EPITOPIC REGIONS FOR ANTIBODIES AGAINST TUMOR-NECROSIS-FACTOR-ALPHA -ANALYSIS BY SYNTHETIC PEPTIDE-MAPPING

The location of biologically relevant epitopes on hu man tumor necrosi s factor alpha (hTNF-alpha) was evaluated by testing the immunoreactiv ity of anti-TNF-alpha antibodies against 149 sequential, overlapping o ctamer peptides. A goat polyclonal antibody raised against recombinant hTNF-alpha, which neutralizes hTNF-alpha biological activities, react ed with oligopeptides corresponding to hTNF-alpha residues 7-11, 17-23 , 30-39, 42-49, 106-112, and 135-142. A possible assembled epitopic re gion (residues 25, 27, and 144) for neutralizing murine monoclonal ant ibodies designated 11D7G4 and 9C4G5 was deduced from the fact that the y bound to tripeptides, mimicking a discontinuous epitope. These antig enic regions were found to include or adjoin poorly conserved amino ac ids and they were located in the turns between beta-sheets on the surf ace of the molecule. Three of the sequential epitopic regions and an a ssembled region were closely related to the receptor binding sites pro posed in several other studies. These antibodies appear to neutralize TNF-alpha activities by directly masking receptor binding sites.