Methods of therapeutic cytokine delivery are addressed. The latent cytokine technology holds significant advantages over other methods of drug delivery by providing simultaneously increased half-life and localised drug delivery without systemic effects. Cytokines that failed clinical trials should be reassessed using this delivery system.

Gene therapy is a powerful approach to treat disease locally. However, if the therapeutic target is intracellular, the therapeutic will be effective only in the cells where the therapeutic gene is delivered. We have engineered a fusion protein containing an intracellular inhibitor of the transcription factor NF-κB pathway that can be effectively secreted from producing cells. This fusion protein is cleaved extracellularly by metalloproteinases allowing release of a protein transduction domain (PTD) linked to the NF-κB inhibitor for translocation into neighboring cells. We show that engineered molecules can be efficiently secreted (>80%); are cleaved with matrix metalloprotease-1; inhibit NF-κB driven transcription in a biological assay with a human reporter cell line; and display significant inhibition in mouse paw inflammation models when delivered by lentivirus or secreting cells. No inhibition of NF-κB transcription or therapeutic effect was seen using molecules devoid of the PTD and NF-κB inhibitory domains. By creating a fusion protein with an endogenous secretion partner, we demonstrate a novel approach to efficiently secrete PTD-containing protein domains, overcoming previous limitations, and allowing for potent paracrine effects.

CitationLatency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-. This is a pre-or post-print of an article published in Mullen, L., Rigby, A., Sclanders, M., Adams, G., Mittal, G., Colston, J., Fatah, R., Subang, C., Foster, J., Francis-West, P., Köster, M., Hauser, H., Layward, L., Vessillier, S., Annenkov, A., Al-Izki, S., Pryce, G., 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-, 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- 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.

In order to generate neural stem cells with increased ability to survive after transplantation in brain parenchyma we developed a chimeric receptor (ChR) that binds to myelin oligodendrocyte glycoprotein (MOG) via its ectodomain and activates the insulin-like growth factor receptor type 1 ‎‎(IGF1R) signalling cascade. Activation of this pro-survival pathway in response to ligand broadly available in the brain might increase neuroregenerative potential of transplanted precursors. The ChR was produced by fusing a MOG-specific single ‎chain antibody with the extracellular boundary of the IGF1R transmembrane segment. The ChR is expressed on the cellular surface, predominantly as a monomer, and is not N-glycosylated. To show MOG-dependent functionality of the ChR, neuroblastoma cells B104 expressing this ChR were stimulated with monolayers of cells expressing recombinant MOG. The ChR undergoes MOG-dependent tyrosine phosphorylation and homodimerisation. It promotes insulin and IGF-independent growth of the oligodendrocyte progenitor cell line CG4. The proposed mode of the ChR activation is by MOG-induced dimerisation which promotes kinase domain transphosphorylation, by-passing the requirement of conformation changes known to be important for IGF1R activation. Another ChR, which contains a segment of the β-chain ectodomain, was produced in an attempt to recapitulate some of these conformational changes, but proved non-functional.