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      Exploiting a natural conformational switch to engineer an Interleukin-2 superkine

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          Abstract

          The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells 13 . Considerable effort has been invested using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary “high affinity” receptor complex consisting of IL-2, IL-2Rα (termed CD25), IL-2Rβ, and γ c 48 . Naïve T cells express only a low density of IL-2Rβ and γ c, and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ andγ c. Here, using in vitro evolution, we eliminated IL-2’s functional requirement for CD25 expression by engineering an IL-2 “superkine” (termed super-2) with increased binding affinity for IL-2Rβ. Crystal structures of super-2 in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, including a flexible helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in super-2 recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation T cells irrespective of CD25 expression. Compared to IL-2, super-2 induced superior expansion of cytotoxic T cells, leading to improved anti-tumor responses in vivo, and elicited proportionally less expansion of T regulatory cells and reduced pulmonary edema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy.

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          Most cited references15

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          Selective stimulation of T cell subsets with antibody-cytokine immune complexes.

          Interleukin-2 (IL-2), which is a growth factor for T lymphocytes, can also sometimes be inhibitory. Thus, the proliferation of CD8+ T cells in vivo is increased after the injection of a monoclonal antibody that is specific for IL-2 (IL-2 mAb), perhaps reflecting the removal of IL-2-dependent CD4+ T regulatory cells (T regs). Instead, we show here that IL-2 mAb augments the proliferation of CD8+ cells in mice simply by increasing the biological activity of preexisting IL-2 through the formation of immune complexes. When coupled with recombinant IL-2, some IL-2/IL-2 mAb complexes cause massive (>100-fold) expansion of CD8+ cells in vivo, whereas others selectively stimulate CD4+ T regs. Thus, different cytokine-antibody complexes can be used to selectively boost or inhibit the immune response.
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            The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design.

            Interleukin-2 and interleukin-15 have pivotal roles in the control of the life and death of lymphocytes. Although their heterotrimeric receptors have two receptor subunits in common, these two cytokines have contrasting roles in adaptive immune responses. The unique role of interleukin-2 is in the elimination of self-reactive T cells to prevent autoimmunity. By contrast, interleukin-15 is dedicated to the prolonged maintenance of memory T-cell responses to invading pathogens. As discussed in this Review, the biology of these cytokines will affect the development of novel therapies for malignancy and autoimmune diseases, as well as the design of vaccines against infectious diseases.
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              Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells.

              IL-2 immunotherapy is an attractive treatment option for certain metastatic cancers. However, administration of IL-2 to patients can lead, by ill-defined mechanisms, to toxic adverse effects including severe pulmonary edema. Here, we show that IL-2-induced pulmonary edema is caused by direct interaction of IL-2 with functional IL-2 receptors (IL-2R) on lung endothelial cells in vivo. Treatment of mice with high-dose IL-2 led to efficient expansion of effector immune cells expressing high levels of IL-2Rbetagamma, including CD8(+) T cells and natural killer cells, which resulted in a considerable antitumor response against s.c. and pulmonary B16 melanoma nodules. However, high-dose IL-2 treatment also affected immune cell lineage marker-negative CD31(+) pulmonary endothelial cells via binding to functional alphabetagamma IL-2Rs, expressed at low to intermediate levels on these cells, thus causing pulmonary edema. Notably, IL-2-mediated pulmonary edema was abrogated by a blocking antibody to IL-2Ralpha (CD25), genetic disruption of CD25, or the use of IL-2Rbetagamma-directed IL-2/anti-IL-2 antibody complexes, thereby interfering with IL-2 binding to IL-2Ralphabetagamma(+) pulmonary endothelial cells. Moreover, IL-2/anti-IL-2 antibody complexes led to vigorous activation of IL-2Rbetagamma(+) effector immune cells, which generated a dramatic antitumor response. Thus, IL-2/anti-IL-2 antibody complexes might improve current strategies of IL-2-based tumor immunotherapy.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                23 February 2012
                25 March 2012
                26 October 2012
                : 484
                : 7395
                : 529-533
                Affiliations
                [1 ]Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA
                [2 ]Department of Molecular and Cellular Physiology, and Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
                [3 ]Laboratory of Applied Immunobiology, University of Zurich, Zurich, CH-8006, Switzerland
                [4 ]Allergy Unit, Department of Dermatology, University Hospital Zurich, Zurich, CH-8091, Switzerland
                [5 ]Stanford University School of Medicine, Department of Medicine, Division of Immunology and Rheumatology, Stanford, CA 94305, USA
                [6 ]Department of Chemistry, Stanford University, Stanford, CA 94305, USA
                Author notes
                []To whom correspondence should be addressed. kcgarcia@ 123456stanford.edu (K.C.G.) or onur.boyman@ 123456uzh.ch (O.B.)
                [*]

                These authors contributed equally to this work

                Article
                NIHMS358623
                10.1038/nature10975
                3338870
                22446627
                142e460b-0ff9-4566-9d8f-e3636fedab9b

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: R01 AI051321-05 || AI
                Funded by: Howard Hughes Medical Institute :
                Award ID: || HHMI_
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