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Abstract

Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).

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References

  1. 1.

    , & The uses and properties of PEG-linked proteins. Crit. Rev. Ther. Drug Carrier Systems 9, 249–304 (1992).

  2. 2.

    , , & An erythropoietin fusion protein comprised of identical repeating domains exhibits enhanced biological properties. J. Biol. Chem. 274, 24773–24778 (1999).

  3. 3.

    , , , & Human erythropoietin dimers with markedly enhanced in vivo activity. Proc. Natl. Acad. Sci. USA 95, 1184–1188 (1998).

  4. 4.

    et al. Dimeric erythropoietin fusion protein with enhanced erythropoietic activity in vitro and in vivo. Blood 97, 3776–3782 (2001).

  5. 5.

    et al. Fusing the carboxy-terminal peptide of the chorionic gonadotropin (CG) β-subunit to the common α-subunit: retention of O-linked glycosylation and enhanced in vivo bioactivity of chimeric human CG. Mol. Endocrinol. 9, 54–63 (1995).

  6. 6.

    et al. Plasminogen activator inhibitor-1 fused with erythropoietin (EPO) mimetic peptide (EMP) enhances the EPO activity of EMP. J. Peptide Res. 56, 59–62 (2000).

  7. 7.

    , , , & A barbourin–albumin fusion protein that is slowly cleared in vivo retains the ability to inhibit platelet aggregation in vitro. Thromb. Haemost. 86, 902–908 (2001).

  8. 8.

    , , & Prolonged in vivo anticoagulant activity of a hirudin–albumin fusion protein secreted from Pichia pastoris. Blood Coag. Fibrinolysis 12, 433–443 (2001).

  9. 9.

    Soluble tumor necrosis factor receptor (p75) fusion protein (ENBREL) as a therapy for rheumatoid arthritis. Rheum. Dis. Clin. North Am. 24, 579–591 (1998).

  10. 10.

    et al. PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimisation of coupling techniques. Int. J. Hematol. 68, 1–18 (1998).

  11. 11.

    et al. The rhGM-CSF-EPO hybrid protein MEN 11300 induces anti-EPO antibodies and severe anaemia in rhesus monkeys. Cytokine 10, 964–969 (1998).

  12. 12.

    et al. Development of antibodies to unprotected glycosylation sites on recombinant human GM-CSF. Lancet 335, 434–437 (1990).

  13. 13.

    Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem. J. 209, 331–336 (1983).

  14. 14.

    & Structural requirements for protein N-glycosylation. Influence of acceptor peptides on cotranslational glycosylation of yeast invertase and site-directed mutagenesis around a sequon sequence. Eur. J. Biochem. 181, 525–529 (1989).

  15. 15.

    & Differences between Asn-Xaa-Thr-containing peptides: a comparison of solution conformation and substrate behavior with oligosaccharyltransferase. Biochemistry 30, 4374–4380 (1991).

  16. 16.

    et al. Erythropoietin: gene cloning, protein structure, and biological properties. Cold Spring Harb. Symp. Quant. Biol. 51, 693–702 (1986).

  17. 17.

    et al. Cloning and expression of the human erythropoietin gene. Proc. Natl. Acad. Sci. USA 82, 7580–7584 (1985).

  18. 18.

    , , & Characterization of recombinant monkey and human erythropoietin. Prog. Clin. Biol. Res. 191, 339–350 (1985).

  19. 19.

    et al. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 77, 1117–1124 (1994).

  20. 20.

    et al. Purification and biologic characterization of plasma-derived megakaryocyte growth and development factor. Blood 86, 540–547 (1995).

  21. 21.

    et al. Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425–432 (1994).

  22. 22.

    et al. Efficiency of signalling through cytokine receptors depends critically on receptor orientation. Nature 395, 511–516 (1998).

  23. 23.

    et al. NMR structure of human erythropoietin and a comparison with its receptor-bound conformation. Nat. Struct. Biol. 5, 861–866 (1998).

  24. 24.

    , , , & Mapping of the active site of recombinant human erythropoietin. Blood 89, 493–502 (1997).

  25. 25.

    et al. Fine-structure epitope mapping of anti-erythropoietin monoclonal antibodies reveals a model of recombinant human erythropoietin structure. Blood 87, 2702–2713 (1996).

  26. 26.

    , & Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis. Anal. Biochem. 162, 485–492 (1987).

