Specific human granulocyte-macrophage colony-stimulating factor antagonists.

Abstract

Human granulocyte-macrophage colonystimulating factor (GM-CSF) is a pleiotropic hemopoietic growth factor and activator of mature myeloid cell function. We have previously shown that residue 21 in the first helix of GM-CSF plays a critical role in both biological activity and high-affinity receptor binding. We have now generated analogues of GM-CSF mutated at residue 21, expressed them in Escherichia coli, and examined them for binding, agonistic, and antagonistic activitles. Binding experiments showed that GM E21A, E21Q, E21F, E21H, E21R, and E21K bound to the GM-CSF receptor a chain with a similar affinity to wild-type GM-CSF and had lost high-affinity binding to the GM-CSF receptor a-chain-common 13-chain complex. From these mutants, only the charge reversal mutants E21R and E21K were completely devoid of agonistic activity. Significantly we found that E21R and E21K antagonized the proliferative effect of GM-CSF on the erythroleukemic cell line TF-1 and primary acute myeloid leukemias, as well as GM-CSF-mediated stimulation of neutrophil superoxide production. This antagonism was specific for GM-CSF in that no antagonism of interleukin 3-mediated TF-1 cell proliferation or tumor necrosis factor a-mediated stimulation of neutrophil superoxide production was observed. E. coli-derived GM E21R and E21K were effective antagonists of both nonglycosylated and glycosylated wild-type GM-CSF. These results show that low-affinity GM-CSF binding can be dissociated from receptor activation and have potential clinical significance for the management of inflammatory diseases and certain leukemias where GM-CSF plays a pathogenic role.

Transient hematologic and clinical effect of E21R in a child with end-stage juvenile myelomonocytic leukemia

“…3,4 E21R has a dual mechanism of action: antagonism of GM-CSF function, via selective binding to the receptor alpha chain, and induction of apoptosis in cells carrying the GM-CSF receptor. In vitro, E21R inhibits GM-CSF activity on myeloid leukemic cells 5 and in vivo, E21R exerts an antileukemic action in a transplanted animal model, 6 prompting us to consider its potential utility in JMML.…”

Construction and Binding Kinetics of a Soluble Granulocyte-Macrophage Colony-stimulating Factor Receptor α-Chain-Fc Fusion Protein

“…It is interesting to speculate on the role of the ␤-subunit (␤ c ) in slowing the dissociation of GM-CSF from the heterodimeric receptor. Studies of GM-CSF mutants support a key role for Glu-21 in binding to the high affinity but not the low affinity receptor (8,(27)(28)(29)(30)(31). These authors would argue for direct binding of the GM-CSF A-helix, centered on residue Glu-21, to the ␤ c .…”

The Receptor Binding Site of Human Interleukin-3 Defined by Mutagenesis and Molecular Modeling

“…Of the seven solventexposed residues in helix A, only Glu 21 was found to play a critical role in binding to the GM-CSF receptor (35,36). Mutagenesis results for solvent-exposed residues in helix D of GM-CSF (36) demonstrated that only Glu 108 and Asp 112 showed significant sensitivity to substitution.…”