Rational cytokine design for increased lifetime and enhanced potency using pH-activated “histidine switching”

Casim A. Sarkar, Ky Lowenhaupt, Thomas Horan, Thomas C. Boone, Bruce Tidor, Douglas A. Lauffenburger

Research output: Contribution to journalArticlepeer-review

139 Scopus citations


We describe a method for the rational design of more effective therapeutic proteins using amino acid substitutions that reduce receptor binding affinity in intracellular endosomal compartments, thereby leading to increased recycling in the ligand-sorting process and consequently resulting in longer half-life in extracellular medium. We demonstrate this approach for granulocyte colony-stimulating factor by using computationally predicted histidine substitutions that switch protonation states between cell-surface and endosomal pH. Molecular modeling of binding electrostatics indicates two different single-histidine mutants that fulfill our design requirements; experimental assays demonstrate that each mutant indeed exhibits an order-of-magnitude increase in medium half-life along with enhanced potency due to increased endocytic recycling.

Original languageEnglish (US)
Pages (from-to)908-913
Number of pages6
JournalNature biotechnology
Issue number9
StatePublished - Sep 2002

Bibliographical note

Funding Information:
Acknowledgments We thank D. Brems and H. Lodish for vital input and encouragement; J. Caravella, E. Fallon, F. Frankel, E. Kangas, K.D. Wittrup, and D. Wu for fruitful discussions; and E. McCulloch for the OCI/AML1 cell line. This work was supported by a Hertz Foundation fellowship to C.A.S., National Institutes of Health grant GM56552 to B.T., and a grant from the Amgen/MIT Partnership to D.A.L.


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