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Summary Interleukin-2 (IL-2) is a cytokine with pleiotropic effects on the immune system. Systemic IL-2 treatment has produced durable responses in melanoma and renal cancer patients, but unfortunately this is effective only in a fraction of patients. Moreover, IL-2 treatment also engenders serious side effects, which limit its clinical utility. It is now appreciated that IL-2 not only stimulates NK and effector T cells but also has a critical role in the generation and maintenance of regulatory T cells, which act to dampen immune responses. Thus, successful immunotherapy of cancers using IL-2 has to address two fundamentally important issues: (1) how to limit side effects yet be active where it is needed, and (2) how to preferentially activate effector T cells while limiting the stimulation of Tregs. Strategies are now being developed to address these critical obstacles that may lead to a renaissance of IL-2 therapy.

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Summary The ability to alter the cytokine microenvironment has the potential to shape immune responses in many physiological settings, including the immunotherapy of tumours. We set out to develop a general approach in which cytokines could be functionally attenuated until activated. We report the development and initial characterization of fusion proteins in which human or mouse interleukin‐2 (IL‐2), a potent growth factor for immune cells, is joined to a specific IL‐2 inhibitory binding component separated by a protease site. The rationale is that upon cleavage by a protease the cytokine is free to dissociate from the inhibitory component and becomes biologically more available. We describe the successful development of two attenuation strategies using specific binding: the first uses the mouse IL‐2 receptor alpha chain as the inhibitory binding component whereas the second employs a human antibody fragment (scFv) reactive with human IL‐2. We demonstrated that the fusion proteins containing a prostate‐specific antigen or a matrix metalloproteinase (MMP) protease cleavage site are markedly attenuated in the intact fusion protein but had enhanced bioactivity of IL‐2 in vitro when cleaved. Further, we showed that a fusion protein composed of the IL‐2/IL‐2 receptor alpha chain with an MMP cleavage site reduced tumour growth in vivo in a peritoneal mouse tumour model. This general strategy should be applicable to other proteases and immune modulators allowing site‐specific activation of immunomodulators while reducing unwanted side‐effects.

SummaryThe tumour microenvironment is complex containing not only neoplastic cells but also a variety of host cells. The heterogeneous infiltrating immune cells include subsets of cells with opposing functions, whose activities are mediated either directly or through the cytokines they produce. Systemic delivery of cytokines such as interleukin-2 ( IL-2) has been used clinically to enhance anti-tumour responses, but these molecules are generally thought to have evolved to act locally in a paracrine fashion. In this study we examined the effect of local production of IL-2 on the growth and the immune response to B16 melanoma cells. We found that the local production of IL-2 enhances the number of interferon-c-expressing CD8 T and natural killer cells in the tumour, as well as inducing expression of vascular cell adhesion molecule 1 on tumour vessels. These responses were largely absent in interferon-c knockout mice. The expression of IL-2 in the tumour microenvironment decreases tumour growth despite also enhancing Foxp3 + CD4 + regulatory T cells and antiinflammatory cytokines such as IL-10. Higher levels of IL-2 in the tumour microenvironment eliminated the progressive growth of the B16 cells in vivo, and this inhibition was dependent on the presence of either T cells or, to a lesser extent, natural killer cells. Surprisingly however, the B16 tumours were not completely eliminated but instead were controlled for an extended period of time, suggesting that a form of tumour dormancy was established.