In vivo phage display screening for tumor vascular targets in glioblastoma identifies a llama nanobody against dynactin-1-p150Glued.
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.
Department of Pathology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands.
Sanne A M
SA
van Lith
Department of Pathology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands.|Modiquest BV, LSP, Molenstraat 110, 5342 CC, Oss, The Netherlands.
Ilse
I
Roodink
Department of Radiotherapy, AMC, 1100 DD, Amsterdam, The Netherlands.
Joost J C
JJ
Verhoeff
Department of Biotechnology and Molecular Medicine, University of Eastern Finland, FI-70211, Kuopio, Finland.
Petri I
PI
Mäkinen
Department of Biotechnology and Molecular Medicine, University of Eastern Finland, FI-70211, Kuopio, Finland.
Jari P
JP
Lappalainen
Department of Biotechnology and Molecular Medicine, University of Eastern Finland, FI-70211, Kuopio, Finland.|Science Service Center and Gene Therapy Unit, Kuopio University Hospital, 70210 Kuopio, Finland.
Seppo
S
Ylä-Herttuala
Modiquest BV, LSP, Molenstraat 110, 5342 CC, Oss, The Netherlands.
Jos
J
Raats
Department of Otorhinolaryngology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands.
Erwin
E
van Wijk
Department of Genetics, RadboudUMC, 6500 HB, Nijmegen,The Netherlands.
Ronald
R
Roepman
Department of Genetics, RadboudUMC, 6500 HB, Nijmegen,The Netherlands.
Stef J
SJ
Letteboer
Department of Pathology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands.
Kiek
K
Verrijp
Department of Pathology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands.
William P J
WP
Leenders
D000818:Animals; D001932:Brain Neoplasms / Q000098:blood supply* / Q000628:therapy; D061505:Cell Surface Display Techniques / Q000379:methods*; D000072159:Dynactin Complex / Q000276:immunology*; D042783:Endothelial Cells / Q000502:physiology; D005909:Glioblastoma / Q000098:blood supply* / Q000628:therapy; D006801:Humans; D007150:Immunohistochemistry; D008264:Macrophages / Q000502:physiology; D051379:Mice; D061905:Single-Domain Antibodies / Q000627:therapeutic use*; D020798:Two-Hybrid System Techniques
D000072159:Dynactin Complex; D061905:Single-Domain Antibodies
glioma; macrophages; nanobody; stroma; targeting
Lab Invest. 2010 Jan;90(1):61-7
19823171
Bioessays. 2012 Jun;34(6):489-97
22442051
N Engl J Med. 2014 Feb 20;370(8):699-708
24552317
J Neurosurg. 2009 Jan;110(1):173-80
18834263
Cancer Cell. 2009 Mar 3;15(3):232-9
19249681
J Neuropathol Exp Neurol. 2002 Mar;61(3):215-25; discussion 226-9
11895036
Int J Radiat Oncol Biol Phys. 2004 Sep 1;60(1):310-21
15337570
Nat Rev Drug Discov. 2009 Dec;8(12):935-48
19949400
Curr Opin Oncol. 2008 Nov;20(6):652-61
18841047
Cancer Res. 2009 Jul 1;69(13):5406-14
19491280
Biochem Soc Trans. 2014 Dec;42(6):1576-83
25399573
Neoplasma. 2015 ;62(4):531-40
25997964
Br J Cancer. 2011 Mar 29;104(7):1106-15
21386847
Cancer Res. 2007 Oct 1;67(19):9507-17
17909061
Immunol Invest. 2011;40(3):299-338
21244216
Nat Med. 1995 Jan;1(1):27-31
7584949
Eur J Cell Biol. 2006 Feb;85(2):61-8
16439306
N Engl J Med. 2004 Jun 3;350(23 ):2335-42
15175435
Front Physiol. 2012 Jul 24;3:294
22934045
Mol Imaging Biol. 2008 May-Jun;10(3):167-75
18297364
Neoplasia. 2014 Mar;16(3):221-8
24726142
Cancer Res. 2003 Sep 1;63(17):5408-13
14500375
Mol Biol Cell. 1996 Aug;7(8):1167-80
8856662
Antioxid Redox Signal. 2009 Sep;11(9):2299-306
19331544
Cancer Lett. 2010 Dec 18;299(1):1-10
20889254
N Engl J Med. 2014 Feb 20;370(8):709-22
24552318
J Natl Cancer Inst. 2005 May 18;97(10):733-47
15900043
BMC Cancer. 2009 Dec 16;9:444
20015387
J Biol Chem. 2008 Nov 28;283(48):33611-9
18812314
Radiographics. 2015 Mar-Apr;35(2):455-74
25763729
Int J Cancer. 2016 Aug 15;139(4):729-35
26934471
Cancer Cell. 2009 Mar 3;15(3):220-31
19249680
Brain Pathol. 2008 Jul;18(3):423-33
18371177
J Pathol. 2011 Apr;223(5):626-34
21341272
Endothelium. 2002;9(2):83-7
12200959
Front Biosci. 2006 Jan 01;11:1057-76
16146797
Int J Cancer. 2006 Nov 1;119(9):2054-62
16804907
Curr Pharm Des. 2011;17(23):2402-10
21827415
N Engl J Med. 2008 May 8;358(19):2039-49
18463380
Nat Rev Mol Cell Biol. 2009 Mar;10(3):165-77
19234476
Ther Deliv. 2013 Oct;4(10 ):1321-36
24116915
Annu Rev Cell Dev Biol. 2004;20:759-79
15473859
J Neurooncol. 1997 May;32(3):253-65
9049887
Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14633-8
20679239
J Clin Invest. 2003 May;111(9):1287-95
12727920
J Clin Invest. 1998 Jul 15;102(2):430-7
9664085
Clin Cancer Res. 2004 Sep 15;10 (18 Pt 1):6222-30
15448011
Eur J Immunol. 2011 Feb;41(2):356-65
21268006
Protein Eng Des Sel. 2004 May;17 (5):433-41
15235124
Mol Cancer Ther. 2014 Apr;13(4):996-1007
24493698
Cancer Res. 2004 Apr 15;64(8):2853-7
15087403
Acta Neuropathol. 2007 Nov;114(5):443-58
17805551
Curr Opin Cell Biol. 2015 Aug;35:69-77
26001269
Methods Enzymol. 2000;316:688-704
10800709
Nat Cell Biol. 2008 Dec;10(12):1470-6
19011622
Cancer Res. 2005 Sep 15;65(18):8317-23
16166308
Nat Biotechnol. 2005 Sep;23(9):1105-16
16151404
Biochim Biophys Acta. 2005 Jul 10;1744(3):316-24
15950296
Mol Cancer Ther. 2008 Jan;7(1):71-8
18187807
Cancer Res. 2003 Aug 15;63(16):5046-53
12941833
PLoS One. 2013;8(3):e58262
23484006
Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):407-12
16384919
Blood. 2007 Oct 1;110(7):2440-8
17536014
Atherosclerosis. 2015 Aug;241(2):297-304
26062989
FASEB J. 2006 May;20(7):979-81
16581960
Int J Cancer. 2014 Jun 1;134(11):2663-73
24222574