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

Abstract

We have engineered a latent mouse interferon β (mIFNβ) using the latency associated peptide (LAP) of transforming growth factor β1 (TGF-β1) 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. IFNβ was fused to the LAP using two flexible linkers flanking a matrix metalloproteinase (MMP) cleavage site for the specific release of IFNβ at disease sites. In order to improve the hydrolysis rate of the cleavage site, 15 different cleavable linkers were introduced in the LAP-mIFNβ 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. © The Author 2005. Published by Oxford University Press. All rights reserved.