Dataset Information

Chemoenzymatic Defucosylation of Therapeutic Antibodies for Enhanced Effector Functions Using Bacterial ?-Fucosidases.

ABSTRACT: Core fucosylation plays a critical role in modulating the effector functions of therapeutic antibodies such as the antibody-dependent cellular cytotoxicity (ADCC) through adversely affecting the affinity of antibodies for Fc? receptors. Thus, a facile method for Fc defucosylation of antibodies is important both for functional studies and for an enhanced therapeutic efficacy. In this chapter, we describe a detailed protocol for chemoenzymatic defucosylation of antibodies using Herceptin (trastuzumab) as a model system. The protocol includes (a) Fc deglycosylation using endoglycosidase S2 (Endo-S2); (b) enzymatic defucosylation of the resulting Fuc?1,6GlcNAc-Herceptin using two distinct bacterial ?-fucosidases, AlfC and BfFuc; (c) transglycosylation of the GlcNAc-Herceptin using an Endo-S2 mutant (Endo-S2 D184M) as the enzyme and a complex N-glycan oxazoline as the donor substrate; and (d) SPR analysis of the binding of antibody glycoforms with the Fc?IIIA receptor. The protocol of enzymatic defucosylation of Herceptin should be equally applicable for the Fc glycan engineering of other mAbs.

SUBMITTER: Li C

PROVIDER: S-EPMC6238083 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Chemoenzymatic Defucosylation of Therapeutic Antibodies for Enhanced Effector Functions Using Bacterial α-Fucosidases.

Li Chao C Li Tiezheng T Wang Lai-Xi LX

Methods in molecular biology (Clifton, N.J.) 20180101

Core fucosylation plays a critical role in modulating the effector functions of therapeutic antibodies such as the antibody-dependent cellular cytotoxicity (ADCC) through adversely affecting the affinity of antibodies for Fcγ receptors. Thus, a facile method for Fc defucosylation of antibodies is important both for functional studies and for an enhanced therapeutic efficacy. In this chapter, we describe a detailed protocol for chemoenzymatic defucosylation of antibodies using Herceptin (trastuzu ...[more]

PMID: 30196507

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Project description:The ability to profile the prevalence and functional activity of endogenous antibodies is of vast clinical and diagnostic importance. Serum antibodies are an important class of biomarkers and are also crucial elements of immune responses elicited by natural disease-causing agents as well as vaccines. In particular, materials for manipulating and/or enhancing immune responses toward disease-causing cells or viruses have exhibited significant promise for therapeutic applications. Antibody-recruiting molecules (ARMs), bifunctional organic molecules that redirect endogenous antibodies to pathological targets, thereby increasing their recognition and clearance by the immune system, have proven particularly interesting. Notably, although ARMs capable of hijacking antibodies against oligosaccharides and electron-poor aromatics have proven efficacious, systematic comparisons of the prevalence and effectiveness of natural anti-hapten antibody populations have not appeared in the literature. Herein we report head-to-head comparisons of three chemically simple antigens, which are known ligands for endogenous antibodies. Thus, we have chemically synthesized bifunctional molecules containing 2,4-dinitrophenyl (DNP), phosphorylcholine (PC), and rhamnose. We have then used a combination of ELISA, flow cytometry, and cell-viability assays to compare these antigens in terms of their abilities both to recruit natural antibody from human serum and also to direct serum-dependent cytotoxicity against target cells. These studies have revealed rhamnose to be the most efficacious of the synthetic antigens examined. Furthermore, analysis of 122 individual serum samples has afforded comprehensive insights into population-wide prevalence and isotype distributions of distinct anti-hapten antibody populations. In addition to providing a general platform for comparing and studying anti-hapten antibodies, these studies serve as a useful starting point for the optimization of antibody-recruiting molecules and other synthetic strategies for modulating human immunity.

Dataset Information

Chemoenzymatic Defucosylation of Therapeutic Antibodies for Enhanced Effector Functions Using Bacterial ?-Fucosidases.

Publications

Chemoenzymatic Defucosylation of Therapeutic Antibodies for Enhanced Effector Functions Using Bacterial α-Fucosidases.

Similar Datasets

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