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Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins

Abstract

Recombinant glycoprotein therapeutics produced in nonhuman mammalian cell lines and/or with animal serum are often modified with the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc; refs. 1,2). This documented contamination has generally been ignored in drug development because healthy individuals were not thought to react to Neu5Gc (ref. 2). However, recent findings indicate that all humans have Neu5Gc-specific antibodies, sometimes at high levels3,4. Working with two monoclonal antibodies in clinical use, we demonstrate the presence of covalently bound Neu5Gc in cetuximab (Erbitux) but not panitumumab (Vectibix). Anti-Neu5Gc antibodies from healthy humans interact with cetuximab in a Neu5Gc-specific manner and generate immune complexes in vitro. Mice with a human-like defect in Neu5Gc synthesis generate antibodies to Neu5Gc after injection with cetuximab, and circulating anti-Neu5Gc antibodies can promote drug clearance. Finally, we show that the Neu5Gc content of cultured human and nonhuman cell lines and their secreted glycoproteins can be reduced by adding a human sialic acid to the culture medium. Our findings may be relevant to improving the half-life, efficacy and immunogenicity of glycoprotein therapeutics.

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Figure 1: ELISA and western blot detection of Neu5Gc on biotherapeutic antibodies by Neu5Gc IgY antibodies from chickens or IgG antibodies from normal human serum.
Figure 2: Effects of Neu5Gc-specific antibodies on the kinetics of therapeutic antibodies in mice with a human-like Neu5Gc deficiency, levels of anti-Neu5Gc IgG in mice after injections of the therapeutic antibodies, and binding of IgG Neu5Gc-specific antibodies from whole human serum to Neu5Gc on the Fab fragment of cetuximab.
Figure 3: An approach to reducing Neu5Gc contamination in biotherapeutic products.

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References

  1. Hokke, C.H. et al. Sialylated carbohydrate chains of recombinant human glycoproteins expressed in Chinese hamster ovary cells contain traces of N-glycolylneuraminic acid. FEBS Lett. 275, 9–14 (1990).

    Article  CAS  Google Scholar 

  2. Noguchi, A., Mukuria, C.J., Suzuki, E. & Naiki, M. Failure of human immunoresponse to N-glycolylneuraminic acid epitope contained in recombinant human erythropoietin. Nephron 72, 599–603 (1996).

    Article  CAS  Google Scholar 

  3. Tangvoranuntakul, P. et al. Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. Proc. Natl. Acad. Sci. USA 100, 12045–12050 (2003).

    Article  CAS  Google Scholar 

  4. 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  Google Scholar 

  5. Aggarwal, S. What′s fueling the biotech engine—2007. Nat. Biotechnol. 26, 1227–1233 (2008).

    Article  CAS  Google Scholar 

  6. 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  Google Scholar 

  7. Durocher, Y. & Butler, M. Expression systems for therapeutic glycoprotein production. Curr. Opin. Biotechnol. 20, 700–707 (2009).

    Article  CAS  Google Scholar 

  8. Higgins, E. Carbohydrate analysis throughout the development of a protein therapeutic. Glycoconj. J. 27, 211–225 (2009).

    Article  Google Scholar 

  9. Galili, U. Immune response, accommodation, and tolerance to transplantation carbohydrate antigens. Transplantation 78, 1093–1098 (2004).

    Article  CAS  Google Scholar 

  10. Varki, A. Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature 446, 1023–1029 (2007).

    Article  CAS  Google Scholar 

  11. Bardor, M., Nguyen, D.H., Diaz, S. & Varki, A. Mechanism of uptake and incorporation of the non-human sialic acid N-glycolylneuraminic acid into human cells. J. Biol. Chem. 280, 4228–4237 (2005).

    Article  CAS  Google Scholar 

  12. Borys, M.C. et al. Effects of culture conditions on N-glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells. Biotechnol. Bioeng. 105, 1048–1057 (2009).

    Google Scholar 

  13. Chung, C.H. et al. Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. N. Engl. J. Med. 358, 1109–1117 (2008).

    Article  CAS  Google Scholar 

  14. Delbaldo, C. et al. Pharmacokinetic profile of cetuximab (Erbitux) alone and in combination with irinotecan in patients with advanced EGFR-positive adenocarcinoma. Eur. J. Cancer 41, 1739–1745 (2005).

    Article  CAS  Google Scholar 

  15. Saadeh, C.E. & Lee, H.S. Panitumumab: a fully human monoclonal antibody with activity in metastatic colorectal cancer. Ann. Pharmacother. 41, 606–613 (2007).

    Article  CAS  Google Scholar 

  16. Diaz, S.L. et al. Sensitive and specific detection of the non-human sialic acid N-glycolylneuraminic acid in human tissues and biotherapeutic products. PLoS ONE 4, e4241 (2009).

    Article  Google Scholar 

  17. Muchmore, E.A., Milewski, M., Varki, A. & Diaz, S. Biosynthesis of N-glycolyneuraminic acid. The primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool. J. Biol. Chem. 264, 20216–20223 (1989).

    CAS  PubMed  Google Scholar 

  18. Hedlund, M. et al. N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution. Mol. Cell. Biol. 27, 4340–4346 (2007).

