Abstract
The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth1,2. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage3,4 at a juxtamembrane site that otherwise lies buried within the quiescent NRR5,6. Subsequent intramembrane proteolysis catalysed by the γ-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer7, making the Notch pathway a compelling target for new drugs. Although γ-secretase inhibitors (GSIs) have progressed into the clinic8, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways9 and cause intestinal toxicity10, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently.
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Acknowledgements
We acknowledge the Notch scientists at Genentech and Exelixis for support throughout this project. In particular, we thank the following contributors at Exelixis: S.-H. Lin, L. Doukhan, S. Weiss, N. Benin and R. Mercer for protein production, purification and analysis, R. Funke for cloning and sequencing and D. Buckley and J. Greve for scientific discussions and guidance. We thank the Genentech protein expression group, R. Vij and J.-A. Hongo for help with antibody characterization, F. Bazan and C. Sanchez-Irizarry for stimulating discussions and critically reading the manuscript, J. Eastham-Anderson for morphometric analysis of the intestines, Y. Shang for making mutant Notch1 expression plasmids, S. Seshagiri for MT-3 sequencing, E. Quan for initial work with T-ALL lines, S. Chaudhuri for cloning and antibody characterization, and G. Plowman for insights and guidance. We appreciate the important contributions of F. de Sauvage, who helped initiate this collaborative project and provided guidance throughout. The Advanced Light Source and Berkeley Center for Structural Biology are supported by the Department of Energy, National Institutes of Health, and the National Institute of General Medical Sciences.
Author Contributions Y.W., Y.C. and S.S. generated the phage display antibodies and performed the in vitro binding experiments. G.P.d.L. purified and crystallized the NRR1-Fab complex. S.G.H. solved and analysed the structure, made the structure figures and wrote the corresponding section of the paper, D.F. and R.V. performed the MT-3 xenograft experiment, X.W. performed the Calu-6 and HM7 xenograft experiments, J.R. performed the HUVEC experiments and M.Y. supervised these experiments; Ji.Z. and C.A.C. analysed intestinal pathology; C.C.-H., L.C. and T.J.H. performed the experiments characterizing antibody activity in vitro and in vivo, C.C.-H. also analysed CD31 staining in the Calu-6 model, L.C. also analysed signalling from mutant receptors, T.J.H. also analysed T-ALL growth, including the HPB-ALL xenograft, L.C. and A.S. analysed T-ALL growth in vitro, G.J.D. performed the domain swap experiments, D.S.-R. performed the neonate retina experiments and R.J.W. supervised these experiments; Je.Z. purified and characterized antigens, R.C. developed in vitro signalling assays, P.H. developed cell-binding assays and L.K. supervised the work at Exelixis. C.W.S. supervised the experiments and wrote the paper.
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[COMPETING INTERESTS: Y.W., C.C.-H., L.C, T.J.H., G.P.d.L., Y.C., D.F., R.V., X.W., J.R., D.S.-R., G.J.D., A.S., S.S., R.J.W., M.Y., Ji.Z., C.A.C., S.G.H. and C.W.S. are or were employed by Genentech, Je.Z., R.C., P.H. and L.K. are employed by Exelixis, and both employers have commercial interests in some of the molecules described.]
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Wu, Y., Cain-Hom, C., Choy, L. et al. Therapeutic antibody targeting of individual Notch receptors. Nature 464, 1052–1057 (2010). https://doi.org/10.1038/nature08878
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DOI: https://doi.org/10.1038/nature08878
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