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A Novel Therapeutic Antibody Targeting Dll4 Modulates Endothelial Cell Function and Angiogenesis in Vivo

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A Novel Therapeutic Antibody Targeting Dll4 Modulates Endothelial Cell Function and Angiogenesis in Vivo Published OnlineFirst June 7, 2012; DOI: 10.1158/1535-7163.MCT-11-1027 Molecular Cancer Therapeutic Discovery Therapeutics MEDI0639: A Novel Therapeutic Antibody Targeting Dll4 Modulates Endothelial Cell Function and Angiogenesis In Vivo David W. Jenkins1, Sarah Ross2, Margaret Veldman-Jones2, Ian N. Foltz3, Brandon C. Clavette3, Kathy Manchulenko4, Cath Eberlein2, Jane Kendrew2, Philip Petteruti1, Song Cho4, Melissa Damschroder4, Li Peng4, Dawn Baker2, Neil R. Smith2, Hazel M. Weir2, David C. Blakey2, Vahe Bedian1, and Simon T. Barry2 Abstract The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand–receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody cross- reacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell–fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway. Mol Cancer Ther; 11(8); 1650–60. Ó2012 AACR. Introduction extracellular and transmembrane subunits. Receptor– The Notch signaling cascade is an evolutionarily con- ligand interactions between neighboring cells triggers a served pathway that has been implicated in cell fate series of proteolytic cleavages by enzymes such as TNFa g determination, stem cell maintenance, and differentiation converting enzyme and -secretase, which results in the in many tissues during development (1, 2). To date, 4 generation of Notch intracellular domain. The Notch Notch receptors (Notch1–4) and 5 ligands [Jagged1/2 and intracellular domain translocates to the nucleus and binds Delta-like ligand (Dll) 1/3/4] have been identified in to transcription factors, ultimately causing the activation mammals. Notch receptors exist in cell membranes as of downstream target genes, including those of the hairy/ heterodimers, composed of 2 noncovalently associated enhancer of split (Hes) and Hes-related (Hey) families (3). An increasing amount of evidence suggests that mul- tiple Notch pathway components are expressed in the Authors' Affiliations: 1Oncology iMED, AstraZeneca R&D Boston, Wal- vasculature and that aberrations in normal Notch sig- tham, Massachusetts; 2Oncology iMED, AstraZeneca, Alderley Park, Mac- naling can result in vascular phenotypes. For example, clesfield, Cheshire, United Kingdom; 3Amgen, Burnaby, British Colum- bia, Canada; and 4MedImmune, One MedImmune Way, Gaithersburg, mutations in Jagged1 and Notch3 result in Alagille syn- Maryland drome and cerebral autosomal dominant arteriopathy Note: Supplementary data for this article are available at Molecular Cancer with subcortical infarcts and leukoencephalopathy Therapeutics Online (http://mct.aacrjournals.org/). (CADASIL) respectively, 2 disorders that exhibit vascu- lar defects (4, 5). Furthermore, genetic deletion of either Current address for D.W. Jenkins: Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, MA. Notch1 or Dll4 in mice both result in embryonic lethality with vascular abnormalities. In addition, deletion of a Corresponding Author: Simon T. Barry, Oncology iMED, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United King- single allele of Dll4, which is the only Notch ligand dom. Phone: 44-1625-513350; Fax: 44-1625-513350; E-mail: expressed predominantly in the endothelium, results in [email protected] embryonic lethality with severe vascular defects in most doi: 10.1158/1535-7163.MCT-11-1027 genetic backgrounds in mice (6–8). This phenotype has Ó2012 American Association for Cancer Research. only previously been reported for VEGF-A and suggests 1650 Mol Cancer Ther; 11(8) August 2012 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst June 7, 2012; DOI: 10.1158/1535-7163.MCT-11-1027 MEDI0639 Targets Dll4 that Dll4 may play a critical role in vascular development HUVECs were cultured in MCDB131 supplemented with (9, 10). 