Simultaneous antagonism of interleukin-5, granulocyte-macrophage colony-stimulating factor, and interleukin-3 stimulation of human eosinophils by targetting the common cytokine binding site of their receptors.
Human interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic cytokines implicated in allergy in general and in the inflammation of the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific alpha chain and a shared subunit (beta(c)). Although binding of IL-5, GM-CSF, and IL-3 to their respective receptor alpha chains is the first step in receptor activation, it is the recruitment of beta(c) that allows high-affinity binding and signal transduction to proceed. Thus, beta(c) is a valid yet untested target for antiasthma drugs with the added advantage of potentially allowing antagonism of all 3 eosinophil-acting cytokines with a single compound. We show here the first development of such an agent in the form of a monoclonal antibody (MoAb), BION-1, raised against the isolated membrane proximal domain of beta(c). BION-1 blocked eosinophil production, survival, and activation stimulated by IL-5 as well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism showed that BION-1 prevented the high-affinity binding of (125)I-IL-5, (125)I-GM-CSF, and (125)I-IL-3 to purified human eosinophils and that it bound to the major cytokine binding site of beta(c). Interestingly, epitope analysis using several beta(c) mutants showed that BION-1 interacted with residues different from those used by IL-5, GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed that BION-1 prevented ligand-induced receptor dimerization and phosphorylation of beta(c), suggesting that ligand contact with beta(c) is a prerequisite for recruitment of beta(c), receptor dimerization, and consequent activation. These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and generate further agents for the treatment of eosinophil-dependent diseases such as asthma.
Sun
Q
Q
Hanson Centre for Cancer Research, the Institute of Medical and Veterinary Science, Adelaide, Australia.
Jones
K
K
McClure
B
B
Cambareri
B
B
Zacharakis
B
B
Iversen
P O
PO
Stomski
F
F
Woodcock
J M
JM
Bagley
C J
CJ
D'Andrea
R
R
Lopez
A F
AF
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004804:Eosinophils / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007377:Interleukin-3 / Q000494:pharmacology; D015848:Interleukin-5 / Q000494:pharmacology*; D007700:Kinetics; D007962:Leukocytes / Q000166:cytology / Q000502:physiology*; D008213:Lymphocyte Activation; D009000:Monocytes / Q000166:cytology / Q000502:physiology; D009504:Neutrophils / Q000166:cytology / Q000502:physiology; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D018123:Receptors, Interleukin / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D016185:Receptors, Interleukin-3 / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D007377:Interleukin-3; D015848:Interleukin-5; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D018123:Receptors, Interleukin; D016185:Receptors, Interleukin-3; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor