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An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis

Abstract

Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl 'decoy receptor' that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high-affinity Axl variant caused structural alterations in side chains across the Gas6-Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc fusion, the engineered decoy receptor bound Gas6 with femtomolar affinity, an 80-fold improvement compared to binding of the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Moreover, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.

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Figure 1: Engineering and characterization of receptor-based Axl antagonists.
Figure 2: Structural basis for high-affinity binding.
Figure 3: Design and characterization of Axl Fc fusion proteins.
Figure 4: MYD1 Fc inhibits Axl activation and downstream signaling in skov3.ip cells.
Figure 5: Sequestration of Gas6 by MYD1 Fc inhibits metastasis.

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Acknowledgements

The authors thank E. Hohenester (Imperial College London) for his generous gift of the Gas6 plasmid and helpful discussions regarding protein production, L.L. Zheng for technical assistance with protein production and purification and the Stanford FACS Core Facility for assistance with the flow cytometric sorting. All in vivo and ex vivo imaging was conducted in the Stanford Small Animal Imaging Facility. Portions of this research were performed at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the US Department of Energy Office of Basic Energy Sciences. This work was supported by the Wallace H. Coulter Translational Research Grant Program (J.R.C. and A.J.G.), the Stanford Chemistry, Engineering & Medicine for Human Health (ChEM-H) Institute (J.R.C. and I.I.M.), Stanford Bio-X and ARCS Graduate Fellowships (M.S.K.), US National Institutes of Health (NIH) grants CA-088480 and CA-67166 (A.J.G. and Y.R.M.), NIH National Institute of General Medical Sciences Training Grant T32 GM008412 and the Siebel Graduate Fellowship (D.S.J.).

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M.S.K., Y.R.M., D.S.J., S.K., I.I.M., A.J.G. and J.R.C. designed the research; M.S.K. and D.S.J. performed protein engineering and characterization; M.S.K., S.K. and I.I.M. did the crystallography; M.S.K. and Y.R.M. conducted the in vivo experiments; all of the authors analyzed the data; and M.S.K. prepared the manuscript with input from all co-authors.

Corresponding authors

Correspondence to Amato J Giaccia or Jennifer R Cochran.

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Competing interests

Stanford University holds patent number US8618254 B2, 'Biologic inhibitors for therapeutic targeting the receptor tyrosine kinase AXL', which is related to the work described in this paper, with M.S.K., Y.R.M., D.S.J., A.J.G. and J.R.C. named as inventors. A.J.G. is a founder of Ruga, a company that discovers and develops targeted therapeutics in oncology that has licensed this patent.

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Kariolis, M., Miao, Y., Jones, D. et al. An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis. Nat Chem Biol 10, 977–983 (2014). https://doi.org/10.1038/nchembio.1636

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