Abstract
BackgroundLigand-targeted approaches have proven successful in improving the therapeutic index of a number of drugs. We hypothesized that the specific targeting of TNF-alpha antagonists to inflamed tissues could increase drug efficacy and reduce side effects.ResultsUsing uteroglobin (UG), a potent anti-inflammatory protein, as a scaffold, we prepared a bispecific tetravalent molecule consisting of the extracellular ligand-binding portion of the human TNF-alpha receptor P75 (TNFRII) and the scFv L19. L19 binds to the ED-B containing fibronectin isoform (B-FN), which is expressed only during angiogenesis processes and during tissue remodeling. B-FN has also been demonstrated in the pannus in rheumatoid arthritis. L19-UG-TNFRII is a stable, soluble homodimeric protein that maintains the activities of both moieties: the immuno-reactivity of L19 and the capability of TNFRII to inhibit TNF-alpha. In vivo bio-distribution studies demonstrated that the molecule selectively accumulated on B-FN containing tissues, showing a very fast clearance from the blood but a very long residence time on B-FN containing tissues. Despite the very fast clearance from the blood, this fusion protein was able to significantly improve the severe symptomatology of arthritis in collagen antibody-induced arthritis (CAIA) mouse model.ConclusionsThe recombinant protein described here, able to selectively deliver the TNF-alpha antagonist TNFRII to inflamed tissues, could yield important contributions for the therapy of degenerative inflammatory diseases.
Highlights
Ligand-targeted approaches have proven successful in improving the therapeutic index of a number of drugs
This paper reports on the production and characterization of the fusion protein L19-UG-TNFRII and its ability to selectively accumulate in vivo in B containing fibronectin isoform (B-FN)-containing tissues and to significantly improve the objective signs of arthritis in the in collagen antibody-induced arthritis (CAIA) mouse model
The purified fusion protein was analyzed in SDSPAGE (Figure 1C) in which it migrated as a monomer of the expected size of about 60 KDa in reducing conditions, while it migrated as a homodimer with an apparent molecular mass of about 120 kDa under nonreducing conditions
Summary
Ligand-targeted approaches have proven successful in improving the therapeutic index of a number of drugs. It has been demonstrated that TNF-alpha plays a role in various inflammatory diseases such as rheumatoid arthritis (RA), ankylosing spondylitis, psoriasis, Crohn’s disease, psoriatic arthritis, and juvenile idiopathic arthritis [1] This observation led to the generation of TNF-alpha antagonists such as Infliximab (trade name REMICADE) a mouse-human chimeric monoclonal, etanercept (trade name ENBREL) a TNF-alpha receptor p75-IgG fusion protein and adalimumab (trade name HUMIRA) a fully human IgG1 monoclonal antibody. While TNF-alpha present in the bloodstream can be annulled without particular problems, its neutralization in inflamed tissues requires the effective penetration and an optimal concentration of the drug in the diseased tissues To achieve this high doses of the TNFalpha inhibitor would be needed, but these would be accompanied by severe side effects [3]. The fast blood clearance of a small molecule is very likely to reduce its therapeutic performance
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