title: Cytokine refacing effect reduces granulocyte macrophage colony-stimulating factor susceptibility to antibody neutralization.
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.
authors: 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.
authors: Pete
authors: P
authors: Heinzelman
authors: Bolder Biotechnology, Boulder, CO 80301, USA.
authors: Sharon J
authors: SJ
authors: Carlson
authors: Bolder Biotechnology, Boulder, CO 80301, USA.
authors: George N
authors: GN
authors: Cox
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology*; D049109:Cell Proliferation; D053648:Cytokine Receptor Common beta Subunit / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007962:Leukocytes / Q000166:cytology; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D053648:Cytokine Receptor Common beta Subunit; D016207:Cytokines; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor
keywords: Alzheimer's disease; Crohn's disease; GM-CSF; immunogenicity; irritable bowel syndrome