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

• Published in: Protein engineering, design and selection Vol. 28; no. 10; pp. 461 - 466
• Main Authors: Heinzelman, Pete, Carlson, Sharon J., Cox, George N.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.10.2015
• Subjects: Antibodies, Neutralizing - immunology; Cell Proliferation; Cytokine Receptor Common beta Subunit - metabolism; Cytokines - metabolism; Diverse Aspects of Antibodies and Fusion Proteins; 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; Crohn's disease; GM-CSF; irritable bowel syndrome; Alzheimer's disease; immunogenicity
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzv019

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.