科研之友
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摘要
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.
- 出版日期2014-9
