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.
Runkel
L
L
Biogen, Inc., Cambridge, MA 02142, USA.
De Dios
C
C
Karpusas
M
M
Baker
D
D
Li
Z
Z
Zafari
M
M
Betzenhauser
M
M
Muldowney
C
C
Miller
S
S
Redlich
P N
PN
Grossberg
S E
SE
Whitty
A
A
Hochman
P S
PS
D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D000068556:Interferon beta-1a; D016899:Interferon-beta / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000378:metabolism*; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D000939:Epitopes; C507964:IFNAR1 protein, human; D008565:Membrane Proteins; D017471:Receptors, Interferon; D053633:Receptor, Interferon alpha-beta; D016899:Interferon-beta; D000409:Alanine; D000068556:Interferon beta-1a