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

Nature Biotechnology volume 20, pages 908–913 (2002)Cite this article

Abstract

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.

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Figure 1: Computational predictions and experimental validation of receptor-binding properties of wild-type GCSF and histidine mutants.
Figure 2: Depletion of wild-type GCSF and histidine mutants and corresponding cell proliferation.

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Acknowledgements

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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Authors and Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, 02139-4307, MA

    Casim A. Sarkar & Douglas A. Lauffenburger

  2. Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, 02139-4307, MA

    Casim A. Sarkar & Douglas A. Lauffenburger

  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139-4307, MA

    Ky Lowenhaupt & Douglas A. Lauffenburger

  4. Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, 02139-4307, MA

    Bruce Tidor & Douglas A. Lauffenburger

  5. Department of Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, 02139-4307, MA

    Bruce Tidor

  6. Department of Process Science, Amgen, Inc., Amgen Center, Thousand Oaks, 91320-1789, CA

    Thomas Horan & Thomas C. Boone

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  1. Casim A. Sarkar
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Correspondence to Bruce Tidor or Douglas A. Lauffenburger.

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Sarkar, C., Lowenhaupt, K., Horan, T. et al. Rational cytokine design for increased lifetime and enhanced potency using pH-activated “histidine switching”. Nat Biotechnol 20, 908–913 (2002). https://doi.org/10.1038/nbt725

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