Engineering of TIMP-3 as a LAP-fusion protein for targeting to sites of inflammation

Abstract

Tissue inhibitor of metalloproteinase (TIMP)-3 is a natural inhibitor of a range of enzymes that degrade connective tissue and are involved in the pathogenesis of conditions such as arthritis and cancer. We describe here the engineering of TIMP-3 using a novel drug-delivery system known as the ‘LAP technology’. This involves creating therapeutic proteins in fusion with the latency-associated peptide (LAP) from the cytokine TGF-? to generate proteins that are biologically inactive until cleavage of the LAP to release the therapy. LAP-TIMP-3 was successfully expressed in mammalian cells and the presence of the LAP resulted in a 14-fold increase in the quantity of recombinant TIMP-3 produced. LAP-TIMP-3 was latent until release from the LAP by treatment with matrix metalloproteinase when it could inhibit proteases of the adamalysins and adamalysins with thrombospondin motifs families, but not matrix metalloproteinases, indicating that this version of TIMP-3 is a more specific inhibitor than the native protein. There was sufficient protease activity in synovial fluid from human joints with osteoarthritis to release TIMP-3 from the LAP fusion. These results demonstrate the potential for development of TIMP-3 as a novel therapy for conditions where upregulation of catabolic enzymes are part of the pathology.