The current work illustrates the feasibility of using proteases to activate cytokines in the context of novel fusion proteins. We demonstrated the protease-activated Selleckchem KU-60019 cytokine approach with mouse and human IL-2 and two specific
binding components, the IL-2Rα and an inhibitory scFv. The specific binding component appears important in this strategy as both of the fusion proteins with the specific binding moieties (IL-2 Rα or the scFv) showed enhancement of IL-2 activity comparing the cleaved with the uncleaved fusion proteins (Figs 2 and 4). In contrast, an approach that relied solely on steric hindrance using IL-2 and Mip1α resulted in a slight decrease in IL-2 activity after protease cleavage, supporting the importance of specific binding (data not shown). Moreover, we could also show that the biological activity of IL-2 is attenuated > 50-fold in the intact fusion protein (IL-2/MMPcs/IL-2Rα fusion protein) when comparing the cleaved find more and uncleaved fusion proteins. We further show that the protease-activated cytokine can function with different protease cleavage sites in a cassette fashion. We successfully used cleavage sites tailored for different proteases, including PSA, MMP9 and MMP2, in the context of an IL-2/IL-2Rα fusion protein. These proteases are relevant to tumour immunotherapy as the MMP family of proteases plays an
important role in the development of a variety of tumours19,39,40 and because tumour cells, as well as host cells such as activated macrophages, can contribute to over-expression of MMPs at the tumour site.41–43 The prostate-expressed protease
PSA is also potentially useful for the protease-activated cytokine approach. It is produced almost exclusively by prostate epithelial cells, and the cancers that arise from them. Whereas PSA can be found in serum, its expression is typically low even in cancer patients (ng/ml range) and it can complex with serum protease inhibitors.44 The prostate is typically removed or ablated as part of the treatment for prostate cancer,45 MG 132 but metatstatic prostate cancer cells often continue to express PSA and so could be targets for a PSA-activated fusion protein. Our finding that cleavage of the fusion protein results in increased biological activity might initially be surprising because the IL-2 could remain bound to the alpha chain or the scFv after cleavage. Moreover, even if dissociated, the inhibitory component could potentially rebind free IL-2. Indeed, others have speculated that IL-2 receptor alpha chain shed by cells such as activated T cells may have a regulatory role in dampening the immune response.46 However, there is probably competition for the free IL-2 derived from the fusion protein by cellular IL-2 receptors. In this light, it is useful to consider that the alpha chain used in the fusion protein has a Kd of approximately 10 nm.
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- The histogram in Figure 4A illustrates the time course and displa