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Biotechnical Methods Section (BTS)

Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin

Leukemia volume 15, pages 1277–1285 (2001)Cite this article

Abstract

Myelopoietins comprise a class of chimeric cytokine receptor agonists consisting of an hIL-3 (human interleukin-3) receptor agonist and an hG-CSF (human granulocyte colony-stimulating factor) receptor agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding receptor. Five amino acid positions in a short region of the hG-CSF receptor agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very ‘deep’ mutagenesis at selected residues: >105 substitution variants were affinity-screened using the hG-CSF receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants.

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Acknowledgements

The authors acknowledge Joe Monahan, Joe Welply, Dave Wood, Keats Nelms, Paul Hippenmeyer, Bernie Violand, Walter Smith, Chuck McWherter, Judy Giri and Maire Caparon for helpful discussions and critical review of the manuscript.

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Author notes

  1. SC Lee

    Present address: Center for Biomedical Engineering, 286 Bevis Hall, 1080 Carnack Road, Columbus, OH, 43210, USA

  2. C Van Valkenburgh

    Present address: Department of Microbiology and Immunology, School of Medicine, Vanderbilt University, 1161 21st Ave S, Nashville, TN, 37232–2363, USA

  3. S Merlin

    Present address: Dept of Pathology, Box 8118, 660 S Euclid Ave, Washington University School of Medicine, Washington University, St Louis, MO 63110–1093, USA

Authors and Affiliations

  1. GD Searle Pharmacia Company, 700 Chesterfield Village Parkway AA4C, St Louis, MO 63196, USA

    SC Lee, R Ibdah, C Van Valkenburgh, E Rowold, A Abegg, A Donnelly, J Klover, S Merlin & JP McKearn

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Lee, S., Ibdah, R., Van Valkenburgh, C. et al. Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin. Leukemia 15, 1277–1285 (2001). https://doi.org/10.1038/sj.leu.2402163

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Keywords

  • chimeric
  • cytokine
  • display
  • hG-CSF
  • hIL-3
  • mutagenesis
  • myelopoietin

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