Comparative analyses of complex formation and binding sites between human tumor necrosis factor-alpha and its three antagonists elucidate their different neutralizing mechanisms

Tumor necrosis factor-alpha (TNF alpha)-blocking therapy, using biologic TNF alpha antagonists, has been approved for the treatment of several diseases including rheumatoid arthritis, psoriasis and Crohn's disease. There have been few detailed studies of binding characterizations for the complex formation by TNF alpha and clinically relevant antagonists, particularly Infliximab (Remicade (R)) and Etanercept (Enbrel (R)). Here we characterized the binding stoichiometry and size of soluble TNF alpha-antagonist complexes and identified energetically important binding sites on TNF alpha for the three antagonists, Etanercept, Infliximab, and the recently developed humanized TNF alpha neutralizing monoclonal antibody, YHB1411-2. Size-exclusion chromatography and dynamic light scattering analyses revealed that the three antagonists formed distinct thermodynamically stable TNF(x-antagonist complexes that exhibited differences in their size and composition. Energetically important binding residues on TNF alpha were identified for each antagonist by a sequence of experiments that consisted of competition binding assays, fragmentations, loop mutations, and single-point mutations using yeast surface-displayed TNF alpha, which was further confirmed for solubly purified TNF alpha mutants by surface plasmon resonance technique. Analyses of the binding geometry based on binding site location, spatial constraints, and valency satisfaction allowed us to interpret the thermodynamically stable complexes as follows: one molecule of Etanercept and one molecule of trimeric TNF alpha (Etanercept(1)-TNF alpha(1)), Infliximab(6)-TNF alpha(3), and YHB1411-2(4) TNF alpha(2). The distinct features of the soluble antagonist-TNF alpha complex formation among the antagonists may give further insights into their different neutralizing mechanisms and pharmacokinetic profiles. (c) 2007 Elsevier Ltd. All rights reserved.