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The use of cytokines as therapeutic agents is important, given their potent biological effects. However, this very potency, coupled with the pleiotropic nature and short half-life of these molecules, has limited their therapeutic use. Strategies to increase the half-life and to decrease toxicity are necessary to allow effective treatment with these molecules.A number of strategies are used to overcome the natural limitations of cytokines, including PEGylation, encapsulation in liposomes, fusion to targeting peptides or antibodies and latent cytokines. Latent cytokines are engineered using the latency-associated peptide of transforming growth factor-β to produce therapeutic cytokines/peptides that are released only at the site of disease by cleavage with disease-induced matrix metalloproteinases. The principles underlying the latent cytokine technology are described and are compared to other methods of cytokine delivery. The potential of this technology for developing novel therapeutic strategies for the treatment of diseases with an inflammatory-mediated component is discussed.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.

Background Latent cytokines are engineered by fusing the latency associated peptide (LAP) derived from transforming growth factor-β (TGF-β) with the therapeutic cytokine, in this case interferon-β (IFN-β), via an inflammation-specific matrix metalloproteinase (MMP) cleavage site. Objectives To demonstrate latency and specific delivery in vivo and to compare therapeutic efficacy of aggrecanase-mediated release of latent IFN-β in arthritic joints to the original MMP-specific release. Methods Recombinant fusion proteins with MMP, aggrecanase or devoid of cleavage site were expressed in CHO cells, purified and characterised in vitro by Western blotting and anti-viral protection assays. Therapeutic efficacy and half-life were assessed in vivo using the mouse collagen-induced arthritis model (CIA) of rheumatoid arthritis and a model of acute paw inflammation, respectively. Transgenic mice with an IFN-regulated luciferase gene were used to assess latency in vivo and targeted delivery to sites of disease. Results Efficient localised delivery of IFN-β to inflamed paws, with low levels of systemic delivery, was demonstrated in transgenic mice using latent IFN-β. Engineering of latent IFN-β with an aggrecanase-sensitive cleavage site resulted in efficient cleavage by ADAMTS-4, ADAMTS-5 and synovial fluid from arthritic patients, with an extended half-life similar to the MMP-specific molecule and greater therapeutic efficacy in the CIA model. Conclusions Latent cytokines require cleavage in vivo for therapeutic efficacy, and they are delivered in a dose dependent fashion only to arthritic joints. The aggrecanase-specific cleavage site is a viable alternative to the MMP cleavage site for the targeting of latent cytokines to arthritic joints.

ObjectiveTo facilitate the targeting to inflammation sites of small anti-inflammatory peptides, with short half-lives, by fusion with the latency-associated peptide (LAP) of transforming growth factor β1 through a cleavable matrix metalloproteinase (MMP) linker. This design improves efficacy, overcoming the limitations to their clinical use.MethodsWe generated latent forms of vasoactive intestinal peptide (VIP), α-melanocyte-stimulating hormone (MSH) and γ3MSH by fusion to LAP through an MMP cleavage site using recombinant DNA technology. The biological activities of these latent therapeutics were studied in vivo using monosodium urate (MSU)-induced peritonitis and collagen-induced arthritis (CIA) models. We assessed gene therapy and purified protein therapy.ResultsThe recruitment of the polymorphonuclear cells induced by MSU injection into mouse peritoneal cavity was reduced by 35% with γ3MSH (1 nmol), whereas administration of a much lower dose of purified latent LAP–MMP–γ3MSH (0.03 nmol) attenuated leucocyte influx by 50%. Intramuscular gene delivery of plasmids coding LAP–MMP–VIP and LAP–MMP–αMSH at disease onset reduced the development of CIA compared with LAP–MMP, which does not contain any therapeutic moiety. Histological analysis confirmed a significantly lower degree of inflammation, bone and cartilage erosion in groups treated with LAP–MMP–VIP or LAP–MMP–αMSH. Antibody titres to collagen type II and inflammatory cytokine production were also reduced in these two groups.ConclusionIncorporation of small anti-inflammatory peptides within the LAP shell and delivered as recombinant protein or through gene therapy can control inflammatory and arthritic disease. This platform delivery can be developed to control human arthritides and other autoimmune diseases.

Cytokines are mediators of cell communication. Their therapeutic use requires frequent high doses to achieve effective local biological levels. However, the clinical use of some cytokines is limited because of their pleiotropism, which can result in unwanted side effects. Here, we review novel protein engineering technologies that overcome these limitations and enable the targeting of cytokines to specific sites. One such technology uses antibody-based recognition to direct the cytokine to a particular tissue, and another creates encapsulated latent cytokines that are released only at the site of disease. The latter method requires the overexpression of matrix-metalloproteinases, thereby exploiting the severity of the pathological process to regulate drug delivery. Because these technologies are based on the expression of fusion proteins, their application can be extended to other biologicals and can be delivered by gene therapy.

Animal models of autoimmune disease have been developed that mimic some aspects of the pathophysiology of human disease. These models have increased our understanding of possible mechanisms of pathogenesis at the molecular and cellular level and have been important in the testing, development and validation of new immunotherapies. The susceptibility to develop disease in the majority of these models is polygenic as is the case in humans. The exceptions to this rule are gene knock outs and transgenic models of particular genes which, in particular genetic backgrounds, have also contributed to the understanding of single gene function and their possible contribution to pathogenesis. Gene therapy approaches that target immune functions are being developed with encouraging results, despite the polygenic nature of these diseases. Basically this novel immuno-genetic therapy harnesses the knowledge of immunology with the myriad of biotechnological breakthroughs in vector design and delivery. Autoimmune disease is the result of genetic dysregulation which could be controlled by gene therapy. Here we summarize the genetic basis of these human diseases as well as some of the best characterized murine models. We discuss the strategies for their treatment using immuno- and gene therapy.