  27. 27.

    et al. Structural requirements for addition of O-linked carbohydrate to recombinant erythropoietin. Biochemistry 33, 11237–11245 (1994).

  28. 28.

    et al. Isolation and characterization of conformation-sensitive anti-erythropoietin monoclonal antibodies: effect of disulfide bonds and carbohydrate on recombinant human erythropoietin structure. Blood 87, 2714–2722 (1996).

  29. 29.

    & Development and characterization of novel erythropoiesis stimulating protein (NESP). Br. J. Cancer 84 (Suppl. 10), 3–10 (2001).

  30. 30.

    et al. Pharmacokinetics of novel erythropoiesis-stimulating protein compared with epoetin alfa in dialysis patients. J. Am. Soc. Nephrol. 10, 2392–2395 (1999).

  31. 31.

    , & Pharmacokinetics of novel erythropoiesis-stimulating protein (NESP) in cancer patients: preliminary report. Br. J. Cancer 84 (Suppl. 6), 11–16 (2001).

  32. 32.

    , , , & Once-weekly compared with three-times-weekly subcutaneous epoetin β: results from a randomized, multicenter, therapeutic-equivalence study. Am. J. Kidney Dis. 40, 119–125 (2002).

  33. 33.

    et al. A dose-finding and safety study of novel erythropoiesis-stimulating protein (NESP) for the treatment of anaemia in patients receiving multicycle chemotherapy. Br. J. Cancer 84 (Suppl. 23), 17–23 (2001).

  34. 34.

    et al. Double-blind, placebo-controlled, randomized phase III trial of darbepoetin alfa in lung cancer patients receiving chemotherapy. J. Natl. Cancer Inst. 94, 1211–1220 (2002).

  35. 35.

    Novel erythropoiesis-stimulating protein for managing the anemia of chronic kidney disease. Am. J. Kidney Dis. 38, 1390–1397 (2001).

  36. 36.

    An overview of the efficacy and safety of novel erythropoiesis-stimulating protein (NESP). Nephrol. Dialysis Transplant. 16, 14–21 (2001).

  37. 37.

    et al. Role of glycosylation on the secretion and biological activity of erythropoietin. Biochemistry 31, 9871–9876 (1992).

  38. 38.

    , , & Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 14, 725–731 (1978).

  39. 39.

    et al. Establishment and characterization of an erythropoietin-dependent subline, UT-7/Epo, derived from human leukemia cell line, UT-7. Blood 82, 456–464 (1993).

  40. 40.

    , , , & Activation of the erythropoietin (EPO) receptor by bivalent anti-EPO receptor antibodies. J. Biol. Chem. 271, 24691–24697 (1996).

  41. 41.

    , , , & Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines. Proc. Natl. Acad. Sci. USA 80, 2931–2935 (1983).

  42. 42.

    et al. Identification and expression cloning of a leptin receptor, OB-R. Cell 83, 1263–1271 (1995).

  43. 43.

    , , & Human erythropoietin receptor: cloning, expression, and biologic characterization. Blood 76, 31–35 (1990).

  44. 44.

    et al. Molecular cloning and characterization of MPL, the human homolog of the v-mpl oncogene: identification of a member of the hematopoietic growth factor receptor superfamily. Proc. Natl. Acad. Sci. USA 89, 5640–5644 (1992).

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Acknowledgements

We thank Glenn Begley for support, encouragement, and help, and Leigh Busse for assistance in the preparation of this manuscript.

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Affiliations

  1. Amgen, One Amgen Center, Thousand Oaks, CA 91320.

    • Steve Elliott
    • , Tony Lorenzini
    • , Sheilah Asher
    • , Ken Aoki
    • , David Brankow
    • , Lynette Buck
    • , Leigh Busse
    • , David Chang
    • , Janis Fuller
    • , James Grant
    • , Natasha Hernday
    • , Martha Hokum
    • , Sylvia Hu
    • , Andrew Knudten
    • , Nancy Levin
    • , Renee Komorowski
    • , Frank Martin
    • , Rachell Navarro
    • , Timothy Osslund
    • , Gary Rogers
    • , Norma Rogers
    • , Geri Trail
    •  & Joan Egrie

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The authors are current or former employees of, and stockholders in, Amgen Inc.

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Correspondence to Steve Elliott.

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https://doi.org/10.1038/nbt799

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