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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) 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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Figure 1: In vitro evolution of human IL-2 variants with high affinity for IL-2Rβ.
Figure 2: Basis of affinity enhancement for IL-2Rβ from structural and molecular dynamics characterization of the D10 IL-2 superkine.
Figure 3: Functional properties of the IL-2 superkine on human NK cells in vitro.
Figure 4: Functional and antitumour activities of the IL-2 superkine in vivo.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under accession codes 3QAZ and 3QB1.

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.

Author information

Authors and Affiliations

Authors

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.

Corresponding authors

Correspondence to Onur Boyman or K. Christopher Garcia.

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Competing interests

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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