Abstract: EphB4 receptor tyrosine kinase and its cognate ligand EphrinB2 regulate induction and maturation of newly forming vessels. Inhibition of their interaction arrests angiogenesis, vessel maturation, and pericyte recruitment. In addition, EphB4 is expressed in the vast majority of epithelial cancers and provides a survival advantage to most. Here, we describe two anti-EphB4 monoclonal antibodies that inhibit tumor angiogenesis and tumor growth by two distinct pathways. MAb131 binds to fibronectin-like domain 1 and induces degradation of human EphB4, but not murine EphB4. MAb131 inhibits human endothelial tube formation in vitro and growth of human tumors expressing EphB4 in vivo. In contrast, MAb47 targets fibronectin-like domain 2 of both human and murine EphB4 and does not alter EphB4 receptor levels, but inhibits angiogenesis and growth of both EphB4-positive and EphB4-negative tumors in a mouse s.c. xenograft model. Combination of MAb47 and bevacizumab enhances the antitumor activity and induces tumor regression. Indeed, humanized antibodies hAb47 and hAb131 showed similar affinity for EphB4 and retained efficacy in the inhibition of primary tumor development and experimental metastasis. EphB4 receptor tyrosine kinase and its cognate ligand EphrinB2 regulate induction and maturation of newly forming vessels. Inhibition of their interaction arrests angiogenesis, vessel maturation, and pericyte recruitment. In addition, EphB4 is expressed in the vast majority of epithelial cancers and provides a survival advantage to most. Here, we describe two anti-EphB4 monoclonal antibodies that inhibit tumor angiogenesis and tumor growth by two distinct pathways. MAb131 binds to fibronectin-like domain 1 and induces degradation of human EphB4, but not murine EphB4. MAb131 inhibits human endothelial tube formation in vitro and growth of human tumors expressing EphB4 in vivo. In contrast, MAb47 targets fibronectin-like domain 2 of both human and murine EphB4 and does not alter EphB4 receptor levels, but inhibits angiogenesis and growth of both EphB4-positive and EphB4-negative tumors in a mouse s.c. xenograft model. Combination of MAb47 and bevacizumab enhances the antitumor activity and induces tumor regression. Indeed, humanized antibodies hAb47 and hAb131 showed similar affinity for EphB4 and retained efficacy in the inhibition of primary tumor development and experimental metastasis. Angiogenesis is a process of new blood vessel sprouting from pre-existing vessels. This process includes primary capillary sprouting, branching, and remodeling into a mature blood vessel network. In the embryo, angiogenesis is preceded by vasculogenesis and represents one of the earliest processes in organ development.1Griffioen AW Molema G Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation.Pharmacol Rev. 2000; 52: 237-268PubMed Google Scholar, 2Yancopoulos GD Davis S Gale NW Rudge JS Wiegand SJ Holash J Vascular-specific growth factors and blood vessel formation.Nature. 2000; 407: 242-248Crossref PubMed Scopus (3285) Google Scholar In adulthood, angiogenesis is induced at sites of tissue repair, tissue remodeling such as during menstruation, and in diseases constituting enhanced angiogenesis.1Griffioen AW Molema G Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation.Pharmacol Rev. 2000; 52: 237-268PubMed Google Scholar Angiogenesis is also induced to varying degrees in cancers.1Griffioen AW Molema G Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation.Pharmacol Rev. 2000; 52: 237-268PubMed Google Scholar Angiogenesis is triggered by protease-mediated degradation of matrix proteins releasing angiogenic factors such as vascular endothelial growth factor (VEGF), epidermal growth factor, and fibroblast growth factor, followed by the migration of endothelial cells to sprout new vessels.1Griffioen AW Molema G Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation.Pharmacol Rev. 2000; 52: 237-268PubMed Google Scholar, 3Kerbel RS Tumor angiogenesis.N Engl J Med. 2008; 358: 2039-2049Crossref PubMed Scopus (1896) Google Scholar One critical step in vessel maturation is the interaction between arterial and venous capillaries leading to the fusion and lumen formation across the two cell types. This step is dependent on the interaction between EphB4 receptor tyrosine kinase expressed on venous endothelial cells and the trans-membrane ligand EphrinB2 expressed on arterial endothelial cells.4Brantley-Sieders DM Chen J Eph receptor tyrosine kinases in angiogenesis: from development to disease.Angiogenesis. 2004; 7: 17-28Crossref PubMed Scopus (126) Google Scholar, 5Wang HU Chen ZF Anderson DJ Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.Cell. 1998; 93: 741-753Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar EphB4 belongs to the Eph family, the largest family of receptor tyrosine kinases. To date, sixteen members of this receptor family have been characterized.6Flanagan JG Vanderhaeghen P The ephrins and Eph receptors in neural development.Annu Rev Neurosci. 1998; 21: 309-345Crossref PubMed Scopus (942) Google Scholar, 7Pasquale EB Eph-ephrin promiscuity is now crystal clear.Nat Neurosci. 2004; 7: 417-418Crossref PubMed Scopus (128) Google Scholar Eph receptors are divided into two subgroups: EphA members (10 in number) that bind to ligands (EphrinA1-6) that lack trans-membrane domains and localize to the cell membrane via glycosylphosphatidyl inositol linkage. EphB receptors (6 members) bind to ligands (EphrinB1-3) that contain a trans-membrane domain.6Flanagan JG Vanderhaeghen P The ephrins and Eph receptors in neural development.Annu Rev Neurosci. 1998; 21: 309-345Crossref PubMed Scopus (942) Google Scholar, 7Pasquale EB Eph-ephrin promiscuity is now crystal clear.Nat Neurosci. 2004; 7: 417-418Crossref PubMed Scopus (128) Google Scholar Receptor-ligand interaction leads to dimerization and phosphorylation of both receptor and ligand, resulting in bidirectional signaling. On stimulation with EphrinB2, Eph receptor "forward" signaling is triggered by autophosphorylation of its intracellular tyrosine kinase domain and downstream signaling.8Heroult M Schaffner F Augustin HG Eph receptor and ephrin ligand-mediated interactions during angiogenesis and tumor progression.Exp Cell Res. 2006; 312: 642-650Crossref PubMed Scopus (146) Google Scholar, 9Cheng N Brantley DM Chen J The ephrins and Eph receptors in angiogenesis.Cytokine Growth Factor Rev. 2002; 13: 75-85Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 10Pasquale EB Eph-ephrin bidirectional signaling in physiology and disease.Cell. 2008; 133: 38-52Abstract Full Text Full Text PDF PubMed Scopus (979) Google Scholar Ephrin-B "reverse" signaling also depends on tyrosine phosphorylation of the Ephrin cytoplasmic region.10Pasquale EB Eph-ephrin bidirectional signaling in physiology and disease.Cell. 2008; 133: 38-52Abstract Full Text Full Text PDF PubMed Scopus (979) Google Scholar Targeted disruption of the EphB4 gene in mice is embryonically lethal secondary to the failure of blood vessel maturation and capillary arrest.11Gerety SS Wang HU Chen ZF Anderson DJ Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.Mol Cell. 1999; 4: 403-414Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar EphrinB2 is the only known ligand for EphB4. Targeted disruption of the EphrinB2 gene has a phenotype similar to EphB4 knockout.5Wang HU Chen ZF Anderson DJ Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.Cell. 1998; 93: 741-753Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar, 11Gerety SS Wang HU Chen ZF Anderson DJ Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.Mol Cell. 1999; 4: 403-414Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar The role of EphB4 in adult vertebrates is not