Elsevier

Cytokine

Volume 45, Issue 2, February 2009, Pages 124-131
Cytokine

Differences in binding and effector functions between classes of TNF antagonists

https://doi.org/10.1016/j.cyto.2008.11.008Get rights and content

Abstract

There are currently two Food and Drug Administration-approved classes of biologic agents that target tumor necrosis factor-α (TNF-α): anti-TNF monoclonal antibodies (mAbs) (adalimumab and infliximab), and soluble TNF receptors (etanercept). This study examined the ability of the TNF antagonists to: (1) bind various polymorphic variants of cell surface-expressed Fc receptors (FcγRs) and the complement component C1q, and (2) mediate Ab-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) killing of cells expressing membrane-bound TNF (mTNF) in vitro. Both mAbs and the soluble TNF receptor demonstrated low-level binding to the activating receptors FcγRI, FcγRIIa, and FcγRIIIa, and the inhibitory receptor FcγRIIb, in the absence of exogenous TNF. However, upon addition of TNF, the mAbs, but not etanercept, showed significantly increased binding, in particular to the FcγRII and FcγRIII receptors. Infliximab and adalimumab induced ADCC much more potently than etanercept. In the presence of TNF, both mAbs bound C1q in in vitro assays, but etanercept did not bind C1q under any conditions. Infliximab and adalimumab also induced CDC in cells expressing mTNF more potently than etanercept. Differences in the ability to bind ligand and mediate cell death may account for the differences in efficacy and safety of TNF antagonists.

Introduction

There are currently two Food and Drug Administration (FDA)-approved classes of biologic therapeutic agents that target tumor necrosis factor (TNF) bioavailability: soluble TNF receptors (etanercept) and anti-TNF monoclonal antibodies (mAbs) (adalimumab and infliximab). Although all three agents bind to TNF, they differ in clinical efficacy and safety [1], [2]. Etanercept is FDA-approved for the treatment of patients with moderate to severe active rheumatoid arthritis (RA), polyarticular juvenile RA, psoriatic arthritis, ankylosing spondylitis, and chronic moderate to severe plaque psoriasis [3]. Adalimumab is indicated for the treatment of moderate to severe RA, polyarticular juvenile RA, psoriatic arthritis, ankylosing spondylitis, psoriasis, and moderate to severe Crohn’s disease [4]. Infliximab is approved for treatment of moderate to severe RA, psoriatic arthritis, ankylosing spondylitis, moderate to severe ulcerative colitis, and moderate to severe Crohn’s disease [5].

All three currently approved agents bear the Fc portion of complement-activating human immunoglobulin G1 (IgG1). The Fc region is a native component of the mAbs, whereas, to construct etanercept, the Fc region is genetically fused to a soluble portion of the TNF receptor p75.

Fc regions bind to Fc receptors (FcγRs), a family of Ig-binding molecules with different patterns of expression on immune cells, including monocytes, macrophages, granulocytes, natural killer (NK) cells, B cells, and platelets [6]. There are several different classes of FcγR in humans: FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (an inhibitory receptor) and FcγRIII (CD16) [7]. Types of FcγRIII include high affinity (V/V) and low affinity (F/F) polymorphic variants, associated with certain autoimmune disorders [8], [9]. FcγRI receptors have high affinity for IgG1 and can bind monomeric IgG1. The other classes have lower affinity and generally bind aggregated antibody–antigen complexes [10], resulting in trapping and clearance of immune complexes. In general, triggering of activating FcγRs leads to effector functions: opsonization, phagocytosis, and downstream cytokine release and immunomodulation. Activation through FcγRIII (CD16) on NK cells can result in triggering of lytic mechanisms through Ab-dependent cellular cytotoxicity (ADCC), in which cells coated with antigen:antibody complexes are targeted for killing by NK cells via lytic and apoptotic cascades [6]. Signal triggering through the inhibitory FcγRIIb receptor results in inhibition of antibody secretion and down-modulation of B cell response and antibody production [7].

The cytoplasmic FcR common gamma chain protein associates with the transmembrane and intracellular regions of FcγRI and FcγRIII [7], and possesses tyrosine-based activation motifs (ITAM motifs) for signaling through the FcγR complex. It also induces optimal cell surface expression of these FcR complexes [7], [11]. FcγRII receptors induce signaling events through ITAM motifs (on activating FcγRIIa) or ITIM motifs (on inhibitory FcγRIIb) within their own gamma-like cytoplasmic domains.

IgG1 Fc also binds to the classical complement component C1q, and can trigger the lytic complement cascade. In addition, C1q has also been shown to increase FcR-based immune complex uptake and phagocytosis [12].

There are many clinically relevant outcomes to individual or combination Fc-mediated events. A balance between stimulation by activating and inhibitory Fc receptors may determine outcome of immune response, based on affinity for receptors, and levels of receptor expression [7], [11]. The role of the TNF antagonist Fc regions in mediating FcγR-related events has not been thoroughly investigated. In previous in vitro studies [13], we have shown that the mAbs, but not the p75 TNF soluble receptor etanercept, form large protein complexes. The close proximity of many Fc regions may enable mAbs to cross-link and activate low affinity FcγRII and FcγRIII. In this report, we investigate the ability of the TNF antagonists to bind to membrane-bound TNF (mTNF), FcγR and C1q, and to induce cell death via ADCC and complement-mediated cytotoxicity (CDC)-mediated pathways.

Section snippets

TNF antagonists

Etanercept was supplied in a buffer containing 25 mM sodium phosphate, 25 mM L-arginine, 98 mM sodium chloride, and 1% sucrose. Adalimumab was supplied in 105 mM sodium chloride, 5.5 mM monobasic sodium phosphate dihydrate, 8.6 mM dibasic sodium phosphate dihydrate, 1 mM sodium citrate, 6.2 mM citric acid monohydrate, 66 mM mannitol, 0.1% polysorbate 80, pH 5.2. Infliximab was supplied in 1.6 mM monobasic sodium phosphate monohydrate, 3.4 mM dibasic sodium phosphate dehydrate, 5% sucrose, 0.005%

Binding of TNF antagonists to mTNF

Disruptions in the normal levels of TNF can be associated with inflammation (elevated TNF) and susceptibility to infection (low levels of TNF). Therefore, differences in mTNF binding characteristics among the TNF antagonists may have implications for the efficacy and safety of these drugs in patients with inflammatory disease. Flow cytometry was used to assess the binding of TNF antagonists to mTNF-expressing MT-3 cells. Both classes of TNF antagonists, the anti-TNF mAbs (adalimumab and

Discussion

Pharmacodynamic differences between the three available anti-TNF drugs may account for differences in their clinical safety and efficacy profiles; however, the complexity of TNF-mediated biologic effects has made it challenging to elucidate the precise mechanisms that may be responsible for these differences. This present study contributes to our understanding of the cellular interactions that may, in part, account for the immunomodulatory effects of these agents.

Consistent with other studies

Conflict of interest disclosures

All authors were employees at Amgen Inc. at the time of writing this manuscript.

Acknowledgments

We thank Julia R. Gage, Ph.D. and Abie Craiu, Ph.D., on behalf of Amgen Inc., for assistance with preparation of the manuscript.

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