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 interferon-beta1a (IFN-beta1a) were mapped with monoclonal antibodies (mAb), grouped on the basis of their specificity and ability to neutralize biologic activity, and alanine scanning mutagenesis (ASM). The binding properties of nine mAb were defined, using ASM-IFN-beta mutants having alanine substituted at targeted, surface-exposed residues. The results were correlated with the 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 binding constitutes their mechanism of neutralization. However, two mAb that bound to sites remote from the IFNAR-2 binding site on IFN-beta also inhibited IFN-beta/IFNAR-2 complex formation and demonstrated potent neutralizing 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 sites.

Systematic mutational mapping of sites on human interferon-beta-1a that are important for receptor binding and functional activity.

A systematic mutational analysis of human interferon-beta-1a (IFN-beta) was performed to identify regions on the surface of the molecule that are important for receptor binding and for functional activity. The crystal structure of IFN-beta-1a was used to design a panel of 15 mutant proteins, in each of which a contiguous group of 2-8 surface residues was mutated, in most instances to alanine. The mutants were analyzed for activity in vitro in antiviral and in antiproliferation assays, and for their ability to bind to the type I IFN (ifnar1/ifnar2) receptor on Daudi cells and to a soluble ifnar2 fusion protein (ifnar2-Fc). Abolition of binding to ifnar2-Fc for mutants A2, AB1, AB2, and E established that the ifnar2 binding site on IFN-beta comprises parts of the A helix, the AB loop, and the E helix. Mutations in these areas, which together define a contiguous patch of the IFN-beta surface, also resulted in reduced affinity for binding to the receptor on cells and in reductions in activity of 5-50-fold in functional assays. A second receptor interaction site, concluded to be the ifnar1 binding site, was identified on the opposite face of the molecule. Mutations in this region, which encompasses parts of the B, C, and D helices and the DE loop, resulted in disparate effects on receptor binding and on functional activity. Analysis of antiproliferation activity as a function of the level of receptor occupancy allowed mutational effects on receptor activation to be distinguished from effects on receptor binding. The results suggest that the binding energy from interaction of IFN-beta with ifnar2 serves mainly to stabilize the bound IFN/receptor complex, whereas the binding energy generated by interaction of certain regions of IFN-beta with ifnar1 is not fully expressed in the observed affinity of binding but instead serves to selectively stabilize activated states of the receptor.