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.

  • Research Article
  • Published:

Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity

Nature Biotechnology volume 17, pages 176–180 (1999)Cite this article

Abstract

The glycosylation pattern of chCE7, an antineuroblastoma chimeric IgG1, was engineered in Chinese hamster ovary cells with tetracycline–regulated expression of β(1,4)–N–acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisected oligosaccharides that have been implicated in antibody–dependent cellular cytotoxicity (ADCC). Measurement of the ADCC activity of chCE7 produced at different tetracycline levels showed an optimal range of GnTIII expression for maximal chCE7 in vitro ADCC activity, and this activity correlated with the level of constant region–associated, bisected complex oligosaccharides determined by matrix–assisted laser desorption/ionization time–of–flight mass spectrometry. The new optimized variants of chCE7 exhibit substantial ADCC activity and, hence, may be useful for treatment of neuroblastoma. The strategy presented here should be applicable to optimize the ADCC activity of other therapeutic IgGs.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Representation of typical human Fc–associated oligosaccharide structures.
Figure 2: Tetracycline–regulated expression of c–myc–tagged GnTIII in CHO cells.
Figure 3: MALDI/TOF–MS spectra of neutral oligosaccharide mixtures from chCE7 samples produced by CHO–tet–GnTIII–chCE7 cell cultures differing in the concentration of tetracycline added to the media and, therefore, expressed the GnTIII gene at different levels.
Figure 4: N–linked oligosaccharide biosynthetic pathway leading to bisected complex and bisected hybrid oligosaccharides via GnTIII–catalyzed reactions.
Figure 5: (A) ADCC activity of different chCE7 samples.

Similar content being viewed by others

References

  1. Deo, Y.M., Graziano, R.F., Repp, R., and van de Winkel, J.G.J. 1997.Clinical significance of IgG Fc receptors and FcgR–directed immunotherapies. Immunol. Today 18:127– 135.

    Article  CAS  Google Scholar 

  2. Dillman, R.O. 1997.Magic bullets at last! Finally—approval of a monoclonal antibody for the treatment of cancer! Cancer Biother. Radiopharm. 12:223–225.

    Article  CAS  Google Scholar 

  3. Frost, J.D., Hank, J.A., Reaman, G.H., Frierdich, S., Seeger, R.C., Gan, J. et al. 1997.A phase I/IB trial of murine monoclonal anti–GD2 antibody 14. 2G2a plus interleukin–2 in children with refractory neuroblastoma. Cancer 80:317–333.

    Article  CAS  Google Scholar 

  4. Surfus, J.E., Hank, J.A., Oosterwijk, E., Welt, S., Lindstrom, M.J., and Albertini, M.R. 1996.Anti–renal–cell carcinoma chimeric antibody G250 facilitates antibody–dependent cellular cytotoxicity with in vitro and in vivo interleukin–2–activated effectors. J. Immunother. 19:184– 191.

    Article  CAS  Google Scholar 

  5. Wright, A. and Morrison, S.L. 1997.Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol. 15:26– 31.

    Article  CAS  Google Scholar 

  6. Wormald, M.R., Rudd, P.M., Harvey, D.J., Chang, S., Scragg, I.G., and Dwek, R.A. 1997.Variations in oligosaccharide–protein interactions in immunoglobulin G determine the site–specific glycosylation profiles and modulate the dynamic motion of the oligosaccharides. Biochemistry 36:1370–1380.

    Article  CAS  Google Scholar 

  7. Lund, J., Takahashi, N., Pound, J.D., Goodall, M., Bentley, T., and Jefferis, R. 1996.Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fcg receptor I and influence the synthesis of its oligosaccharide chains. J. Immunol. 157:4963–4969 .

    CAS  PubMed  Google Scholar 

  8. Bergweff, A.A., Stroop, C.J., Murray, B., Holtorf, A.P., Pluschke, G., Van Oostrum, J., et al. 1995.Variation in N–linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones. Glycoconj. J. 12:318–330.

    Article  Google Scholar 

  9. Lifely, R.M., Hale, C., Boyce, S., Keen, M.J., and Phillips, J. 1995.Glycosylation and biological activity of CAMPATH–1H expressed in different cell lines and grown under different culture conditions. Glycobiology 5: 813–822.

    Article  CAS  Google Scholar 

  10. Amstutz, H., Rytz, C., Novak–Hofer, I., Spycher, M., Schubiger, P.A., Blaser, K., and Morgenthaler, J.J. 1993.Production and characterization of a mouse/human chimeric antibody directed against human neuroblastoma. Int. J. Cancer 53:147–152.

    Article  CAS  Google Scholar 

  11. Dürr, U., Haldemann, A.R., Leibundgut, K., Novak–Hofer, I., Amstutz, H., Wagner, H.P., et al. 1993.First clinical results with the chimeric antibody chCE7 in neuroblastoma. Targeting features and biodistribution data. Eur. J. Nucl. Med. 20: 858.

    Google Scholar 

  12. Schachter, H. 1986.Biosynthetic controls that determine the branching and microheterogeneity of protein–bound oligosaccharides. Biochem. Cell Biol. 64:163–181.

