Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-β

Abstract

Objectives:Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-β with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes.Methods:Mature cytokines cloned downstream of LAP and a MMP cleavage site were expressed in 293T cells and assessed for latency and biological activity by Western blotting and bioassay.Results:We demonstrate here that fusion proteins of TGF-β, erythropoietin, IL-1ra, IL-10, IL-4, BMP-7, IGF1 and IL-17 were rendered latent by fusion to LAP, requiring cleavage to become active in respective bioassays. As further proof of principle, we also show that delivery of engineered TGF-β can inhibit experimental autoimmune encephalomyelitis and that this approach can be used to efficiently deliver cytokines to the brain and spinal cord in mice with this disease.Conclusions: The latent cytokine approach can be successfully applied to a range of molecules, including cytokines of different molecular structure and mass, growth factors and a cytokine antagonist.