SFB-824 Symposium Alpbach Anti-angiogenic strategies: from basic science to clinical applications Dr. med. Andreas Fischer Joint Research Division Vascular Biology Medical Faculty Mannheim, University of Heidelberg, and German Cancer Research Center (DKFZ) Heidelberg (Director: Prof. Dr. Hellmut G. Augustin) Alpbach, 13.6.2010 fischer@angiogenese.de E15.5 mouse embryos Coll. with E. Lang, P.M. Jakob; Dep. of Physics, Würzburg [Bruker Avance 500 spectrometer (11.7 Tesla; 500 MHz)]
Physiological angiogenesis and blood vessels National Cancer Institute www.cancer.gov Pathophysiological angiogenesis and blood vessels In situ tumor www.targetvegf.com tumor stimulates angiogenesis Tumor growth and metastasis
Strategies to inhibit tumor angiogeneis More than 100 substances are currently in different clinical trials Anti-angiogenic substances Substances for vascular targeting MMP Inhibitors Pharmacological inhibitor Signalling antiinvasive substances Antiadhesive substances substances with unknown targets Thalidomid 2-Methoxyestradiol AGM1470 CAI α-vegf signalling α-integrin Endogenous inhibitors Angiostatin Endostatin Tumstatin Interferon-α Interleukin-12 Thrombospondin α-vegf-r; α-basalmembrane Immunotargeting-substances Pharmacological substances Combretastatin CM101 VEGF and its receptors VEGF-A: Receptors 1 & 2 Angiogenesis VEGF-B & PlGF: Receptor 1 Pathological angiogenesis (?) VEGF-C & VEGF-D: Receptor 3 (& 2) Lymphangiogenesis
upstream factors: mtor inhibitors Torisel (Temsirolismus) Afinitor (Everolimus) Strategies to inhibit VEGF VEGF production: (Antisense, Aptamer) Macugen (Pegaptanib) Veglin (sirna) Aflibercept (VEGF-Trap) Tumor cell Growth factors produced by tumors, e.g. VEGF Avastin (Bevacizumab) Lucentis (Ranibizumab) soluble receptors antibodies VEGF-receptors Sutent (Sunitinib) Nexavar (Sorafenib) Votrient (Pazopanib) Recentin (Cediranib) Axitinib Vatalanib XL184 X Blockade of receptor activation DC101 IMC-18F1 Endothelial cells VEGF neutalizing antibody Avastin (Bevacizumab) Avastin is a recombinant humanized antibody against VEGF-A VEGF Avastin binds and neutralizes VEGF Avastin Yuan et al., PNAS 1996
VEGF neutralizing antibody Avastin (Bevacizumab) Phase III trial (Roche and Genentech) 815 patients with advanced colorectal carcinomas Irinotecan/5-FU/FA (IFL) ± Avastin Increase of survival rate from 15.6 months to 20.3 months Progression free survival survival Hurwitz et al, NEJM, 2003 Bevacizumab therapy in metastatic breast cancer Avastin increases progression-free survival but not overall survival Miller et al NEJM 2007; 357: 2666
Approved indications for Bevacizumab (Avastin ) Late stage: colorectal cancer breast cancer non-small cell lung cancer kidney cancer (RCC) gliobastoma (only in USA) A series of phase III studies for other indications are ongoing. Soluble VEGF receptors - VEGF-Trap (Aflibercept) Fusion protein consisting of VEGFR1 and VEGFR2 domains binds to VEGF-A, VEGF-B, PlGF Aflibercept (VEGF-Trap ) (Regeneron / Bayer-Schering) Results of Phase III studies for lung and colorectal cancer expected in 2011 Phase III study for macula degeneration ongoing Holash et al., PNAS. 2002
Blocking VEGF-receptors VEGF VEGF binding domain Sunitinib Inhibition by: cell membrane P- P- -P -P Tyrosine kinase activity Sorafenib (Nexavar,Bayer-Schering) Sunitinib (Sutent, Pfizer) Pazopanib (Votrient, GSK) Cediranib (Recentin, AstraZeneca) Vatalanib (Novartis/Schering) Axitinib (Pfizer) XL184 (Exelixis) Angiogenesis Problems with Receptor tyrosine kinase inhibitors: Specificity BRK Kit PDGFR VEGFR courtesy: Christoph Schächtele (www.proqinase.com)