fully understood, in part because of a lack of appropriate conditional knock-out lines. EphB4 and EphrinB2 are induced in newly forming venous and arterial vessels, respectively, and are thus expected to play an important role in adult angiogenesis.9Cheng N Brantley DM Chen J The ephrins and Eph receptors in angiogenesis.Cytokine Growth Factor Rev. 2002; 13: 75-85Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 12Gale NW Baluk P Pan L Kwan M Holash J DeChiara TM McDonald DM Yancopoulos GD Ephrin-B2 selectively marks arterial vessels and neovascularization sites in the adult, with expression in both endothelial and smooth-muscle cells.Dev Biol. 2001; 230: 151-160Crossref PubMed Scopus (321) Google Scholar, 13Kuijper S Turner CJ Adams RH Regulation of angiogenesis by Eph-ephrin interactions.Trends Cardiovasc Med. 2007; 17: 145-151Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar This hypothesis is supported by the observation that the soluble monomeric form of the extracellular domain of EphB4 receptor (sEphB4) that blocks EphrinB2 interaction with its cognate receptors, blocks bidirectional signaling and inhibits angiogenesis at sites of neovascularization in the adult.14Martiny-Baron G Korff T Schaffner F Esser N Eggstein S Marme D Augustin HG Inhibition of tumor growth and angiogenesis by soluble EphB4.Neoplasia. 2004; 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119: 1236-1248Crossref PubMed Scopus (61) Google Scholar prostate,24Xia G Kumar SR Masood R Zhu S Reddy R Krasnoperov V Quinn DI Henshall SM Sutherland RL Pinski JK Daneshmand S Buscarini M Stein JP Zhong C Broek D Roy-Burman P Gill PS EphB4 expression and biological significance in prostate cancer.Cancer Res. 2005; 65: 4623-4632Crossref PubMed Scopus (133) Google Scholar, 25Lee YC Perren JR Douglas EL Raynor MP Bartley MA Bardy PG Stephenson SA Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma.BMC Cancer. 2005; 5: 119Crossref PubMed Scopus (44) Google Scholar and ovary.26Castellano G Reid JF Alberti P Carcangiu ML Tomassetti A Canevari S New potential ligand-receptor signaling loops in ovarian cancer identified in multiple gene expression studies.Cancer Res. 2006; 66: 10709-10719Crossref PubMed Scopus (30) Google Scholar, 27Kumar SR Masood R Spannuth WA Singh J Scehnet J Kleiber G Jennings N Deavers M Krasnoperov V Dubeau L Weaver FA Sood AK Gill PS The receptor tyrosine kinase EphB4 is overexpressed in ovarian cancer, provides survival signals and predicts poor outcome.Br J Cancer. 2007; 96: 1083-1091Crossref PubMed Scopus (84) Google Scholar EphB4 directly supports tumor cell survival by inhibiting apoptosis in many of these cancers.17Kumar SR Singh J Xia G Krasnoperov V Hassanieh L Ley EJ Scehnet J Kumar NG Hawes D Press MF Weaver FA Gill PS Receptor tyrosine kinase EphB4 is a survival factor in breast cancer.Am J Pathol. 2006; 169: 279-293Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 18Kumar SR Scehnet JS Ley EJ Singh J Krasnoperov V Liu R Manchanda PK Ladner RD Hawes D Weaver FA Beart RW Singh G Nguyen C Kahn M Gill PS Preferential induction of EphB4 over EphB2 and its implication in colorectal cancer progression.Cancer Res. 2009; 69: 3736-3745Crossref PubMed Scopus (108) Google Scholar, 21Xia G Kumar SR Stein JP Singh J Krasnoperov V Zhu S Hassanieh L Smith DL Buscarini M Broek D Quinn DI Weaver FA Gill PS EphB4 receptor tyrosine kinase is expressed in bladder cancer and provides signals for cell survival.Oncogene. 2006; 25: 769-780Crossref PubMed Scopus (80) Google Scholar, 22Berclaz G Karamitopoulou E Mazzucchelli L Rohrbach V Dreher E Ziemiecki A Andres AC Activation of the receptor protein tyrosine kinase EphB4 in endometrial hyperplasia and endometrial carcinoma.Ann Oncol. 2003; 14: 220-226Crossref PubMed Scopus (48) Google Scholar, 23Masood R Kumar SR Sinha UK Crowe DL Krasnoperov V Reddy RK Zozulya S Singh J Xia G Broek D Schonthal AH Gill PS EphB4 provides survival advantage to squamous cell carcinoma of the head and neck.Int J Cancer. 2006; 119: 1236-1248Crossref PubMed Scopus (61) Google Scholar, 24Xia G Kumar SR Masood R Zhu S Reddy R Krasnoperov V Quinn DI Henshall SM Sutherland RL Pinski JK Daneshmand S Buscarini M Stein JP Zhong C Broek D Roy-Burman P Gill PS EphB4 expression and biological significance in prostate cancer.Cancer Res. 2005; 65: 4623-4632Crossref PubMed Scopus (133) Google Scholar, 27Kumar SR Masood R Spannuth WA Singh J Scehnet J Kleiber G Jennings N Deavers M Krasnoperov V Dubeau L Weaver FA Sood AK Gill PS The receptor tyrosine kinase EphB4 is overexpressed in ovarian cancer, provides survival signals and predicts poor outcome.Br J Cancer. 2007; 96: 1083-1091Crossref PubMed Scopus (84) Google Scholar Tumor cell–expressed EphB4 also induces angiogenesis by direct interaction with EphrinB2 on tumor vessels.28Noren NK Lu M Freeman AL Koolpe M Pasquale EB Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth.Proc Natl Acad Sci USA. 2004; 101: 5583-5588Crossref PubMed Scopus (218) Google Scholar In this study, we describe novel EphB4-specific monoclonal antibodies that inhibit formation and maturation of newly forming vessels and inhibit tumor growth in vivo. Phosphotyrosine antibody (clone 4G10) was from Upstate (Lake Placid, NY). β-actin antibody was from Sigma Chemical Co. (St Louis, MO). Ki-67 antibody was from Abcam (Cambridge, MA). CD31, PDGFR, and NG2 antibodies were obtained from R&D systems (Minneapolis, MN). Hypoxyprobe-1 and hypoxyprobe antibody were from Chemicon International (Temecula, CA). TdT-mediated dUTP nick-end labeling (TUNEL) assay kit was from Roche (Piscataway, NJ). Human cancer cell line Hey was obtained from Dr. L. Dubeau of University of Southern California (USC, Los Angeles, CA). PC3M was from Dr. P. Roy-Burman of USC. KS-SLK was obtained from Dr. Rubinstein.29Siegal B Levinton-Kriss S Schiffer A Sayar J Engelberg I Vonsover A Ramon Y Rubinstein E Kaposi's sarcoma in immunosuppression.Possibly the result of a dual viral infection Cancer. 1990; 65: 492-498Google Scholar MCF7, HT29, and SCC-15 were from American Type Culture Collection (Manassas, VA). All cells were propagated in RPMI-1640, supplemented with 10% Fetal Bovine Serum, 100 units/ml of penicillin and 100 μg/ml streptomycin from Cellgro (Manassas, VA). Extracellular domain of hEphB4 (aa 16-537) – hEphB4-ECD was purified as previously described16Kertesz N Krasnoperov V Reddy R Leshanski L Kumar SR Zozulya S Gill PS The soluble extracellular domain of EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and inhibits tumor growth.Blood. 2006; 107: 2330-2338Crossref PubMed Scopus (134) Google Scholar and used as an antigen. Five female Swiss Webster mice from Charles River Laboratories (Wilmington, MA) were immunized three times (every second week) intraperitoneally (i.p.) with 50 μg of hEphB4-ECD per mouse. Antigen was injected as 1:1 mixture with Complete Freund's Adjuvant (Sigma, St. Louis, MO) in the first immunization, and with incomplete Freund's Adjuvant (Sigma) in the second and third doses. Mice were given a final boost with 20 μg of hEphB4-ECD through tail-vein injection, and splenocytes were harvested 4 days later for fusion with myeloma cell line NS0 from Lonza (Walkersville, MD). Hybridoma supernatants were screened for antibodies that immunoprecipitate hEphB4-ECD fused to alkaline phosphatase (AP). Selected monoclonal antibodies (MAb) were produced in ascites as previously described.30Harlow E Lane D Antibodies: A Laboratory Manual.in: Harlow E Lane D Cold Spring Harbor Laboratory Press, New York1988: 274-277Google Scholar MAbs were purified using three-step protocol: Ammonium sulfate precipitation, hydroxyapatite chromatography, followed by purification on anion-exchange resin. Estimated purity of MAbs was higher than 