    Article  CAS  Google Scholar 

  19. Hedlund, M., Padler-Karavani, V., Varki, N.M. & Varki, A. Evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression. Proc. Natl. Acad. Sci. USA 105, 18936–18941 (2008).

    Article  CAS  Google Scholar 

  20. Tahara, H. et al. Immunological property of antibodies against N-glycolylneuraminic acid epitopes in cytidine monophospho-n-acetylneuraminic acid hydroxylase-deficient mice. J. Immunol. 184, 3269–3275 (2010).

    Article  CAS  Google Scholar 

  21. Taylor, R.E. et al. Novel mechanism for the generation of human xeno-auto-antibodies against the non-human sialic acid N-glycolylneuraminic acid. J. Exp. Med. published online, doi: 10.1084/jem.20100575 (12 July 2010).

  22. 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  Google Scholar 

  23. Axworthy, D.B. et al. Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity. Proc. Natl. Acad. Sci. USA 97, 1802–1807 (2000).

    Article  CAS  Google Scholar 

  24. Pham, T. et al. Evidence for a novel human-specific xeno-auto-antibody response against vascular endothelium. Blood 114, 5225–5235 (2009).

    Article  CAS  Google Scholar 

  25. Jahn, E.M. & Schneider, C.K. How to systematically evaluate immunogenicity of therapeutic proteins—regulatory considerations. New Biotechnol. 25, 280–286 (2009).

    Article  CAS  Google Scholar 

  26. Galili, U. et al. Enhancement of antigen presentation of influenza virus hemagglutinin by the natural human anti-Gal antibody. Vaccine 14, 321–328 (1996).

    Article  CAS  Google Scholar 

  27. Benatuil, L. et al. The influence of natural antibody specificity on antigen immunogenicity. Eur. J. Immunol. 35, 2638–2647 (2005).

    Article  CAS  Google Scholar 

  28. Abdel-Motal, U.M., Wigglesworth, K. & Galili, U. Mechanism for increased immunogenicity of vaccines that form in vivo immune complexes with the natural anti-Gal antibody. Vaccine 27, 3072–3082 (2009).

    Article  CAS  Google Scholar 

  29. 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  Google Scholar 

  30. Shankar, G., Pendley, C. & Stein, K.E. A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs. Nat. Biotechnol. 25, 555–561 (2007).

    Article  CAS  Google Scholar 

  31. Wilson, J.M. Medicine. A history lesson for stem cells. Science 324, 727–728 (2009).

    Article  CAS  Google Scholar 

  32. Martin, M.J., Muotri, A., Gage, F. & Varki, A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 11, 228–232 (2005).

    Article  CAS  Google Scholar 

  33. Martin, M.J., Muotri, A., Gage, F. & Varki, A. Response to Cerdan et al.: Complement targeting of nonhuman sialic acid does not mediate cell death of human embryonic stem cells. Nat. Med. 12, 1115 (2006).

    Article  CAS  Google Scholar 

  34. Van Hoeyveld, E. & Bossuyt, X. Evaluation of seven commercial ELISA kits compared with the C1q solid-phase binding RIA for detection of circulating immune complexes. Clin. Chem. 46, 283–285 (2000).

    Article  CAS  Google Scholar 

  35. Campagnari, A.A., Gupta, M.R., Dudas, K.C., Murphy, T.F. & Apicella, M.A. Antigenic diversity of lipooligosaccharides of nontypable Haemophilus influenzae. Infect. Immun. 55, 882–887 (1987).

    Article  CAS  Google Scholar 

  36. Greiner, L.L. et al. Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans. Infect. Immun. 72, 4249–4260 (2004).

    Article  CAS  Google Scholar 

  37. Gagneux, P. et al. Proteomic comparison of human and great ape blood plasma reveals conserved glycosylation and differences in thyroid hormone metabolism. Am. J. Phys. Anthropol. 115, 99–109 (2001).

    Article  CAS  Google Scholar 

  38. Debeire, P., Montreuil, J., Moczar, E., van Halbeek, H. & Vliegenthart, J.F.G. Primary structure of two major glycans of bovine fibrinogen. Eur. J. Biochem. 151, 607–611 (1985).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health grants R01-GM32373 and R01-CA38701 to A.V. and The International Sephardic Education Foundation for V.P.-K. Haemophilus influenzae strain 2019 was a generous gift from M. Apicella, Department of Microbiology, University of Iowa.

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All authors helped design the studies; D.G. and S.D. performed the research; R.E.T. and V.P.-K. generated crucial reagents; D.G. and A.V. wrote the paper; and all authors read the paper.

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Correspondence to Ajit Varki.

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Competing interests

None of the authors has a personal financial interest in any of the companies whose products are mentioned. A.V. is a co-founder of, and shareholder in Sialix, Inc. (formerly Gc-Free, Inc.), a startup biotech company focused on solving problems arising from Neu5Gc contamination of foods and drugs. D.G. is currently an employee of Sialix, Inc.

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Ghaderi, D., Taylor, R., Padler-Karavani, V. et al. Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat Biotechnol 28, 863–867 (2010). https://doi.org/10.1038/nbt.1651

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