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) cells1,2,3. Considerable effort has been invested in 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 IL-2Rγ4,5,6,7,8. Naive T cells express only a low density of IL-2Rβ and IL-2Rγ, and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ and IL-2Rγ. Here, using in vitro evolution, we eliminated the functional requirement of IL-2 for CD25 expression by engineering an IL-2 ‘superkine’ (also called super-2) with increased binding affinity for IL-2Rβ. Crystal structures of the IL-2 superkine 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, reducing the flexibility of a helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in the IL-2 superkine recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation of T cells irrespective of CD25 expression. Compared to IL-2, the IL-2 superkine induced superior expansion of cytotoxic T cells, leading to improved antitumour responses in vivo, and elicited proportionally less expansion of T regulatory cells and reduced pulmonary oedema. 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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Change history
16 April 2012
In the legend to Supplementary Movie 2 'wild-type IL-2' was changed to 'D10 IL-2 superkine'.
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Acknowledgements
The authors gratefully acknowledge W. Leonard, R. Levy and R. Schwendener for reagents and discussion. This work was supported by NIH-RO1AI51321 (to K.C.G.), PP00P3-128421 from the Swiss National Science Foundation and KFS-02672-08-2010 from the Swiss Cancer League (both to O.B.), NIH R01-GM062868 (to V.S.P.), MRI-R2 (this award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)) (to V.S.P.), NIH-AR050942 (to J.T.L.), NIH U01 DK078123 (to C.G.F.), and NIH U19 AI 082719 (to C.G.F.). A.M.R. was supported by the Stanford Medical Scientist Training Program (NIH-GM07365). K.C.G. is an Investigator of the Howard Hughes Medical Institute.
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Contributions
A.M.L. performed in vitro evolution and contributed to preparation of the manuscript. D.L.B. produced recombinant proteins, determined crystal structures, and carried out surface plasmon resonance analysis. A.M.R. carried out cellular and signalling assays, biophysical measurements and contributed to preparation of the manuscript. C.K. carried out in vivo experiments, analysed data and contributed to preparation of the manuscript; M.E.R. carried out in vivo experiments in mice. I.M. analysed cell-signalling data. G.R.B., P.N. and V.S.P. carried out and analysed molecular dynamics simulations. J.T.L., L.S. and C.G.F. performed and analysed T-cell signalling experiments. O.B. designed and supervised in vivo experiments, analysed data and contributed to preparation of the manuscript. K.C.G. conceived of the project, analysed data, supervised execution of the project, and prepared the manuscript.
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K.C.G., A.M.L. and A.M.R. declare competing financial interests due to submission of a pending patent application describing the IL-2 superkine. O.B. declares competing financial interests due to being a shareholder of Nascent Biologics Inc.
Supplementary information
Supplementary Information
This file contains Supplementary Table 1, Supplementary Figures 1-12 and the legend for Supplementary Movies 1-2. (PDF 6476 kb)
Supplementary Movie 1
In this movie we see atomistic molecular dynamics simulations of wild-type IL-2 (see Supplementary Information file for movie legend). (MOV 956 kb)
Supplementary Movie 2
In this movie we see atomistic molecular dynamics simulations of D10 IL-2 superkine (see Supplementary Information file for movie legend). (MOV 499 kb)
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Levin, A., Bates, D., Ring, A. et al. Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’. Nature 484, 529–533 (2012). https://doi.org/10.1038/nature10975
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DOI: https://doi.org/10.1038/nature10975
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