PEDS Advance Access originally published online on February 11, 2005
Protein Engineering Design and Selection 2004 17(12):829-835; doi:10.1093/protein/gzh097
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Latent cytokines: development of novel cleavage sites and kinetic analysis of their differential sensitivity to MMP-1 and MMP-3
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
1 To whom correspondence should be addressed. E-mail: y.chernajovsky{at}qmul.ac.uk
We have engineered a latent mouse interferon ß (mIFNß) using the latency associated peptide (LAP) of transforming growth factor ß1 (TGF-ß1) 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. IFNß was fused to the LAP using two flexible linkers flanking a matrix metalloproteinase (MMP) cleavage site for the specific release of IFNß at disease sites. In order to improve the hydrolysis rate of the cleavage site, 15 different cleavable linkers were introduced in the LAP–mIFNß 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.
Received August 24, 2004; revised December 17, 2004; accepted December 21, 2004.
Edited by Gideon Schreiber