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PEDS Advance Access published online on February 11, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzh097
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© The Author (2005). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org
Received August 24, 2004
Revised December 17, 2004
Accepted December 21, 2004

Article

Latent cytokines: development of novel cleavage sites and kinetic analysis of their differential sensitivity to MMP-1 and MMP-3

Sandrine Vessillier 1, Gill Adams 1, and Yuti Chernajovsky 1*

1 Bone and Joint Research Unit, William Harvey Research Institute, Barts and the London, Queen Mary's School of Medicine and Dentistry, University of London, Charterhouse Square, London EC1M 6BQ, UK

* To whom correspondence should be addressed.
Yuti Chernajovsky, E-mail: y.chernajovsky{at}qmul.ac.uk


  Abstract

We have engineered a latent mouse interferon {beta} (mIFN{beta}) using the latency associated peptide (LAP) of transforming growth factor {beta}1 (TGF-{beta}1) to protect the cytokine and avoid its interaction with its receptors. This approach improves the pharmacokinetic properties and reduces the pleiotropic effects limiting the current therapeutic use of cytokines. IFN{beta} was fused to the LAP using two flexible linkers flanking a matrix metalloproteinase (MMP) cleavage site for the specific release of IFN{beta} at disease sites. In order to improve the hydrolysis rate of the cleavage site, 15 different cleavable linkers were introduced in the LAP-mIFN{beta} construct. The kinetic parameters relative to the linker cleavage by MMP-1 and MMP-3 were measured by an ELISA method. Among the modifications done, one of the constructs bearing the activation site of pro-MMPs was the best substrate for both enzymes. The introduction of a hydrophilic sequence derived from the furin cleavage site of the anthrax toxin protective antigen increased the sensitivity to MMP-3 to up to 29-fold. These data suggest that this strategy could be useful for improving the effectiveness of the delivery and targeting of protein therapeutics.

Keywords: cytokine targeting; inflammatory disease; kinetic parameters; matrix metalloproteinase; specific activation.
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