Mapping of IFN-beta epitopes important for receptor binding and biologic activation: Comparison of results achieved using antibody-based methods and alanine substitution mutagenesis

The epitopes important for receptor binding and activation of human interfe ron-beta 1a (IFN-beta 1a) were mapped with monoclonal antibodies (mAb), gro uped on the basis of their specificity and ability to neutralize biologic a ctivity, and alanine scanning mutagenesis (ASM). The binding properties of nine mAb were defined, using ASM-IFN-beta mutants having alanine substitute d at targeted, surface-exposed residues. The results were correlated with t he mAb neutralizing potency. Of six mAb that bound either at or adjacent to the IFNAR-2 receptor chain binding site defined by the ASM epitopes, only three had measurable neutralizing activity. Two of these inhibited IFN-beta /IFNAR-2 complex formation, suggesting that steric hindrance of receptor b inding constitutes their mechanism of neutralization. However, two mAb that bound to sites remote from the IFNAR-2 binding site on IFN-beta also inhib ited IFN-beta /IFNAR-2 complex formation and demonstrated potent neutralizi ng activity. Thus, neutralizing mAb may employ mechanisms other than steric blockade to inhibit directly the binding of receptor by cytokine, limiting their usefulness as tools to define precise receptor-ligand interaction si tes.