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

Nature Protocols volume 12, pages 1702–1721 (2017)Cite this article

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Abstract

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.

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Figure 1: General scheme of chemoenzymatic synthesis of glycoengineered IgGs and gsADCs.
Figure 2
Figure 3
Figure 4: Semisynthesis of N-glycan oxazolines (Steps 1–44).
Figure 5
Figure 6: Synthesis of dual-payload ADC (28) (Steps 96–108).
Figure 7: LC–MS profiles (deconvolution data) from IgG deglycosylation monitoring.
Figure 8: SDS-PAGE and LC–MS characterization of glycoengineered Herceptin (5a–e) bearing non-natural N-glycans26.
Figure 9: SDS-PAGE and LC–MS characterization of gsADC (7) (ref. 26).

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (NNSFC, nos. 21372238 and 21572244 to W.H.), the SIMM Institute Fund (CASIMM0120153004 to W.H.), and the 'Personalized Medicines: Molecular Signature-based Drug Discovery and Development' Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDA12020311 to W.H.).

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Authors and Affiliations

  1. 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

    Feng Tang & Wei Huang

  2. University of Chinese Academy of Sciences, Beijing, China

    Feng Tang & Wei Huang

  3. Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, USA

    Lai-Xi Wang

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Contributions

F.T. and W.H. designed the research and developed the methods; F.T. performed the experiments; F.T. and W.H. wrote the manuscript; and L.-X.W. developed the original IgG glycoengineering approach and revised the manuscript.

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Correspondence to Wei Huang.

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Tang, F., Wang, LX. & Huang, W. Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody–drug conjugates. Nat Protoc 12, 1702–1721 (2017). https://doi.org/10.1038/nprot.2017.058

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