Abstract
Tumor necrosis factor is a major pro-inflammatory cytokine which triggers various physiological consequences by binding to and trimerizing its receptors, and has been the single most sought-after drug target for intervening autoimmune diseases such as rheumatoid arthritis and psoriasis. However, current TNF-α blockers, including soluble receptor-Fc fusion and therapeutic antibodies, are all dimeric in structure, whereas their target TNF-α itself is homotrimeric in nature. Here we describe the development of a trivalent soluble TNF receptor and show that it is a more potent than the dimeric TNF receptor decoys in inhibiting TNF-α signaling both in vitro and in vivo. The process involves gene fusion between a soluble receptor TNFRII with a ligand binding domain and a trimerization tag from the C-propeptide of human collagen (Trimer-Tag), which is capable of self-assembly into a covalently linked trimer. We show that the homotrimeric soluble TNF receptor (TNFRII-Trimer) produced with such method is more potent in ligand binding kinetics and cell based bioassays, as well as more efficacious in attenuating collagen-induced arthritis (CIA) in a mouse model than its dimeric TNFRII-Fc counterpart. Thus, this work demonstrates the proof of concept of Trimer-Tag and provides a new platform for rational designs of next generation biologic drugs.
Highlights
Tumor necrosis factor (TNF-α) is a major pro-inflammatory cytokine produced by human body to combat infections, and it provides necessary protections for the host[1]
The result clearly revealed that TNFRII-Trimer formed a disulfide bond-linked homotrimer as predicted by its higher molecular weight under non-reducing condition and by size-exclusion HPLC (SEC-HPLC) than that of TNFRII-Fc (Fig. 2c,d) and its purity reached over 95% (Fig. 2d)
The results indicated that TNFRII-Trimer and TNFRII-Fc when administrated via i.p., with dosages being both at 12.5 mg/kg showed similar pharmacokinetic profiles as shown in the drug concentration-time profile in semi-logarithmic scale (Fig. 5a,b)
Summary
Tumor necrosis factor (TNF-α) is a major pro-inflammatory cytokine produced by human body to combat infections, and it provides necessary protections for the host[1]. Such trimeric receptor decoys theoretically should have a much higher affinity to their trimeric ligands than their dimeric counterparts such as etanercept (Fig. 1) Such rationally designed soluble trimeric receptor analogs could increase the clinical benefits as well as potentially lower the amount or frequency of the drug injections for each patient. One of the feasible plans to improve the current design of the TNF-α antagonist molecule may be creating a secreted form of homo-trimeric soluble receptors, or proteins with similar biological activities, containing canonical docking sites, with greater affinities for the natural homotrimeric ligands than that of dimeric TNFRII-Fc. we have developed a soluble trimeric TNFR antagonist which acquires greater affinity to its natural homo-trimeric ligands in the hope for a significant increase of its clinical benefits
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