Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Glycosylation as a strategy to improve antibody-based therapeutics

Nature Reviews Drug Discovery volume 8, pages 226–234 (2009)Cite this article

Key Points

  • Natural and recombinant immunoglobulin G (IgG) antibodies consist of an IgG Fab (antigen-binding fragment) region and IgG Fc (crystallizable fragment) regions. On antigen binding, antibodies form multimeric complexes that can initiate inflammatory processes leading to their elimination and destruction.

  • Glycosylation of the IgG Fc region is essential for the activation of downstream effector functions. There is heterogeneity in the oligosaccharides that are attached, resulting in multiple glycoforms that can differ from each other in biological efficacy.

  • Production cell lines may be engineered to produce selected glycoforms with enhanced biological properties and to provide maximum efficacy for a given disease indication.

  • It may be possible to further improve the effector function of currently approved therapeutic antibodies, without increasing their immunogenicity, by generating particular glycoforms.

  • Improved production vehicles can offer lower cost of goods and contribute to lower cost of treatment.

Abstract

To date, more than 20 recombinant immunoglobulin G (IgG) antibody therapeutics are licensed for the treatment of various diseases. The mechanism of action of recombinant monoclonal antibodies (rMAbs) has been extensively investigated and several distinct pathways have been defined; selective activation of specific pathways may optimize clinical outcomes for different diseases, such as cancer and chronic inflammation. Human IgG is a glycoprotein with oligosaccharides attached at a single site. These are essential to the mode of action of rMAbs, and the antibody efficacy can vary depending on the particular oligosaccharide that is attached. Methods are now becoming available that allow the production of rMAbs bearing pre-selected oligosaccharides — glycoforms — to provide maximum efficacy for a given disease indication. This Review summarizes current knowledge of these methods and avenues for their exploitation in the clinic.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The α-carbon backbone structure of the immunoglobulin G (IgG) molecule.
Figure 2: IgG Fc diantennary-complex oligosaccharide composition.

Similar content being viewed by others

References

  1. Carter, P. J. Potent antibody therapeutics by design. Nature Rev. Immunol. 6, 343–357 (2006).

    Article  CAS  Google Scholar 

  2. Moutel, S. & Perez, F. Antibodies — Europe. Engineering the next generation of antibodies. Biotechnol. J. 3, 298–300 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Reichert, J. M. & Valge-Archer, V. E. Development trends for monoclonal antibody cancer therapeutics. Nature Rev. Drug Discov. 6, 349–356 (2007).

    Article  CAS  Google Scholar 

  4. Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975).

    Article  CAS  PubMed  Google Scholar 

  5. Mirik, G. R., Bradt, B. M., Denardo, S. J. & Denardo, G. L. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words. Q. J. Nucl. Med. Mol. Imaging 48, 251–257 (2004).

    Google Scholar 

  6. Koren, E. et al. Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products. J. Immunol. Methods 333, 1–9 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Aarden, L., Ruul, S. R. & Wolbink, G. Immunogenicity of anti-tumor necrosis factor antibodies-toward improved methods of anti-antibody measurement. Curr. Opin. Immunol. 20, 431–435 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Klitgaard, J.L. et al. Reduced susceptibility of recombinant polyclonal antibodies to inhibitory anti-variable domain antibody responses. J. Immunol. 177, 3782–3790 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Woof, J. M. & Burton, D.R. Human antibody–Fc receptor interactions illuminated by crystal structures. Nature Rev. Immunol. 4, 89–99 (2004).

    Article  CAS  Google Scholar 

  10. Nezlin, R. & Ghetie, V. Interactions of immunoglobulins outside the antigen-combining site. Adv. Immunol. 82, 155–215 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Jefferis, R. Antibody therapeutics: isotype and glycoform selection. Expert Opin. Biol. Ther. 7, 1401–1413 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. van Sorge, N. M., van der Pol, W. L. & van de Winkel, J. G. FcγR polymorphisms: implications for function, disease susceptibility and immunotherapy. Tissue Antigens 61, 189–202 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Nimmerjahn, F. & Ravetch, J. Fcγ receptors as regulators of immune responses. Nature Rev. Immunol. 8, 34–47 (2008).

    Article  CAS  Google Scholar 

  14. Jefferis, R. Glycosylation of recombinant antibody therapeutics. Biotechnol. Prog. 21, 11–16 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Sinclair, A. M. & Elliott, S. J. Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. J. Pharm. Sci. 94, 1626–1635 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Shields, R. L. et al. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733–26740 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Shinkawa, T. et al. 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, 3466–3473 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Niwa, R. et al. IgG subclass-independent improvement of antibody-dependent cellular cytotoxicity by fucose removal from Asn297-linked oligosaccharides. J. Immunol. Methods 306, 151–160 (2005). A demonstration that non-fucosylated glycoforms of all four IgG subclasses can bind and activate FcγRIIIa.

