Addition of a Viral Immunomodulatory Domain to Etanercept Generates a Bifunctional Chemokine and TNF Inhibitor

Abstract

The inhibition of tumor necrosis factor (TNF) through the use of either antibodies or soluble receptors is a highly effective strategy for the clinical control of chronic inflammatory conditions such as rheumatoid arthritis. Different viruses have similarly exploited this concept by expressing a set of specifically tailored secreted TNF decoy receptors to block host inflammatory responses. Poxviruses have been shown to encode at least two distinct molecules, termed Cytokine response modifier D (CrmD) and CrmB, in which a TNF inhibitor is combined with a chemokine inhibitor on the same molecule. The ectromelia virus CrmD protein was found to be a critical determinant of virulence in vivo, being able to control local inflammation to allow further viral spread and the establishment of a lethal infection. Strikingly, both the TNF and the chemokine inhibitory domains are required for the full activity of CrmD, suggesting a model in which inhibition of TNF is supported by the concomitant blockade of a reduced set of chemokines. Inspired by this model, we reasoned that a similar strategy could be applied to modify the clinically used human TNF receptor (etanercept), producing a generation of novel, more effective therapeutic agents. Here we show the analysis of a set of fusion proteins derived from etanercept by addition of a viral chemokine-binding protein.A bifunctional inhibitor capable of binding to and blocking the activity of TNF as well as a set of chemokines is generated that is active in the prevention of arthritis in a murine disease model.

Deriving Immune Modulating Drugs from Viruses—A New Class of Biologics

“…CrmD expression specifically in intestinal epithelial cells reduces inflammatory cell migration in vitro and decreased gut inflammation in a transgenic model of Crohn's like inflammatory bowel disease [107]. In recent work, the SECRET domain from ECTV was fused to the clinically used human TNF receptor Ig Fc fusion protein (Etanercept) and demonstrated its efficacy in a mouse model of arthritis, and possible improvement under certain conditions [108]. This recent work demonstrates that functional domains of virus-derived immune modulators can be exploited in conjunction with other therapeutic proteins to produce synergistic treatments.…”

The binding and specificity of chemokine binding proteins, through the lens of experiment and computation

“… SECRET Domain Organism: CrmB (variola virus) and CrmD (ectromelia virus). CC CXC C CX3C Inhibits arthritis when combined with a TNF binding protein [ 39 ]. Transgenic expression CrmD attenuates gut inflammation in a mouse model of Crohn's disease, likely due both to the TNF-binding ability and the chemokine binding ability of CrmD [ 40 ].…”

“…As described in section 4 , many studies have investigated amino acids on the chemokine that affect its binding to a CBP, particularly with vCCI [ 61 , 63 , 64 ]. Perhaps more relevant for the practical use of CBP in medical applications are investigations to add chemokine binding functionality to this medically relevant protein itself [ 39 ] or to study changes in CBP that affect their ability to bind particular chemokines. Structural studies have guided mutations in vCCI-like proteins to determine important features for chemokine binding [ 32 ] as well as to allow the successful construction of a variant (R89A) that increases the potency of vCCI [ 34 ].…”

“…Overall, the long co-evolution of viruses and parasites with their hosts has led to a unique family of proteins, CBP, that subvert the mammalian immune system by binding to chemokines, thereby impeding the function of some host immune cells. Due to this binding ability, CBP have been shown to have remarkable anti-inflammatory properties in vitro and in vivo , with work ongoing in several labs to translate their function into useful therapeutics [ 19 , 24 , 39 ]. These CBP often share structural features and bind conserved regions of the chemokines, particularly basic residues and those residues involved in receptor binding and GAG binding.…”

TNF Decoy Receptors Encoded by Poxviruses

“…This chimeric protein TNFR2-SECRET showed high affinity for both TNF and chemokines, achieved protection from TNF effects, and was able to prevent chemokine-induced migration. Notably, this fusion protein was tested in a mouse model of arthritis with similar results to etanercept in delaying the development of clinical signs, even when using a smaller dose than the one of the established drug [ 103 ]. This report constitutes a proof of concept for new therapies involving etanercept-based bifunctional fusion constructs that develop a coordinated blockade of TNF and chemokines.…”