Comparative Analyses of Complex Formation and Binding Sites between Human Tumor Necrosis Factor-alpha and its Three Antagonists Elucidate their Different Neutralizing Mechanisms

Abstract

Tumor necrosis factor-alpha (TNFα)-blocking therapy, using biologic TNFα 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 TNFα and clinically relevant antagonists, particularly Infliximab (Remicade®) and Etanercept (Enbrel®). Here we characterized the binding stoichiometry and size of soluble TNFα–antagonist complexes and identified energetically important binding sites on TNFα for the three antagonists, Etanercept, Infliximab, and the recently developed humanized TNFα neutralizing monoclonal antibody, YHB1411-2. Size-exclusion chromatography and dynamic light scattering analyses revealed that the three antagonists formed distinct thermodynamically stable TNFα–antagonist complexes that exhibited differences in their size and composition. Energetically important binding residues on TNFα 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 TNFα, which was further confirmed for solubly purified TNFα 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 TNFα (Etanercept₁––TNFα₁), Infliximab₆–TNFα₃, and YHB1411–2₄-TNFα₂. The distinct features of the soluble antagonist–TNFα complex formation among the antagonists may give further insights into their different neutralizing mechanisms and pharmacokinetic profiles.

Introduction

Tumor necrosis factor-alpha (TNFα), also called cachectin, is a pleiotropic cytokine with a broad range of biological activities including cytotoxicity, immune cell proliferation, inflammation, tumorigenesis, and viral replication.1, 2 Like other TNF family ligands³, TNFα (∼17 kDa) exists as a rigid homotrimeric molecule in solution, with each monomer being entirely composed of two antiparallel β-pleated sheets and loops in a standard jellyroll topology.⁴ The homotrimeric form of TNFα is thought to be essential for its biological activity by binding to two distinct TNF receptors (TNFRs), TNFR1 (p55) and TNFR2 (p75), on the cell surface and subsequent signal transduction through the receptor's intracellular domain.³

Even though TNFα is important for normal immunity, elevated TNFα is a key pathological factor in autoimmune inflammatory diseases, such as rheumatoid arthritis, psoriasis, and Crohn's disease.1, 2 Thus blocking pro-inflammatory TNFα from binding to TNFRs has been proven to be an effective therapeutic strategy for these diseases.1, 2 Three biologic TNFα antagonists are clinically available1, 2, Infliximab (Remicade®), Etanercept (Enbrel®), and Adalimumab (Humira®). Infliximab, a chimeric anti-TNFα immunoglobulin (Ig)G1 monoclonal antibody (mAb), has binding specificity for human TNFα.5, 6 Etanercept, a recombinant human soluble TNF receptor, is a dimeric fusion protein generated by linking the extracellular domains of human TNFR2 to the Fc portion of human IgG1.7, 8 Adalimumab is a fully human IgG1 mAb generated from a human anti-TNFα single-chain variable fragment.1, 9 The primary function of these antagonists is to specifically bind to both soluble and/or membrane-bound TNFα, acting as a competitive inhibitor for TNFα binding to cell surface TNFRs and eventually neutralizing its cytotoxic effects in vivo and in vitro.2, 6, 7, 9 To date, there have been few detailed studies of binding characterizations for the complex formation by TNFα and the antagonists, in particular Infliximab and Etanercept.¹⁰ Adalimumab has been reported to form a most stable Adalimumab₃–TNFα₃ complex composed of three molecules of Adalimumab and three molecules of trimeric TNFα.⁹

Here, we characterized the binding stoichiometry and size of three soluble TNFα–antagonist complexes and identified energetically important binding sites on TNFα for the antagonists, Etanercept, Infliximab, and YHB1411-2, to obtain further insight into their specific neutralizing mechanisms. We have recently developed a humanized anti-human TNFα IgG1 mAb, designated as YHB1411-2, from a murine mAb which was produced by the immunization of BALB/c mice with recombinant human TNFα†. Like Infliximab and Etanercept, YHB1411-2 protected L929 cells from TNFα-induced cytotoxicity in vitro and significantly mitigated the development of rheumatoid arthritis in transgenic 197 mice†. Size-exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses revealed dramatically different size distributions of the TNFα–antagonist complexes depending on their relative molar ratios. Energetically important binding sites for the three antagonists on TNFα were mapped through a sequential approach consisting of competition binding assays, fragmentations, loop mutations, and single-point mutation analysis using yeast surface-displayed TNFα, which were further confirmed for solubly purified TNFα mutants by surface plasmon resonance (SPR) analysis. Binding geometry analyses of the antagonists based on the topographically and energetically distinct binding sites within the spatial constraints made it possible to interpret the differences in size and composition of the soluble antagonist–TNFα complexes, which may give further insights into the differences in neutralizing mechanisms.