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.

Keywords::

• bioassay
• cytokines
• latency-associated peptide
• protein design
• targeted delivery

Declaration of interest

We thank our collaborators H. Nagase and A. Mustafa. This work has been funded in part by grants from MRC (G0800795), BHF (PG/09/093), AR UK (19454 and 17559), NMSS (USA) Promise 2010. Yuti Chernajovsky is the Founder and Director of Stealthyx Therapeutics Ltd that develops the latent cytokine technology. He has not received any remuneration in that role.