    Article  CAS  PubMed  Google Scholar 

  19. Ferrara, C. et al. Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of Golgi enzyme localization domain and co-expression of heterologous β1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Biotechnol. Bioeng. 93, 851–861 (2006).

    Article  CAS  PubMed  Google Scholar 

  20. Krapp, S. et al. Structural analysis of human IgG glycoforms reveals a correlation between oligosaccharide content, structural integrity and Fcγ-receptor affinity. J. Mol. Biol. 325, 979–989 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Deisenhofer, J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-Å resolution. Biochemistry 20, 2361–2370 (1981). This early paper showed that oligosaccharides are integral to an antibody's three-dimensional structure.

    Article  CAS  PubMed  Google Scholar 

  22. Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M. & Dwek, R. A. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu. Rev. Immunol. 25, 21–50 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Mizuochi, T., Taniguchi, T. Shimizu, A. & Kobata, A. Structural and numerical variations of the carbohydrate moiety of immunoglobulin G. J. Immunol. 129, 2016–2020 (1982).

    CAS  PubMed  Google Scholar 

  24. Routier, F. H. et al. Quantitation of the oligosaccharides of human serum IgG from patients with rheumatoid arthritis: a critical evaluation of different methods. J. Immunol. Methods 213, 13–30 (1998).

    Article  Google Scholar 

  25. Kaneko, Y., Nimmerjahn, F. & Ravetch, J. V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313, 670–673 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Scallon, B., Tam, S. H., McCarthy, S. G., Cai, A. N. & Raju, T. S. Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality. Mol. Immunol. 44, 1524–1534 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Nimmerjahn, F. & Ravetch, J. V. The antiinflammatory activity of IgG: the intravenous IgG paradox. J. Exp. Med. 204, 11–15 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Anthony, R. M. et al. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320, 373–376 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jefferis, R. et al. A comparative study of the N-linked oligosaccharide structures of human IgG subclass proteins. Biochem. J. 268, 529–537 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Farooq, M., Takahashi N., Arrol, H., Drayson, M. & Jefferis, R. Glycosylation of polyclonal and paraprotein IgG in multiple myeloma. Glycoconjugate J. 14, 489–492 (1997).

    Article  CAS  Google Scholar 

  31. Youings, A. et al. Site-specific glycosylation of human immunoglobulin G is altered in four rheumatoid arthritis patients. Biochem. J. 314, 621–630 (1996).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Holland, M. et al. Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA associated systemic vasculitis. Biochim. Biophys. Acta 1760, 669–677 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Mimura, Y., Ashton, P. R., Takahashi, N., Harvey, D. J. & Jefferis, R. Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry. J. Immunol. Methods 326, 116–126 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Mimura, Y. et al. The influence of glycosylation on the thermal stability and effector function expression of human IgG1-Fc: properties of a series of truncated glycoforms. Mol. Immunol. 37, 697–706 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Birch, J. R. & Racher, A. J. Antibody production. Adv. Drug Deliv. Rev. 58, 671–685 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Imai-Nishiya, H. et al. Double knockdown of α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC. BMC Biotechnol. 7, 84 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Macher, B. A. & Galili, U. The Galα1,3Galβ1,4GlcNAc-R (α-Gal) epitope: a carbohydrate of unique evolution and clinical relevance. Biochim. Biophys. Acta 1780, 75–88 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Padler-Karavani, V. et al. Diversity in specificity, abundance and composition of anti-Neu5Gc antibodies in normal humans: potential implications for disease. Glycobiology 18, 818–830 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Qian, J. et al. Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion. Anal. Biochem. 364, 8–18 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Chung, C. H. et al. Cetuximab-induced anaphylaxis and IgE specific for galactose-α-1,3-galactose. N. Engl. J. Med. 358, 1109–1117 (2008). Important report of hypersensitivity reactions to a recombinant therapeutic due to production in Sp2/0 cells in which gal a (1–3) gal is added.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Huang, L., Biolosi, S., Bales, K. R. & Kuchibhotla, U. Impact of variable domain glycosylation on antibody clearance: an LC/MS characterization. Anal. Biochem. 349, 197–207 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Carter, P. et al., Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc. Natl Acad. Sci. USA 89, 4285–4289 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Lim, A., Reed-Bogan, A. & Harmon, B. J. Glycosylation profiling of a therapeutic recombinant monoclonal antibody with two N-linked glycosylation sites using liquid chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer. Anal. Biochem. 375, 163–172 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Desjarlais, J. R., Lazar, G. A., Zhukovsky E. A. & Chu, S. Y. Optimizing engagement of the immune system by anti-tumor antibodies: an engineer's perspective. Drug Discov. Today 12, 898–910 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Sondermann, P., Huber, R., Oosthuizen, V. & Jacob, U. The 3.2-Å crystal structure of the human IgG1 Fc–FcγRIII complex. Nature 406, 267–273 (2000). First structural confirmation of the interaction site of IgG Fc for an Fcγ receptor. This has allowed in silico modelling of other FcγRs.

