Exploiting a Natural Conformational Switch to Engineer an interleukin-2 'Superkine'

Nature. 2012 Mar 25;484(7395):529-33. doi: 10.1038/nature10975.

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. 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-2Rγ. 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.

Publication types

  • Research Support, American Recovery and Reinvestment Act
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Cell Line
  • Cell Proliferation
  • Crystallography, X-Ray
  • Directed Molecular Evolution*
  • Humans
  • Immunotherapy
  • Interleukin-2 / chemistry*
  • Interleukin-2 / genetics
  • Interleukin-2 / immunology*
  • Interleukin-2 / pharmacology
  • Interleukin-2 Receptor alpha Subunit / chemistry
  • Interleukin-2 Receptor alpha Subunit / deficiency
  • Interleukin-2 Receptor alpha Subunit / immunology
  • Interleukin-2 Receptor alpha Subunit / metabolism
  • Interleukin-2 Receptor beta Subunit / chemistry
  • Interleukin-2 Receptor beta Subunit / metabolism
  • Killer Cells, Natural / immunology
  • Mice
  • Mice, Inbred C57BL
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Mutant Proteins / chemistry*
  • Mutant Proteins / genetics
  • Mutant Proteins / immunology*
  • Mutant Proteins / pharmacology
  • Mutation
  • Neoplasm Transplantation
  • Neoplasms / drug therapy
  • Neoplasms / immunology
  • Phosphorylation
  • Protein Conformation
  • Protein Engineering*
  • STAT5 Transcription Factor / metabolism
  • Surface Plasmon Resonance
  • T-Lymphocytes / cytology
  • T-Lymphocytes / immunology

Substances

  • Interleukin-2
  • Interleukin-2 Receptor alpha Subunit
  • Interleukin-2 Receptor beta Subunit
  • Mutant Proteins
  • STAT5 Transcription Factor

Associated data

  • PDB/3QAZ
  • PDB/3QB1