Neutralizing and nonneutralizing monoclonal antibodies to the human granulocyte-macrophage colony-stimulating factor receptor alpha-chain

Abstract

A panel of monoclonal antibodies was raised against the low-affinity human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor alpha-chain expressed as recombinant protein on murine FDC-P1 cells. All the selected antibodies were of the IgG2A isotype and bound to protein A. They each recognized both native and recombinant receptors by indirect surface immunofluorescence and by immunoprecipitation. Several of the antibodies also recognized presumably denatured receptors as detected by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three different epitopes on the extracellular domain of the GM-CSF receptor alpha-chain were defined by these antibodies, and two of the epitopes did not appear to be involved in binding hGM-CSF or in interactions with the beta-chain of the GM-CSF receptor that are required for high-affinity binding of GM-CSF. On the other hand, the epitope recognized by antibody 2B7–17-A appeared to be critically involved in the binding of GM-CSF because this antibody completely abrogated both high- and low- affinity binding of GM-CSF to native and recombinant receptors. Antibody 2B7–17-A had a relatively high affinity for the GM-CSF receptor alpha-chain (kd = 3 nmol/L) and slow dissociation kinetics (kd = 0.002 min-1). These properties made the 2B7–17-A antibody a potent inhibitor of hGM-CSF biologic action in several different bioassays, with a half-maximal inhibitory dose of about 6 nmol/L (1 microgram/mL). This antibody could prove useful in alleviating any pathologic states mediated by excess GM-CSF levels and in defining the domains of the GM- CSF receptor required for ligand binding.

Trial Watch: Immunostimulation with recombinant cytokines for cancer therapy

“…184 These findings are at odds with previous results from prospective Phase II and non-prospective Phase III studies suggesting that recombinant GM-CSF (alone or combined with other immunotherapeutics) provides a clinical benefit to patients with melanoma at high risk for recurrence. [185][186][187][188][189][190][191] Of note, it has recently been demonstrated that >90% of melanoma patients receiving adjuvant therapy with GM-CSF develop GM-CSF-targeting antibodies, [192][193][194] which in >40% of the cases are neutralizing. 195 This may explain, at least in part, the limited therapeutic efficacy of GM-CSF in some clinical settings.…”