title: One-pot N-glycosylation remodeling of IgG with non-natural sialylglycopeptides enables glycosite-specific and dual-payload antibody-drug conjugates.
abstract: Chemoenzymatic transglycosylation catalyzed by endo-S mutants is a powerful tool for in vitro glycoengineering of therapeutic antibodies. In this paper, we report a one-pot chemoenzymatic synthesis of glycoengineered Herceptin using an egg-yolk sialylglycopeptide (SGP) substrate. Combining this one-pot strategy with novel non-natural SGP derivatives carrying azido or alkyne tags, glycosite-specific conjugation was enabled for the development of new antibody-drug conjugates (ADCs). The site-specific ADCs and semi-site-specific dual-drug ADCs were successfully achieved and characterized with SDS-PAGE, intact antibody or ADC mass spectrometry analysis, and PNGase-F digestion analysis. Cancer cell cytotoxicity assay revealed that small-molecule drug release of these ADCs relied on the cleavable Val-Cit linker fragment embedded in the structure. These results represent a new approach for glycosite-specific and dual-drug ADC design and rapid synthesis, and also provide the structural requirement for their biologic activities.
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn and University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Tang
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn and iHuman Institute, ShanghaiTech University, 99 Haike Road, Pudong, Shanghai, 201210 China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn and Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China.
authors.forename: Yubo
authors.initials: Y
authors.lastname: Tang
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn.
authors.forename: Shuai
authors.initials: S
authors.lastname: Tang
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn and University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China.
authors.forename: Liyun
authors.initials: L
authors.lastname: Yang
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn and iHuman Institute, ShanghaiTech University, 99 Haike Road, Pudong, Shanghai, 201210 China.
authors.forename: Bingyang
authors.initials: B
authors.lastname: Sun
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn and iHuman Institute, ShanghaiTech University, 99 Haike Road, Pudong, Shanghai, 201210 China.
authors.forename: Bofeng
authors.initials: B
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Dong
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn.
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Huang
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn.
authors.forename: Mei-Yu
authors.initials: MY
authors.lastname: Geng
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China 201203. huangwei@simm.ac.cn and iHuman Institute, ShanghaiTech University, 99 Haike Road, Pudong, Shanghai, 201210 China.
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
mesh_terms: D045744:Cell Line, Tumor; D006020:Glycopeptides / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009584:Nitrogen / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D006020:Glycopeptides; D018796:Immunoconjugates; D007074:Immunoglobulin G; D009584:Nitrogen