    Article  CAS  PubMed  Google Scholar 

  46. Radaev, S. et al. The structure of human type FcγIII receptor in complex with Fc. J. Biol. Chem. 276, 16469–16477 (2001).

    Article  CAS  PubMed  Google Scholar 

  47. Mimura, Y. et al. The role of oligosaccharide residues of IgG1-Fc in FcγIIb binding. J. Biol. Chem. 276, 5539–45547 (2001).

    Article  Google Scholar 

  48. Matsumiya, S. et al. Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1. J. Mol. Biol. 368, 767–779 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Watier, H. Variability factors in the clinical response to recombinant antibodies and IgG Fc-containing fusion proteins. Expert Opin. Biol. Ther. 5 (Suppl. 1), S1–S8 (2005).

    Article  Google Scholar 

  50. Marcus, R. & Hagenbeek, A. The therapeutic use of rituximab in non-Hodgkin's lymphoma. Eur. J. Haematol. 78 (Suppl. 67), 5–14 (2007).

    Article  Google Scholar 

  51. Lifely, M. R., Hale, G. & Boyse, S. Glycosylation and biological activity of CAMPATH-1H expressed in different cell lines and grown under different culture conditions. Glycobiology 5, 813–822 (1995).

    Article  CAS  PubMed  Google Scholar 

  52. Umana, P. et al. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nature Biotechnol. 17, 176–180 (1999). First report of a cell line glycoengineered to produce a recombinant antibody with enhanced efficacy for killing cancer cells.

    Article  CAS  Google Scholar 

  53. Davies, J. et al. Expression of GTIII in a recombinant anti-CD20 CHO production cell line: expression of antibodies of altered glycoforms leads to an increase in ADCC thro' higher affinity for FcRIII. Biotechnol. Bioeng. 74, 288–294 (2001).

    Article  CAS  PubMed  Google Scholar 

  54. Ymane-Ohuki, N. et al. Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol. Bioeng. 87, 614–622 (2004).

    Article  Google Scholar 

  55. Iida, S. et al. Non-fucosylated therapeutic IgG antibody can evade the inhibitory effect of serum IgG on antibody-dependent cellular cytotoxicity through its binding to FcγRIIIa. Clin. Cancer Res. 12, 2879–2887 (2006).

    Article  CAS  PubMed  Google Scholar 

  56. Preithner, S. et al. High concentrations of therapeutic IgG1 antibodies are needed to compensate for the inhibition of ADCC by excess endogenous immunoglobulin G. Mol. Immunol. 43, 1183–1193 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Nechansky, A. et al. Compensation of endogenous IgG mediated inhibition of antibody-dependent cellular cytotoxicity by glyco-engineering of therapeutic antibodies. Mol. Immunol. 44, 1815–1817 (2007).

    Article  CAS  PubMed  Google Scholar 

  58. Suzuki, E. et al. Nonfucosylated anti-HER2 antibody augments antibody-dependent cellular cytotoxicity in breast cancer patients. Clin. Cancer Res. 13, 1875–1882 (2007).

    Article  CAS  PubMed  Google Scholar 

  59. Peipp, M. et al. Antibody fucosylation differentially impacts cytotoxicity mediated by NK and PMN effector cells. Blood 112, 2390–2399 (2008). Potentially important finding that optimal IgG Fc glycoforms for cellular activation may differ between PBMCs and PMNs.

    Article  CAS  PubMed  Google Scholar 

  60. Ferarra, C., Stuart, F., Sondermann, P., Brunker, P. & Umana, P. The carbohydrate at FcγaRIIIa Asn-162. An element required for high affinity binding to non-fucosylated IgG glycoforms. J. Biol. Chem. 281, 5032–5036 (2006).

    Article  Google Scholar 

  61. Boyd, P. N., Lines, A. C. & Patel, A. K. The effect of the removal of sialic acid, galactose and total carbohydrate on the functional activity of Campath-1H. Mol. Immunol. 32, 1311–1318 (1995).

    Article  CAS  PubMed  Google Scholar 

  62. US Food and Drug Administration (FDA). Memorandum. FDA web site [online], (1997).

  63. Malhotra, R. et al. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nature Med. 1, 237–243 (1995).

    Article  CAS  PubMed  Google Scholar 

  64. Abadeh, S. et al. Remodelling the oligosaccharide of human IgG antibodies: effects on biological activities. Biochem. Soc. Trans. 25, S661 (1997).

