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

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

Author information

1
Department of Chemical, Biological & Materials Engineering, University of Oklahoma, Sarkeys Energy Center, 100 East Boyd Street, Room T-301, Norman, OK 73019, USA proteinpete@gmail.com.
2
Bolder Biotechnology, Boulder, CO 80301, USA.

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

PMID:
25855658
PMCID:
PMC4596277
DOI:
10.1093/protein/gzv019
[Indexed for MEDLINE]
Free PMC Article

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