title: Exploiting a natural conformational switch to engineer an interleukin-2 'superkine'.
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.
authors.affiliation: Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Aron M
authors.initials: AM
authors.lastname: Levin
authors.forename: Darren L
authors.initials: DL
authors.lastname: Bates
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Ring
authors.forename: Carsten
authors.initials: C
authors.lastname: Krieg
authors.forename: Jack T
authors.initials: JT
authors.lastname: Lin
authors.forename: Leon
authors.initials: L
authors.lastname: Su
authors.forename: Ignacio
authors.initials: I
authors.lastname: Moraga
authors.forename: Miro E
authors.initials: ME
authors.lastname: Raeber
authors.forename: Gregory R
authors.initials: GR
authors.lastname: Bowman
authors.forename: Paul
authors.initials: P
authors.lastname: Novick
authors.forename: Vijay S
authors.initials: VS
authors.lastname: Pande
authors.forename: C Garrison
authors.initials: CG
authors.lastname: Fathman
authors.forename: Onur
authors.initials: O
authors.lastname: Boyman
authors.forename: K Christopher
authors.initials: KC
authors.lastname: Garcia
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D049109:Cell Proliferation; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D006801:Humans; D007167:Immunotherapy; D007376:Interleukin-2 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D053645:Interleukin-2 Receptor alpha Subunit / Q000737:chemistry / Q000172:deficiency / Q000276:immunology / Q000378:metabolism; D053646:Interleukin-2 Receptor beta Subunit / Q000737:chemistry / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D009154:Mutation; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000188:drug therapy / Q000276:immunology; D010766:Phosphorylation; D011487:Protein Conformation; D015202:Protein Engineering; D050799:STAT5 Transcription Factor / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology
chemical_list: D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D053646:Interleukin-2 Receptor beta Subunit; D050505:Mutant Proteins; D050799:STAT5 Transcription Factor