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Title: Engineering tailor-made Interleukin-2 variants through in vitro evolution: potent super agonists with improved developability profiles
Authors: Gertrudis Rojas1*, Ernesto Relova-Hernández1, Annia Pérez-Riverón1, Camila Castro-Martínez1, Osmany Diaz-Bravo1, Yanelys Cabrera Infante1, Tania Gómez1, Joaquín Solozábal1, Dainelys Jimenez1, Maren Schubert2, Marlies Becker2, Beatriz Pérez-Massón1, Dayana Pérez-Martínez1, Osmany Guirola3, Glay Chinea3, Luis Graca4, Stefan Dübel2, Kalet León1, Tania Carmenate1
Gertrudis Rojas: grojas@cim.sld.cu
Author affiliations:
1Center of Molecular Immunology, calle 216 esq 15, apartado 16040, Atabey, Playa, CP 11300, La Habana, Cuba
2Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Department of Biotechnology, Spielmannstraße 7, 38106 Braunschweig, Germany
3 Center of Genetic Engineering and Biotechnology, Ave 31 e/ 158 y 190, apartado 6162, Playa, CP 11300, La Habana, Cuba
4Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, Centro Académico de Medicina de Lisboa, Lisbon, Portugal
Interleukin-2 (IL-2) plays a pivotal role between immune effector responses and tolerance. Protein engineering has created molecules strongly biased towards one of these functions, like H9 superkine, wich has increased reactivity to the beta subunit of the IL-2 receptor and promotes anti-tumor immune responses. The current work was aimed at obtaining a new generation of IL-2-derived super agonists from large libraries of phage-displayed mutated variants. Diverse molecules, representing different structural solutions for improved beta chain binding, were obtained. A common feature was the accumulation of negatively charged amino acids in the segment 81-87, which seems to optimize the electrostatic complementarity with the positively charged beta chain. The new molecules exhibit a wide range of receptor affinities, above the one of the wild-type IL-2, and some even superior to H9 superkine (by up to ten-fold) due to faster association kinetics. These binders are distinguished from H9 by a greatly improved developability profile (high expression levels in mammalian host cells, reduced aggregation propensity and improved thermal stability). The molecular bases behind these favorable properties were tracked to the amino acid at position 92 of IL-2 variants, being Leu the ideal one. Receptor binding capacity and intrinsic stability of the selected IL-2 variants are translated into preferential expansion of effector T cells over T regulatory populations, and enhanced anti-tumor effects in vivo. Phage display is an optimal platform for in vitro evolution of new cytokine variants with fine-tuned receptor binding and overcomes the practical challenges associated with production of these difficult-to-express proteins.
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