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Enhancement of therapeutic protein in vivo activities through glycoengineering

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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Figure 1: Construction and testing of EPO glycosylation analogs.
Figure 2: Molecular comparison of rHuEPO (A) and darbepoetin alfa (B).
Figure 3: In vivo activities of glycoengineered proteins.
Figure 4: Construction and testing of Mpl-ligand glycosylation analogs.
Figure 5: Construction and testing of leptin glycosylation analogs.

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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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Correspondence to Steve Elliott.

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

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Elliott, S., Lorenzini, T., Asher, S. et al. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat Biotechnol 21, 414–421 (2003). https://doi.org/10.1038/nbt799

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