A Method to Detect the Binding of Hyper-Glycosylated Fragment Crystallizable (Fc) Region of Human IgG1 to Glycan Receptors

  • Patricia Blundell
  • Richard PleassEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1904)


Engineering the fragment crystallizable (Fc) of human IgG can bring improved effector functions to monoclonal antibodies and Fc-fusion-based medicines and vaccines. Such Fc-effector functions are largely controlled by posttranslational modifications (PTMs) within the Fc, including the addition of glycans that introduce structural and functional heterogeneity to this class of therapeutic. Here, we describe a detailed method to allow the detection of hyper-sialylated Fcs to glycan receptors that will facilitate the future development of new mAbs and Fc-fragment therapies and vaccines.

Key words

IgG Glycans Glycosylation Fc-receptors ADCC ADCP CDC Effector function Therapeutic antibodies 


  1. 1.
    Czajkowsky DM, Hu J, Shao Z et al (2012) Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med 4:1015–1028CrossRefGoogle Scholar
  2. 2.
    Lund J, Takahashi N, Pound JD et al (1996) Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fcy receptor I and influence the synthesis of its oligosaccharide chains. J Immunol 157:4963–4969PubMedGoogle Scholar
  3. 3.
    Dalziel M, Crispin M, Scanlan CN et al (2014) Emerging principles for the therapeutic exploitation of glycosylation. Science 343:1235681. Scholar
  4. 4.
    Frank M, Walker RC, Lanzilotta WN et al (2014) Immunoglobulin G1 Fc domain motions: implications for Fc engineering. J Mol Biol 426:1799–1811CrossRefGoogle Scholar
  5. 5.
    Subedi GP, Hanson QM, Barb AW (2014) Restricted motion of the conserved immunoglobulin G1 N-glycan is essential for efficient Fc g RIIIa binding. Structure 22:1478–1488CrossRefGoogle Scholar
  6. 6.
    Schwab I, Nimmerjahn F (2013) Intravenous immunoglobulin therapy: how does IgG modulate the immune system? Nat Rev Immunol 13:176–189CrossRefGoogle Scholar
  7. 7.
    Debre M, Bonnet MC, Fridman WH et al (1993) Infusion of Fc gamma fragments for treatment of children with acute immune thrombocytopenic purpura. Lancet 342:945–949CrossRefGoogle Scholar
  8. 8.
    Washburn N, Schwab I, Ortiz D et al (2015) Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity. Proc Natl Acad Sci U S A 112:E1297–E1306CrossRefGoogle Scholar
  9. 9.
    Anthony RM, Kobayashi T, Wermeling F et al (2011) Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway: commentary. Nature 475:110–113CrossRefGoogle Scholar
  10. 10.
    Anthony RM, Wermeling F, Karlsson MCI et al (2008) Identification of a receptor required for the anti-inflammatory activity of IVIG. Proc Natl Acad Sci U S A 105:19571–19578CrossRefGoogle Scholar
  11. 11.
    Liu L (2015) Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins. J Pharm Sci 104:1866–1884CrossRefGoogle Scholar
  12. 12.
    Li H, Sethuraman N, Stadheim TA et al (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24:210–215CrossRefGoogle Scholar
  13. 13.
    St-Amour I, Pare I, Alata W et al (2013) Brain bioavailability of human intravenous immunoglobulin and its transport through the murine blood–brain barrier. J Cereb Blood Flow Metab 33:1983–1992CrossRefGoogle Scholar
  14. 14.
    Finke JM, Ayres KR, Brisbin RP et al (2017) Antibody blood-brain barrier efflux is modulated by glycan modification. Biochim Biophys Acta 1861:2228–2239CrossRefGoogle Scholar
  15. 15.
    Zhang G, Lopez PHH, Li CY et al (2004) Anti-ganglioside antibody-mediated neuronal cytotoxicity and its protection by intravenous immunoglobulin: implications for immune neuropathies. Brain 127:1085–1100CrossRefGoogle Scholar
  16. 16.
    Fiebiger BM, Maamary J, Pincetic A et al (2015) Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs. Proc Natl Acad Sci 112:E2385–E2394. Scholar
  17. 17.
    Blundell PA, Le NPL, Allen J et al (2017) Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors. J Biol Chem 292:12994–13007CrossRefGoogle Scholar
  18. 18.
    van de Bovenkamp FS, Hafkenscheid L, Rispens T et al (2016) The emerging importance of IgG Fab glycosylation in immunity. J Immunol 196:1435–1441CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Liverpool School of Tropical MedicineLiverpoolUK

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