Comparisons of affinities, avidities, and complement activation of adalimumab, infliximab, and etanercept in binding to soluble and membrane tumor necrosis factor
Introduction
The central role of TNF in the pathogenesis of several immune-mediated inflammatory diseases has been established by the therapeutic efficacies of the TNF antagonists adalimumab, infliximab, and etanercept. Adalimumab and infliximab are IgG1 mAb — fully human and chimeric, respectively. Etanercept is a fusion protein composed of a human IgG1 Fc fragment linked to 2 extra-cellular portions of the human p75 TNF receptor (TNF-R)2. Adalimumab and etanercept are administered subcutaneously; infliximab is administered intravenously.
Adalimumab, infliximab, and etanercept have demonstrated efficacy in the treatment of rheumatoid arthritis (RA), psoriatic arthritis, ankylosing spondylitis, and psoriasis. By contrast, only adalimumab and infliximab have demonstrated efficacy for Crohn's disease [1]. This difference in clinical efficacy profiles has attracted great interest, but its scientific basis remains uncertain [2], [3], [4], [5], [6]. The possible explanations for the difference may be conceived as relating either to differences in the abilities of TNF antagonists to achieve adequate serum concentrations (i.e., pharmacokinetic differences); differences in their respective abilities to achieve therapeutic concentrations in inflammatory microenvironments (i.e., differences in tissue penetration); or differences in their abilities to bind to TNF and induce therapeutic effects (i.e., mechanistic differences). In addition, only etanercept binds to lymphotoxin [7].
TNF molecules are homotrimers that exist either as membrane TNF (mTNF) on cell surfaces, or, following cleavage of mTNF by TNF-alpha-converting enzyme (TACE), as free molecules in solution. Both mTNF and soluble TNF can be bound by adalimumab, infliximab, and etanercept, and all 3 agents can inhibit binding of mTNF or soluble TNF to TNF-R [8], [9], [10], [11]. When these agents bind to mTNF, they have the potential to induce Fc-mediated effects, such as complement-dependent cytotoxicity (CDC) [10], [11], [12], although these effects have not been demonstrated in patients. In addition, TNF antagonists may trigger intracellular signals in mTNF-bearing cells. This “reverse signaling” may inhibit cell proliferation, modify cytokine production, induce apoptosis, or, conversely, promote activation [10], [11], [13], [14], [15], [16], [17], as reviewed by Tracey et al. [6]. The binding properties of TNF antagonists may be different for soluble TNF vs. mTNF.
The understanding of binding interactions between TNF and TNF antagonists has been advanced by many in vitro experiments, but several uncertainties remain. First, studies using current technology to directly compare the binding rates and affinities of adalimumab, infliximab, and etanercept for soluble TNF and mTNF have not been reported. Second, studies of binding to mTNF, and of its functional consequences, have often used transfected cell lines that express a TACE-resistant, mutated form of mTNF. Such cells might not accurately reflect the behavior of normal human cells. Finally, studies of binding properties have not always clearly distinguished between univalent and multivalent interactions for binding to soluble TNF or mTNF.
Gaps in knowledge have made it difficult to assess how the binding properties of adalimumab, infliximab, and etanercept, as well as their respective abilities to activate complement, relate to effects observed with these agents in patients. The present studies were conducted to address these topics. The results indicate that adalimumab, infliximab, and etanercept have similar binding properties for both soluble TNF and mTNF, and that none of them induced CDC in normal, activated human mononuclear cells. These findings elucidate the mechanistic effects of these agents, and suggest that TNF-binding properties, per se, do not suffice in explaining their different clinical efficacy profiles.
Section snippets
TNF antagonists, antibody reagents, and normal cells
Infliximab (Remicade® lot 03E023A; Centocor, Inc., Malvern, PA, USA) and etanercept (Enbrel®, lot D040590; Immunex Corporation, Thousand Oaks, CA, USA) were purchased in powder form and reconstituted according to the manufacturers' instructions. Adalimumab was obtained from the Abbott Laboratories' Drug Supply Group in a 50-mg/ml-solution prepared for clinical use (Humira® lot AYP23H; Abbott Laboratories, Abbott Park, IL, USA). Adalimumab, infliximab, and etanercept were each radio-labeled with
Intrinsic affinity of binding to soluble TNF
Intrinsic binding affinities of TNF antagonists were determined by a surface plasmon resonance instrument, which measured the rates at which soluble TNF bound to and dissociated from single Ag-binding sites of surface-bound adalimumab or infliximab, and single molecules of surface-bound etanercept (Fig. 1A). The Kon for etanercept was 5.64 × 106 M− 1 s− 1, which was greater (i.e., faster) than for the association rates for either adalimumab or infliximab by a factor of 3.3 and 2.6, respectively (
Discussion
This study compared the TNF-binding properties of adalimumab, infliximab, and etanercept. Biologically active TNF is a homotrimer, with soluble and membrane-anchored forms, both of which were studied here. For each form of TNF, 2 experimental approaches were used to evaluate binding by TNF antagonists: first, one that focused on molecular interactions between single molecules of TNF antagonist and TNF; and, second, one that used physical conditions or cell types more representative of the in
Disclosures
All authors are employees of Abbott Laboratories.
Acknowledgments
The authors thank Drs. S. J. Rozzo, P. B. Sugerman, M. M. Okun, and M. J. McIlraith for their critical involvement and helpful suggestions. They also thank Dr. Ling Santora for the preparation and characterization of reagents used in this study. The authors acknowledge Michael A. Nissen, ELS, for his editorial support in the development of this manuscript. All acknowledged are employees of Abbott Laboratories.
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