Latent cytokines: development of novel cleavage sites and kinetic analysis of their differential sensitivity to MMP-1 and MMP-3.
We have engineered a latent mouse interferon beta (mIFNbeta) using the latency associated peptide (LAP) of transforming growth factor beta1 (TGF-beta1) 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. IFNbeta was fused to the LAP using two flexible linkers flanking a matrix metalloproteinase (MMP) cleavage site for the specific release of IFNbeta at disease sites. In order to improve the hydrolysis rate of the cleavage site, 15 different cleavable linkers were introduced in the LAP-mIFNbeta 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.
Vessillier
Sandrine
S
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.
Adams
Gill
G
Chernajovsky
Yuti
Y
D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D016207:Cytokines / Q000096:biosynthesis* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006868:Hydrolysis; D016899:Interferon-beta / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry; D007700:Kinetics; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism*; D020782:Matrix Metalloproteinases / Q000378:metabolism; D051379:Mice; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014162:Transfection
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D016207:Cytokines; D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; C030325:anthrax toxin; D016899:Interferon-beta; D020782:Matrix Metalloproteinases; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1