    Article  CAS  Google Scholar 

  13. Stanley, P. and Campbell, C.A. 1984.A dominant mutation to ricin resistance in Chinese hamster ovary cells induces UDP–GlcNac: glycopeptide b–4–N–Acetylglucosaminyl–transferase III activity. J. Biol. Chem. 261:13370– 13378.

    Google Scholar 

  14. Nishikawa, A., Ihara, Y., Htakeyama, M., Kangawa, K., and Taniguchi, N. 1992.Purification, cDNA cloning, and expression of UDP–N–acetylglucosamine:b–D–mannoside b–1,4N–acetylglucosaminyltransferase III from rat kidney. J. Biol. Chem. 267:18199–18204.

    CAS  PubMed  Google Scholar 

  15. Gossen, M. and Bujard, H. 1992.Tight control of gene expression in mammalian cells by tetracycline–responsive promoters. Proc. Natl. Acad. Sci. USA 89:5547– 5551.

    Article  CAS  Google Scholar 

  16. Field, M., Papac, C., and Jones, A. 1996.The use of high–performance anion–exchange chromatography and matrix–assisted laser desorption/ionization time–of–flight mass spectrometry to monitor and identify oligosaccharide degradation. Anal. Biochem. 239:92–98.

    Article  CAS  Google Scholar 

  17. Harvey, D.J. 1993.Quantitative aspects of the matrix–assisted laser desorption mass spectrometry of complex oligosaccharides. Rapid Commun. Mass Spectrom. 7:614– 619.

    Article  CAS  Google Scholar 

  18. Knster, B., Wheeler, S.F., Hunter, A.P., Dwek, R.A., and Harvey, D.J. 1997.Sequencing of N–linked oligosaccharides directly from protein gels: in–gel deglycosylation followed by matrix–assisted laser desorption/ionization mass spectrometry and normal–phase high–performance liquid chromatography. Anal. Biochem. 250:82–101 .

    Article  Google Scholar 

  19. Naven, T.J.P. and Harvey, D.J. 1996.Effect of structure on the signal strength of oligosaccharides in matrix–assisted laser desorption/ionization mass spectrometry on time–of–flight and magnetic sector instruments. Rapid Commun. Mass Spectrom. 10:1361–1366.

    Article  CAS  Google Scholar 

  20. Yu Ip, C.C., Miller, W.J., Silberklang, M., Mark, G.E., Ellis, R.W., Huang, L. et al. 1994.Structural characterization of the N–glycans of a humanized anti–CD18 murine immunoglobulin G. Arch. Biochem. Biophys. 308:387–399.

    Article  Google Scholar 

  21. Bibila, T.A. and Robinson, D.K. 1995.In pursuit of the optimal fed–batch process for monoclonal antibody production. Biotechnol. Prog. 11:1– 13.

    Article  CAS  Google Scholar 

  22. Trill, J.J., Shatzman, A.R., and Ganguly, S. 1995.Production of monoclonal antibodies in COS and CHO cells. Curr. Opin. Biotechnol. 6: 553–560.

    Article  CAS  Google Scholar 

  23. Nilsson, T., Pypaert, M., Hoe, M.H., Slusarewicz, P., Berger, E.G., and Warren, G. 1993.Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells. J. Cell Biol. 120:5–13.

    Article  CAS  Google Scholar 

  24. Reff, M.E., Carner, K., Chambers, K.S., Chinn, P.C., Leonard, J.E., Raab, R. et al. 1994.Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood 83: 435–445.

    CAS  Google Scholar 

  25. Clackson, T., Güssow, D., and Jones, P.T. 1991. General applications of PCR to gene cloning and manipulation, pp. 187– 214, in PCR: a practical approach. McPherson, M.J., Quirke, P., Taylor G.R. (eds.). Oxford University Press, Oxford, UK.

    Google Scholar 

  26. Kolber, M.A., Quinones, R.R., Gress, R.E., and Henkart, P.A. 1988.Measurement of cytotoxicity by target cell release and retention of the fluorescent dye bis–carboxyethyl–carboxyfluorescein (BCECF). J. Immunol. Methods. 108:255– 264.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by the Swiss Priority Program in Biotechnology (SPP BioTech). We would like to thank Ilse Novak for comparing the binding of different chCE7 samples to neuroblastoma cells, Heidi Ernst for DNA sequencing, and Naoyuki Taniguchi for providing us with the rat GnTIII cDNA.

Author information

Authors and Affiliations

  1. ZLB Central Laboratory, Blood Transfusion Service, Swiss Red Cross, Bern, CH–3000 22, Switzerland

    Radmila Moudry & Hanspeter Amstutz

  2. Institute of Biotechnology, ETH Zürich, Zürich, CH–8093, Switzerland

    Pablo Umaña, Joël Jean–Mairet & James E. Bailey

Authors
  1. Pablo Umaña
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Joël Jean–Mairet
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Radmila Moudry
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Hanspeter Amstutz
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. James E. Bailey
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence to James E. Bailey.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Umaña, P., Jean–Mairet, J., Moudry, R. et al. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17, 176–180 (1999). https://doi.org/10.1038/6179

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/6179

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