    Article  CAS  PubMed  Google Scholar 

  65. Arnold, J. N., Dwek, R. A., Rudd, P. M. & Sim, R. B. Mannan binding lectin and its interaction with immunoglobulins in health and in disease. Immunol. Lett. 106, 103–110 (2006).

    Article  CAS  PubMed  Google Scholar 

  66. Garred, P. et al. J. Two edged role of mannose binding lectin in rheumatoid arthritis: a cross sectional study. J. Rheumatol. 27, 26–34 (2000).

    CAS  PubMed  Google Scholar 

  67. Sato, R. et al. Substances reactive with mannose-binding protein (MBP) in sera of patients with rheumatoid arthritis. Fukushima J. Med. Sci. 43, 99–111 (1997).

    CAS  PubMed  Google Scholar 

  68. Saevarsdottir, S., Vikingsdottir, T. & Valdimarsson, H. The potential role of mannan-binding lectin in the clearance of self-components including immune complexes. Scand. J. Immunol. 60, 23–29 (2004).

    Article  CAS  PubMed  Google Scholar 

  69. Taylor, M. E. & Drickamer, K. Paradigms for glycan-binding receptors in cell adhesion. Curr. Opin. Cell Biol. 19, 572–577 (2007).

    Article  CAS  PubMed  Google Scholar 

  70. Vyas, J. M., Van der Veen, A. G., & Ploegh, H. L. The known unknowns of antigen processing and presentation. Nature Rev. Immunol. 8, 607–618 (2008).

    Article  CAS  Google Scholar 

  71. Roopenian, D. C. & Akilesh, S. FcRn: the neonatal Fc receptor comes of age. Nature Rev. Immunol. 7, 715–725 (2007).

    Article  CAS  Google Scholar 

  72. Harris, R. J. Heterogeneity of recombinant antibodies: linking structure to function. Dev. Biol. (Basel) 122, 117–127 (2005).

    CAS  Google Scholar 

  73. Jones, A. J. et al. Selective clearance of glycoforms of a complex glycoprotein pharmaceutical caused by terminal N-acetylglucosamine is similar in humans and cynomolgus monkeys. Glycobiology 17, 529–540 (2007).

    Article  CAS  PubMed  Google Scholar 

  74. Keck, R. et al. Characterization of a complex glycoprotein whose variable metabolic clearance in humans is dependent on terminal N-acetylglucosamine content. Biologicals 36, 49–60 (2008).

    Article  CAS  PubMed  Google Scholar 

  75. Edberg, J. C. & Kimberly, R. P. Cell type-specific glycoforms of Fc gamma RIIIa (CD16): differential ligand binding. J. Immunol. 159, 3849–3857 (1997).

    CAS  PubMed  Google Scholar 

  76. Watt, G. et al. Synthesis of homogeneous neoglycoforms of IgG-Fc molecules and their functional properties. Hum. Antibodies 11, 29–30 (2002).

    Article  Google Scholar 

  77. Zhou, Q. et al. Development of a simple and rapid method for producing non-fucosylated oligomannose containing antibodies with increased effector function. Biotechnol. Bioeng. 99, 652–665 (2008).

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Immunity and Infection, University of Birmingham, School of Medicine, Edgbaston, B15 2TT, Birmingham, UK

    Roy Jefferis

Authors
  1. Roy Jefferis
    View author publications

    You can also search for this author in PubMed Google Scholar

Ethics declarations

Competing interests

Roy Jefferis has consultancy contracts with Novartis, Merck and Gilde.

Glossary

Diantennary oligosaccharide

An oligosaccharide that has two mannose 'arms'; as opposed to triantennary, tetra-antennary and so on.

Differential scanning micro-calorimetry

A thermoanalytic technique in which the difference in the amount of heat required to increase the temperature of a sample and a reference is measured as a function of temperature.

Alanine scanning

A genetic manipulation that sequentially replaces wild-type amino acids with alanine to determine the impact on the protein's structure and function.

Conformer

A discrete, defined conformation in three-dimensional space.

Antibody-dependent cellular cytotoxicity

(ADCC). Cell death that results when the Fc fragment of an antibody bound to a target cell interacts with Fc receptors on monocytes, macrophages or natural killer cells that are consequently activated to kill the target.

Complement-dependent cytotoxicity

(CDC). Cell death that results when the IgG Fc regions of an antibody bound to a target cell activate the C1 component of complement, initiating a cascade of reactions that lead to the formation of a complex that disrupts the cell membrane.

Granzyme

An apoptosis-inducing serine protease released from cytoplasmic granules by cytotoxic T cells and NK cells.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jefferis, R. Glycosylation as a strategy to improve antibody-based therapeutics. Nat Rev Drug Discov 8, 226–234 (2009). https://doi.org/10.1038/nrd2804

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrd2804

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing