In vivo phage display screening for tumor vascular targets in glioblastoma identifies a llama nanobody against dynactin-1-p150Glued
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Sanne A.M. van Lith1,*, Ilse Roodink1,5,*, Joost J.C. Verhoeff2, Petri I. Mäkinen3, Jari P. Lappalainen3, Seppo Ylä-Herttuala3,4, Jos Raats5, Erwin van Wijk6, Ronald Roepman7, Stef J. Letteboer7, Kiek Verrijp1, William P.J. Leenders1
1Department of Pathology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands
2Department of Radiotherapy, Amsterdam Medical Center, 1100 DD, Amsterdam, The Netherlands
3Department of Biotechnology and Molecular Medicine, University of Eastern Finland, FI-70211, Kuopio, Finland
4Science Service Center and Gene Therapy Unit, Kuopio University Hospital, 70210 Kuopio, Finland
5Modiquest BV, LSP, Molenstraat 110, 5342 CC, Oss, The Netherlands
6Department of Otorhinolaryngology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands
7Department of Genetics, RadboudUMC, 6500 HB, Nijmegen,The Netherlands
*These authors contributed equally to this work
William Leenders, email: [email protected]
Keywords: glioma, stroma, targeting, nanobody, macrophages
Received: June 30, 2016 Accepted: September 19, 2016 Published: September 26, 2016
Diffuse gliomas are primary brain cancers that are characterised by infiltrative growth. Whereas high-grade glioma characteristically presents with perinecrotic neovascularisation, large tumor areas thrive on pre-existent vasculature as well. Clinical studies have revealed that pharmacological inhibition of the angiogenic process does not improve survival of glioblastoma patients. Direct targeting of tumor vessels may however still be an interesting therapeutic approach as it allows pinching off the blood supply to tumor cells. Such tumor vessel targeting requires the identification of tumor-specific vascular targeting agents (TVTAs).
Here we describe a novel TVTA, C-C7, which we identified via in vivo biopanning of a llama nanobody phage display library in an orthotopic mouse model of diffuse glioma. We show that C-C7 recognizes a subpopulation of tumor blood vessels in glioma xenografts and clinical glioma samples. Additionally, C-C7 recognizes macrophages and activated endothelial cells in atherosclerotic lesions. By using C-C7 as bait in yeast-2-hybrid (Y2H) screens we identified dynactin-1-p150Glued as its binding partner. The interaction was confirmed by co-immunostainings with C-C7 and a commercial anti-dynactin-1-p150Glued antibody, and via co-immunoprecipitation/western blot studies. Normal brain vessels do not express dynactin-1-p150Glued and its expression is reduced under anti-VEGF therapy, suggesting that dynactin-1-p150Glued is a marker for activated endothelial cells.
In conclusion, we show that in vivo phage display combined with Y2H screenings provides a powerful approach to identify tumor-targeting nanobodies and their binding partners. Using this combination of methods we identify dynactin-1-p150Glued as a novel targetable protein on activated endothelial cells and macrophages.
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