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Targeting cytokines to inflammation sites

Nature Biotechnology volume 21, pages 1314–1320 (2003)Cite this article

Abstract

To increase the half-life of a cytokine and target its activation specifically to disease sites, we have engineered a latent cytokine using the latency-associated protein (LAP) of transforming growth factor-β1 (TGF-β1) fused via a matrix metalloproteinase (MMP) cleavage site to interferon (IFN)-β at either its N or C terminus. The configuration LAP-MMP-IFN-β resembles native TGF-β and lacks biological activity until cleaved by MMPs, whereas the configuration IFN-β-MMP-LAP is active. LAP provides for a disulfide-linked shell hindering interaction of the cytokine with its cellular receptors, conferring a very long half-life of 55 h in vivo. Mutations of the disulfide bonds in LAP abolish this latency. Samples of cerebrospinal fluid (CSF) or synovial fluid from patients with inflammatory diseases specifically activate the latent cytokine, whereas serum samples do not. Intramuscular injection in arthritic mice of plasmid DNA encoding these constructs demonstrated a greater therapeutic effect of the latent as compared to the active forms.

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Figure 1: Schematic representation of fusion proteins and their putative folding.
Figure 2: Detection and characterization of fusion proteins by immunoprecipitation.
Figure 3: Kinetics of IFN activity after incubation of cell supernatants in medium alone or with synovial fluid from rheumatoid arthritis patients.
Figure 4: Affinity chromatography of LAP-IFN-β and pharmacokinetics.
Figure 5: Inhibition of established collagen-induced arthritis by DNA injection with LAP-IFN-β.

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Notes

  1. *Note: In Figure 3b, the zero time point for IFN-LAP + RASF was omitted. This mistake has been corrected in the HTML version and will appear correctly in print. The correction has been appended to the PDF version available online.

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Acknowledgements

This work was funded by the Multiple Sclerosis Society of Great Britain and Northern Ireland, the Arthritis Research Campaign (UK) and Kinetique Biomedical Research Seed Fund UK. We thank Irene Theoharidou for her expert secretarial assistance, G. Scott and G. Giovannoni for clinical samples, S. Amor for samples from rhesus monkeys, S. Mather and D. Ellison for help with protein iodination, J. Wilson and A. Mustafa for technical help, A. Johnston for the analysis of pharmacokinetic data, L. Layward, D. Willoughby, O.L. Podhajcer and P. Colville-Nash for reviewing the manuscript and H. Nagase for helpful and encouraging discussions.

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  1. Hanna Dreja

    Present address: Institut de Génétique Moléculaire, CNRS, 1919 route de Mende, 34293, Montpellier, Cedex 05, France

  2. Gill Adams and Sandrine Vessillier: These authors contributed equally to this work.

Authors and Affiliations

  1. Bone and Joint Research Unit, William Harvey Research Institute, St. Bartholomew's and Royal London School of Medicine and Dentistry, Queen Mary, University of London, Charterhouse Square, London, EC1M 6BQ, UK

    Gill Adams, Sandrine Vessillier & Yuti Chernajovsky

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Correspondence to Yuti Chernajovsky.

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G.A., H.D. and Y.C. are shareholders in Stealtnyx Therapeutics, Ltd., which holds patents based on this technology.

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Adams, G., Vessillier, S., Dreja, H. et al. Targeting cytokines to inflammation sites. Nat Biotechnol 21, 1314–1320 (2003). https://doi.org/10.1038/nbt888

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