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 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 (trastuzumab) as a model system. The protocol includes (a) Fc deglycosylation using endoglycosidase S2 (Endo-S2); (b) enzymatic defucosylation of the resulting Fucα1,6GlcNAc-Herceptin using two distinct bacterial α-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 FcγIIIA receptor. The protocol of enzymatic defucosylation of Herceptin should be equally applicable for the Fc glycan engineering of other mAbs.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ecker DM, Jones SD, Levine HL (2015) The therapeutic monoclonal antibody market. MAbs 7(1):9–14. https://doi.org/10.4161/19420862.2015.989042
Adams GP, Weiner LM (2005) Monoclonal antibody therapy of cancer. Nat Biotechnol 23(9):1147–1157
Jefferis R (2005) Glycosylation of recombinant antibody therapeutics. Biotechnol Prog 21(1):11–16. https://doi.org/10.1021/bp040016j
Shields RL, Lai J, Keck R, O'Connell LY, Hong K, Meng YG, Weikert SH, Presta LG (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem 277(30):26733–26740. https://doi.org/10.1074/jbc.M202069200
Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M, Yamasaki M, Hanai N, Shitara K (2003) The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 278(5):3466–3473. https://doi.org/10.1074/jbc.M210665200
Nimmerjahn F, Ravetch JV (2005) Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science 310(5753):1510–1512. https://doi.org/10.1126/science.1118948
Beck A, Reichert JM (2012) Marketing approval of mogamulizumab. MAbs 4(4):419–425. https://doi.org/10.4161/mabs.20996
Gagez AL, Cartron G (2014) Obinutuzumab: a new class of anti-CD20 monoclonal antibody. Curr Opin Oncol 26(5):484–491. https://doi.org/10.1097/CCO.0000000000000107
Huang W, Giddens J, Fan SQ, Toonstra C, Wang LX (2012) Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions. J Am Chem Soc 134(29):12308–12318. https://doi.org/10.1021/ja3051266
Li T, Tong X, Yang Q, Giddens JP, Wang LX (2016) Glycosynthase mutants of endoglycosidase S2 show potent transglycosylation activity and remarkably relaxed substrate specificity for antibody glycosylation remodeling. J Biol Chem 291(32):16508–16518. https://doi.org/10.1074/jbc.M116.738765
Wang LX, Amin MN (2014) Chemical and chemoenzymatic synthesis of glycoproteins for deciphering functions. Chem Biol 21(1):51–66. https://doi.org/10.1016/j.chembiol.2014.01.001
Tsai TI, Li ST, Liu CP, Chen KY, Shivatare SS, Lin CW, Liao SF, Lin CW, Hsu TL, Wu YT, Tsai MH, Lai MY, Lin NH, Wu CY, Wong CH (2017) An effective bacterial fucosidase for glycoprotein remodeling. ACS Chem Biol 12(1):63–72. https://doi.org/10.1021/acschembio.6b00821
Lin CW, Tsai MH, Li ST, Tsai TI, Chu KC, Liu YC, Lai MY, Wu CY, Tseng YC, Shivatare SS, Wang CH, Chao P, Wang SY, Shih HW, Zeng YF, You TH, Liao JY, Tu YC, Lin YS, Chuang HY, Chen CL, Tsai CS, Huang CC, Lin NH, Ma C, Wong CH (2015) A common glycan structure on immunoglobulin G for enhancement of effector functions. Proc Natl Acad Sci U S A 112(34):10611–10616. https://doi.org/10.1073/pnas.1513456112
Giddens JP, Wang LX (2015) Chemoenzymatic Glyco-engineering of monoclonal antibodies. Methods Mol Biol 1321:375–387. https://doi.org/10.1007/978-1-4939-2760-9_25
Yang Q, Wang LX (2017) Chemoenzymatic glycan remodeling of natural and recombinant glycoproteins. Methods Enzymol 597:265–281. https://doi.org/10.1016/bs.mie.2017.06.006
Becerra JE, Coll-Marques JM, Rodriguez-Diaz J, Monedero V, Yebra MJ (2015) Preparative scale purification of fucosyl-N-acetylglucosamine disaccharides and their evaluation as potential prebiotics and antiadhesins. Appl Microbiol Biotechnol 99(17):7165–7176. https://doi.org/10.1007/s00253-015-6666-2
Rodriguez-Diaz J, Carbajo RJ, Pineda-Lucena A, Monedero V, Yebra MJ (2013) Synthesis of fucosyl-N-acetylglucosamine disaccharides by transfucosylation using alpha-L-fucosidases from lactobacillus casei. Appl Environ Microbiol 79(12):3847–3850. https://doi.org/10.1128/AEM.00229-13
Rodriguez-Diaz J, Monedero V, Yebra MJ (2011) Utilization of natural fucosylated oligosaccharides by three novel alpha-L-fucosidases from a probiotic Lactobacillus casei strain. Appl Environ Microbiol 77(2):703–705. https://doi.org/10.1128/AEM.01906-10
Li C, Zhu S, Ma C, Wang LX (2017) Designer alpha1,6-fucosidase mutants enable direct core fucosylation of intact N-glycopeptides and N-glycoproteins. J Am Chem Soc 139(42):15074–15087. https://doi.org/10.1021/jacs.7b07906
Huang W, Li J, Wang LX (2011) Unusual transglycosylation activity of Flavobacterium meningosepticum Endoglycosidases enables convergent chemoenzymatic synthesis of core fucosylated complex N-glycopeptides. Chembiochem 12(6):932–941. https://doi.org/10.1002/cbic.201000763
Champion T, Beck A (2013) Capture of the human IgG1 antibodies by protein A for the kinetic study of h-IgG/FcgammaR interaction using SPR-based biosensor technology. Methods Mol Biol 988:331–343. https://doi.org/10.1007/978-1-62703-327-5_21
Acknowledgment
We thank other members of the Wang Lab for technical assistance and helpful discussions. This work was supported by the National Institutes of Health (NIH grants R01GM080374 and R01GM096973 to LXW).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Li, C., Li, T., Wang, LX. (2018). Chemoenzymatic Defucosylation of Therapeutic Antibodies for Enhanced Effector Functions Using Bacterial α-Fucosidases. In: Nevoltris, D., Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 1827. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8648-4_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8648-4_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8647-7
Online ISBN: 978-1-4939-8648-4
eBook Packages: Springer Protocols