10% fetal calf serum (FCS) or in endothelial growth media Dll4 expression has also been reported on the vascula- and normal human dermal fibroblasts were cultured in ture of several tumor types, including clear-cell renal cell fibroblast growth media. Each vial of primary cells was carcinomas, glioblastomas, Ewings sarcoma, and cancers used for no more than 6 passages. No further authenti- of the breast and bladder (11–15). In addition, expression cation was conducted. Cells were maintained in 5% CO2 on a small proportion of tumor cells has also been at 37C. Angiogenesis kits and tubule staining kits were reported (16, 17). In normal physiology, Dll4-Notch obtained from TCS CellWorks Ltd. signaling appears to regulate angiogenesis by modulat- ing the number of endothelial tip cells and hence angio- Immunization genic sprouting (18). The effect of blocking Dll4 signaling Immunization protocol is shown in Supplementary on tumor growth has been evaluated in several preclin- Materials and Methods. ical xenograft models of human cancer in which either tumor cell lines or primary human tumors are grown Determining binding affinities of MEDI0639 subcutaneously in immunodeficient mice (17, 19, 20). In The binding affinities of MEDI0639 were estimated these studies, reductions in tumor growth have been using fluorescence-activated cell-sorting (FACS) techni- reported. Agents disrupting Dll4-Notch signaling can ques. HEK293 cells overexpressing either human or cyno- have 2 modes of action. First, antagonists can affect molgus monkey Dll4 were seeded at approximately survival of tumor-initiating stem cells (17). Second, 85,000 to 104,000 cells/well into 96-well nonadherent antagonists increase the number of endothelial tip cells plates in PBS and incubated with titrations of purified in the tumor, stimulating an angiogenic response, lead- antibody for 5 hours at 4C. The cells were then washed ing to the formation of immature angiogenic vessels. and incubated with goat anti-human IgG-Fc-Cy5 þ These vessels are not well perfused and poorly function- 5 mg/mL 7-aminoactinomycin (7AAD) for 30 minutes at al, reducing the effective blood flow. This increases 4C. Bound Dll4 was detected using FACS analysis and tumor hypoxia thereby driving a reduction in tumor data were fitted to the following equation: growth (19–21). qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi These data suggest that, in addition to its role in vas- 2 ðÞKD þ LT þ n  M ÀðÞKD þ LT þ n  M À4n  M  LT cular development, Dll4 may also play a role in develop- F ¼ P0 þ B 2 ment of the tumor vasculature. In this report, we describe F ¼ L ¼ the in vitro and in vivo characterization of a human anti- In this equation, mean fluorescence, T total molar 0 Dll4–specific monoclonal antibody, MEDI0639, which mAb concentration, P ¼ proportionality constant that targets a novel epitope on Dll4 and is a potent modulator relates arbitrary fluorescence units to bound mAb, M ¼ of Dll4-Notch signaling. cell concentration in molarity, n ¼ number of receptors per B ¼ K ¼ cell, background signal, and D equilibrium dis- sociation constant. For each mAb titration curve estimates Materials and Methods P0 n B K for , , , and D are obtained by nonlinear analysis Reagents and cell culture fitting of experimental data to the above equation. Recombinant human Notch1-Fc, human Dll4, anti- human Jagged1, anti-human Dll1, and anti-mouse/ Receptor–ligand binding assays human Dll4 antibodies were from R&D Systems. Anti- To determine the potency of the panel of antibodies at Human IgG-Fc-fluorescein isothiocyanate (FITC), anti- inhibiting receptor–ligand binding, parental 293T cells or human IgG-Fc-Cy5, and anti-mouse IgG-Fc-Cy5 were cells transiently transfected with human Dll4 were resus- from Jackson ImmunoResearch Laboratories, Inc. Anti- pended in PBS containing 2% FCS and added at a con- goat IgG phycoerythrin (PE) was from Invitrogen. centration of 25,000 cells/well to a nonadherent 96-well Anti-cleaved Notch1 (Val1744) was from Cell Signalling plate containing purified antibodies at a final concentra- Technologies and anti-GAPDH from Ambion. Protease tion of 10, 1, or 0.1 mg/mL. After incubation for 1 hour at inhibitor cocktail tablets were from Roche Diagnostics 4 C, Alexa-647–labeled human Notch1-Fc was added at a Ltd. DAPT, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-
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