STRUCTURE-FUNCTION STUDY OF THE EXTRACELLULAR DOMAIN OF THE HUMAN IFN-ALPHA RECEPTOR (HIFNAR1) USING BLOCKING MONOCLONAL-ANTIBODIES - THE ROLE OF DOMAIN-1 AND DOMAIN-2

We have performed a structure-function analysis of extracellular domai n regions of the human IFN-alpha receptor (hIFNAR1) using mAbs generat ed by immunizing mice with a soluble hIFNAR1-IgG. Five mAbs described in this study recognize different epitopes as determined by a competit ive binding ELISA and by alanine substitution mutant analyses of the h IFNAR1-IgG. Two mAbs, 2E1 and 4A7, are able to block IFN-stimulated ge ne factor 3 (ISGF3) formation and inhibit the antiviral cytopathic eff ect induced by several IFN-alpha (IFN-alpha 2/1, -alpha 1, -alpha 2, - alpha 5, and -alpha 8). None of these anti-IFNAR1 mAbs were able to bl ock activity of IFN-beta. mAb 4A7 binds to a domain 1-hIFNAR1-IgG but not to a domain 2-hIFNAR1-IgG, which suggests that its binding region is located in domain 1, The binding of the most potent blocking mAb, 2 E1, requires the presence of domain 1 and domain 2, The most critical residue for 2E1 binding is a lysine residue at position 249, which is in domain 2. These findings suggest that both domain 1 and domain 2 ar e necessary to form a functional receptor and that a region in domain 2 is important. IFN-beta recognizes regions of the hIFNAR complex that are distinct from those important for the IFN-alpha.