A Comparative Study Of Matrix Metalloproteinase And Aggrecanase Mediated Release Of Latent Cytokines At Arthritic Joints

Background Latent cytokines are engineered by fusing the latency associated peptide (LAP) derived from transforming growth factor-beta (TGF-beta) with the therapeutic cytokine, in this case interferon-beta (IFN-beta), via an inflammation-specific matrix metalloproteinase (MMP) cleavage site.Objectives To demonstrate latency and specific delivery in vivo and to compare therapeutic efficacy of aggrecanase-mediated release of latent IFN-beta in arthritic joints to the original MMP-specific release.Methods Recombinant fusion proteins with MMP, aggrecanase or devoid of cleavage site were expressed in CHO cells, purified and characterised in vitro by Western blotting and anti-viral protection assays. Therapeutic efficacy and half-life were assessed in vivo using the mouse collagen-induced arthritis model (CIA) of rheumatoid arthritis and a model of acute paw inflammation, respectively. Transgenic mice with an IFN-regulated luciferase gene were used to assess latency in vivo and targeted delivery to sites of disease.Results Efficient localised delivery of IFN-beta to inflamed paws, with low levels of systemic delivery, was demonstrated in transgenic mice using latent IFN-beta. Engineering of latent IFN-beta with an aggrecanase-sensitive cleavage site resulted in efficient cleavage by ADAMTS-4, ADAMTS-5 and synovial fluid from arthritic patients, with an extended half-life similar to the MMP-specific molecule and greater therapeutic efficacy in the CIA model.Conclusions Latent cytokines require cleavage in vivo for therapeutic efficacy, and they are delivered in a dose dependent fashion only to arthritic joints. The aggrecanase-specific cleavage site is a viable alternative to the MMP cleavage site for the targeting of latent cytokines to arthritic joints.