Cytokine refacing effect reduces granulocyte macrophage colony-stimulating factor susceptibility to antibody neutralization

Protein Eng Des Sel. 2015 Oct;28(10):461-6. doi: 10.1093/protein/gzv019. Epub 2015 Apr 7.

Abstract

Crohn's Disease (CD) afflicts over half a million Americans with an annual economic impact exceeding $10 billion. Granulocyte macrophage colony-stimulating factor (GM-CSF) can increase patient immune responses against intestinal microbes that promote CD and has been effective for some patients in clinical trials. We have made important progress toward developing GM-CSF variants that could be more effective CD therapeutics by virtue of being less prone to neutralization by the endogenous GM-CSF autoantibodies that are highly expressed in CD patients. Yeast display engineering revealed mutations that increase GM-CSF variant binding affinity by up to ∼3-fold toward both GM-CSF receptor alpha and beta subunits in surface plasmon resonance experiments. Increased binding affinity did not reduce GM-CSF half-maximum effective concentration (EC50) values in conventional in vitro human leukocyte proliferation assays. Affinity-enhancing mutations did, however, promote a 'refacing effect' that imparted all five evaluated GM-CSF variants with increased in vitro bioactivity in the presence of GM-CSF-neutralizing polyclonal antisera. The most improved variant, H15L/R23L, was 6-fold more active than wild-type GM-CSF. Incorporation of additional known affinity-increasing mutations could augment the refacing effect and concomitant bioactivity improvements described here.

Keywords: Alzheimer's disease; Crohn's disease; GM-CSF; immunogenicity; irritable bowel syndrome.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antibodies, Neutralizing / immunology*
  • Cell Proliferation
  • Cytokine Receptor Common beta Subunit / metabolism
  • Cytokines / metabolism*
  • Granulocyte-Macrophage Colony-Stimulating Factor / chemistry
  • Granulocyte-Macrophage Colony-Stimulating Factor / genetics*
  • Granulocyte-Macrophage Colony-Stimulating Factor / immunology*
  • Humans
  • Leukocytes / cytology
  • Models, Molecular
  • Mutation
  • Protein Engineering*
  • Protein Structure, Secondary
  • Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology

Substances

  • Antibodies, Neutralizing
  • Cytokine Receptor Common beta Subunit
  • Cytokines
  • Receptors, Granulocyte-Macrophage Colony-Stimulating Factor
  • Recombinant Proteins
  • Granulocyte-Macrophage Colony-Stimulating Factor