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

A. M. Levin, D. L. Bates, A. M. Ring, J. T. Lin, L. Su, C. Krieg, G. R. Bowman, P. Novick, V. S. Pande, H. E. Kohrt, O. Boyman, C. G. Fathman, K. C. Garcia

Nature, volume 484, pages 529-33 (2012)

It has long been known that a protein called IL-2 can help stimulate an immune response to fight AIDS or cancer, so in theory giving people with diseases like immune deficiencies IL-2 could be tremendously helpful. In practice, however, giving them IL-2 often leads to severe heart and lung problems. To find a better solution, collaborators at Stanford designed a variant of IL-2 that can stimulate an immune response without causing any side effects. However, they couldn’t understand how it worked because the two proteins had almost identical structures! Using Folding@home, we showed that IL-2 is a relatively floppy protein while our collaborators’ variant is locked into a structure that is poised to stimulate an immune response.

Dr. Garcia’s lab was the driving force behind this work, but we are very excited to be able to make a contribution to it! For more information, please see http://medicalxpress.com/news/2012-03-scientists-boost-potency-side-effects.html

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.