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P007. Differential cleavage of anti-tumor necrosis factor-alpha agents by matrix metalloproteinase (MMP)-10 and MMP-12 in inflammatory bowel disease

P. Biancheri1, A. Di Sabatino1, I. Joe-Njoku2, L. Rovedatti1, N. Ahmad2, G.R. Corazza1, T.T. MacDonald2

1First Department of Medicine, Fondazione IRCCS Policlinico S. Matteo, University of Pavia, Pavia, Italy; 2Centre for Immunology and Infectious Disease, BICMS, Barts and The London School of Medicine and Dentistry, London, United Kingdom

Background and Aims: A considerable proportion of inflammatory bowel disease (IBD) patients fail to respond to anti-tumor necrosis factor (TNF)-alpha treatment. MMPs cleave human IgG proximally to the hinge region releasing a single chain 32kDa Fc monomer. Anti-TNF-alpha agents are thought to mediate their activity in inflamed mucosa, a site rich in activated MMPs. We therefore investigated the cleavage of anti-TNF-alpha agents by MMP-10 (stromelysin-2) and MMP-12 (macrophage metalloelastase).

Methods: Activated recombinant human MMP-10 or MMP-12 were incubated with infliximab, adalimumab, certolizumab pegol or etanercept. IgG Fc portion or Ig kappa light chains were detected by immunoblotting on the supernatants collected at 0, 3, 6, 10 and 24 h. The ability of MMP-10- or MMP-12-treated antibodies to neutralise TNF-alpha was tested using a luciferase reporter cell line. Mucosal homogenates from inflamed areas of 6 IBD patients and from normal gut of 6 control subjects were depleted of endogenous IgG and incubated for 24 h with the four drugs, then immunoblotted for human IgG Fc portion or kappa light chains.

Results: Certolizumab pegol was not cleaved by both MMP-12 and MMP-10. Infliximab and adalimumab were degraded only by MMP-12, releasing 32kDa Fc monomers and F(ab)2. Etanercept was cleaved by both MMP-10 and MMP-12, although Fc monomers were found only after MMP-12 digestion. All the four drugs inhibited luciferase production in TNF-alpha-stimulated HeLa 57A cells. When the anti-TNF-alpha agents were pre-incubated with activated MMP-10 or MMP-12, only etanercept lost its ability to inhibit luciferase production. None of the anti-TNF-alpha agents were degraded by the control mucosa, whereas only etanercept was cleaved by IBD tissue.

Conclusions: Adalimumab and infliximab are functioning as F(ab)2 fragments after degradation by MMP-12, whereas etanercept is unable to neutralise soluble TNF-alpha after MMP-cleavage. These findings provide a possible explanation for the clinical inefficacy of etanercept in IBD patients.