Sorafenib Sorafenib (Nexavar, BAY 43-9006, Bayer-Schering / Onyx) Inhibits VEGFR2, VEGFR3, PDGFRβ, Raf kinase (MAPK pathway), c-kit, SCF, Flt-3, RET Approved for renal cell carcinoma and hepatocellular carcinoma Wilhelm et al., Nat Reviews Drug Discovery, (5) 2006 Drevs. UNI-MED Verlag Sorafenib Phase-III study (advanced renal cell carcinoma): Sorafenib prolongs progression-free survival Wilhelm et al. Nature Reviews Drug Discovery 5, 835 844 (October 2006) doi:10.1038/nrd2130
mtor inhibitors mtor is critical for cell division, survival, metabolism and also for induction of VEGF expression Inhibitors of mtor should affect tumor cells and angiogenesis Everolimus (Afinitor (oncology), Certican (transplantation), RAD001, Novartis) approved for advanced renal cell carcinoma (EU 8/2009) oral application also used on coronary stents to prevent stenosis Temsirolimus (Torisel, CCI-779, Wyeth) approved for advanced renal cell carcinoma (EU 11/2007) i.v. application VTA: vascular targeting agents Concept: Directed against existing tumor vessels VTAs cause disruption or occlusion of tumor vessels esp. in the center of the tumor massive hypoxia and necrosis of tumor cells. However: not all tumor cells are dying target endothelial tubulin Thorpe., Clin. Cancer Res.; Vol. 10, 415-427; 2004
Effects of anti-angiogenic therapy in humans Treatment of malignant diseases: 1) Monotherapy leads to decrease in vessel density but is almost ineffective in terms of survival, progression-free survival WHY? 2) In contrast, anti-angiogenic therapy can synergize with classical chemotherapy (and radiation). WHY? Normal colorectal mucosa Adjacent colorectal tumor From: Molls and Vaupel, eds. Blood Perfusion and Microenvironment of Human Tumors, 2002 Vessel normalization If anti-angiogenic therapy would lead to massive destruction of tumor vessels: -more hypoxia - less perfusion decreased delivery of chemotherpeutics to tumor cells However: the rate of perfusion increases! during anti-angiogenic treatment Concept: Anti-angiogenic therapy leads to pruning of immature vessels, but also to a normalization of the remain vessels Jain, Science, Vol 307, 7 Jan 2005
Vessel normalization Please note: VEGF is a potent permeability factor! Jain, Science, Vol 307, 7 Jan 2005 Jain, Science, Vol 307, 7 Jan 2005
Resistance against anti-angiogenic therapy Still poorly understood, possible reasons: Production of higher VEGF levels and/or of other vascular growth factors or receptors: VEGF-B, PlGF, FGF, PDGF, VEGFR3, Nrp1, Nrp2, inflammatory cytokines, Normalized vessels (J Exp Med. 2010 Mar 15;207(3):491-503.) Tumor infiltration with bone marrow derived myeloid cells (CD11b + Gr1 + ) Targets: Angiopoetins Hypoxia sensors Notch signaling Pericytes PlGF MMPs Integrins Some novel strategies Fischer and Gessler, Nucl Acids Res. 2007 Inhibits endothelial proliferation, migration, sprouting, branching, tip cell formation Promotes arterial fate, represses venous fate
The tip cell / stalk cell concept tip cells: sense a VEGF gradient with their filopodia migrate towards the source of VEGF do rarely proliferate form no lumen stalk cells: follow the tip cells proliferate to elongate the sprout form a lumen Gerhardt et al., The Journal of Cell Biology, Volume 161, Number 6, June 23, 2003 1163 1177 Which cell will become the tip cell? Why do the neighbouring cells become stalk cells? VEGF induces the Dll4 gene in tip cells Dll4 activates the Notch receptor in neighbouring cells Notch signaling prevents the adoption of a tip cell fate Phng and Gerhardt; Dev Cell. 2009 Feb;16(2):196-208. Roca and Adams; Genes Dev. 2007 Oct 15;21(20):2511-24.
