Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’

• Published in: Nature (London) Vol. 484; no. 7395; pp. 529 - 533
• Main Authors: Levin, Aron M., Bates, Darren L., Ring, Aaron M., Krieg, Carsten, Lin, Jack T., Su, Leon, Moraga, Ignacio, Raeber, Miro E., Bowman, Gregory R., Novick, Paul, Pande, Vijay S., Fathman, C. Garrison, Boyman, Onur, Garcia, K. Christopher
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 26-04-2012; Nature Publishing Group
• Subjects: 631/154/309/2144; 631/250/127; 631/45/535; Animals; Binding Sites; Biological and medical sciences; Cell Line; Cell Proliferation; CHEM; Crystallography, X-Ray; Cytokines; Directed Molecular Evolution; Engineers; Health aspects; Humanities and Social Sciences; Humans; Immunomodulators; Immunotherapy; INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Interleukin-2; 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; Interleukins; Killer Cells, Natural - immunology; letter; Medical sciences; Methods; Mice; Mice, Inbred C57BL; Models, Molecular; Molecular Dynamics Simulation; multidisciplinary; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - immunology; Mutant Proteins - pharmacology; Mutation; Neoplasm Transplantation; Neoplasms - drug therapy; Neoplasms - immunology; Pharmacology. Drug treatments; Phosphorylation; Physiological aspects; Protein Conformation; Protein Engineering; Science; STAT5 Transcription Factor - metabolism; Surface Plasmon Resonance; T-Lymphocytes - cytology; T-Lymphocytes - immunology; Tissue engineering; Yeast; United States; Antineoplastic agent; Biological fixation; Immunostimulant agent; Immunostimulation; Interleukin 2; Toxicity; Cytokine; Affinity; Genetic engineering; Property structure relationship; Conformation; Biological receptor
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature10975

Although IL-2 has been studied for its immune-stimulating activity against metastatic cancer, its side effects have limited its clinical use; here, an engineered IL-2 ‘superkine’ is shown to have increased activity, particularly in inducing antitumour T cells, but fewer side effects.
Engineering an interleukin-2 'superkine'
Chris Garcia and colleagues elucidate the molecular mechanism that underlies the sensitization of T cells to the immunostimulatory cytokine interleukin-2 (IL-2). They use this information to engineer a single-chain IL-2 superkine that functions independent of its α-receptor (IL-2Rα or CD25). This new superkine is more efficacious than IL-2 in inducing antitumour T-cell responses and has fewer toxic side effects.
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
1
,
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,
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-2Rγ
4
,
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,
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,
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,
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.