first deglycosylated with a wild-type endoglycosidase. Endoglycosidase-catalyzed in vitro glycoengineering technology is a powerful tool for IgG Fc (fragment cystallizable) N-glycosylation remodeling. Glycoengineered therapeutic antibodies and glycosite-specific antibody–drug conjugates (gsADCs) have generated great interest among researchers because of their therapeutic potential.
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