Notch signaling during cardiovascular development Hey1/L double knockout Loss of Hey2: - cardiac hypertrophy, ectopic ANF expression - increased length of the AV canal -congentialheart defects Loss of Hey1/L -congentialheart defects - decreased EMT Loss of Dll4, Notch1 or Hey1/2 - lethal angiogenesis defects - defective arterial differentiation - decreased EMT -heartdefects Hey1/2 double knockout control Gessler et al., Curr Biol., 2002; Fischer et al., Genes & Dev. 2004; Fischer et al., Mamm Genome, 2004; Fischer et al., Mol Cell Biol, 2005; Ruthenberg, Fischer et al, Development, 2006; Diez et al, Exp Cell Res, 2007; Fischer et al, Circ Res, 2007 Is Dll4-Notch signaling a potential target for antiangiogenic therapy? Nature. Vol 444 21/28 December 2006
Tools to block Notch signaling Gamma-secretase inhibitors Monoclonal antibodies against ligands and receptors Soluble ligands Nature, Vol 464 15 April 2010 Nature. 2010 Feb 11;463(7282):E6-7. Pathological angiogenesis Tumors Rheumatoid arthritis Ischemic Retinopathies Age-dependent macular degeneration Chronic transplant rejection Psoriasis Atherosclerosis Restenosis Obesity Pulmonary hypertension Chronic respiratory diseases Cerebral ischemia Dementia Vascular malformations Newsletter Anti-Angiogenese, Nr. 2, 6/2008
anti-angiogenic therapy of non-malignant diseases Age-related macula degeneration [subfoveal choroidal neovascularisation] Ranibizumab (Lucentis, Novartis): recombinant, humanized, monoclonal antibody (Fab fragment). Monthly intravitreal injections: reduced risk of visual acuity loss Effect of VEGF inhibition on neovascular AMD in patients Rosenfeld et al NEJM October 6th 2006 Alternative: Pegaptanib (Macugen, Pfizer, OSI Pharmaceuticals) Aptamer against VEGF Intravitreal injections every 6 weeks
Obesity and potential new applications: Y. Cao and potential new applications: Vascular malformations Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagic telangiectasia. (Nat Med. 2010 Apr;16(4):420-8.) Sorafenib as a potential drug for cerebral cavernous malformations (Wüstehube et al., PNAS, in press) radiopaedia.org
CCM: Cerebral cavernous malformations berry-like bulk of enlarged microvessels often lacking pericytes 0.5% of human population is affected (range 1:200 1:1000) symptoms: headaches, seizures, focal neurological deficits, hemorrhage Cause: loss of CCM1, CCM2 or CCM3 in endothelial cells Lancet Neurol 2007; 6: 237 44 J. Medical Genetics CCM: A novel animal model Xenotransplantation of human endothelial cells (HUVEC) into SCID mice (Alajati et al.2008 Nature Methods and Laib et al.2009 Nature Protocols) Stable lentiviral shrna mediated knockdown of CCM1 in HUVEC
CCM: Anti-angiogenic therapy? Summary Anti-angiogenic therapy is effective in non-malignant and malignant diseases For tumor treatment anti-angiogenic therapy works best in combination with classical chemotherapy Anti-angiogenic therapy causes partial regression of tumor vessels (and partially also healthy vessels, esp. fenestrated capillaries) Anti-angiogenic therapy leads to vessel normalization better perfusion of tumors, less hypoxia, better drug delivery
Fischer laboratory Joycelyn Wüstehube Sven Liebler Gordian Adam Caroline Berger Rene Brütsch Stefanie Herberich Wen-Jen Yang Anja Telzerow DKFZ Hellmut Augustin Arne Bartol Univ. of Würzburg Manfred Gessler Svenja Meierjohann Toni Wagner Neurosurgery, Essen Ulrich Sure Yuan Zhu Human Genetics, Greifswald Ute Felbor Grenoble Daniel Bouvard Corinne Albiges-Rizo Funding: DFG (Fi1568/1-1; SFB/TR23; GRK880) Universität Heidelberg (Med. Fak. Mannheim; Exzellenzinitiative) DKFZ - Israel - Kooperation