95% based on HPLC analysis. Both mouse MAb47 and mouse MAb131 were humanized using Composite Human Antibody technology (manuscript in preparation). Fc domain was replaced with human IgG1 subclass. Affinity of humanized antibodies for hEphB4-ECD was determined as described for murine clones. Chinese hamster ovarian cell lines stably expressing humanized antibodies (hAb47 and hAb131) were established and maintained in chemically defined serum-free medium (Irvine Scientific, Irvine, CA). Antibodies were purified from conditioned culture medium using Protein A-Agarose beads (GE health care, Piscataway, NJ). The set of recombinant EphB ECDs fused to AP were incubated with MAbs immobilized on Protein A-Agarose (100 ng/well) for 30 minutes. Unbound proteins were removed by washing three times with PBS and precipitated AP activity was detected with para-nitro-phenyl-phosphate. Negative controls included unrelated IgG (IgG control) or AP alone (No EphB). The set of recombinant EphA ECDs fused to Fc from R&D Systems (Minneapolis, MN) were immobilized (100 ng/well) overnight on 96-well plate from Thermo Scientific (Rockford, IL). Wells were blocked with 0.5% BSA in PBS for 40 minutes, followed by application of each monoclonal antibody at a concentration of 1 μg/ml for 40 minutes. Antibodies specifically bound to EphA receptor proteins were detected with anti-mouse-IgG-HRP (Thermo Scientific). Negative controls included wells without addition of EphA proteins ("No EphA") and wells without the addition of MAb47 or MAb131 ("No AB"). As positive control, EphA receptor proteins were detected with biotinylated EphrinA1-Fc/Streptavidin-HRP. Biotinylation of antibodies and mEphrinA1-Fc protein was performed using EZ-Link Biotin Hydrazide (Pierce, Rockford, IL) through oxidation of carbohydrate chains according to manufacturer's protocol. Biotinylated proteins were stored at −80°C until use. Retention of the binding capacity of biotinylated proteins was confirmed using competition assay. Biotinylated MAb47 and MAb131 were immobilized on Streptavidin Agarose from Thermo Scientific at final density 10 μg of IgG per 1 ml of beads. Beads were then preblocked with 0.5% BSA in PBS and used for binding with extracellular fragment of human EphB4 fused to Alkaline Phosphatase (EphB4-AP). Sixteen different concentrations ranging from 50 pmol/L to 5000 pmol/L of EphB4-AP in triplicate were used to obtain a saturation plot. After binding for 40 minutes, all beads were washed three times with PBS followed by application of para-nitro-phenyl-phosphate. Total values minus nonspecific background (no MAb added) were converted into coordinates of a Scatchard plot.31Scatchard G The attractions of proteins for small molecules and ions.Ann NY Acad Sci. 1949; 51: 660-672Crossref Scopus (17799) Google Scholar 250 μl of Matrigel (Becton-Dickinson, Laguna Hills, CA) was placed in each well of an ice-cold 24-well plate. The plate was incubated at room temperature for 15 minutes and at 37°C for 30 minutes to allow Matrigel to solidify.17Kumar SR Singh J Xia G Krasnoperov V Hassanieh L Ley EJ Scehnet J Kumar NG Hawes D Press MF Weaver FA Gill PS Receptor tyrosine kinase EphB4 is a survival factor in breast cancer.Am J Pathol. 2006; 169: 279-293Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar 2 × 104 human umbilical vein endothelial cells in EGM2-MV medium from Clonetics (Allendale, NJ) were mixed with 5 μg of test MAb and plated in Matrigel-coated wells in triplicate. After 8 to 24 hours incubation, pictures were taken and analyzed by ImageJ software (National Institutes of Health, Bethesda, MD). Proteins extracted from cells or tissue homogenate were resolved on 4% to 20% SDS-PAGE and transferred onto 0.2 μm nitrocellulose membrane (BioRad, Hercules, CA). Membrane was blocked with 5% nonfat dry milk in TBS and 0.05% Tween-20 (TBST) for 40 minutes. The membrane was incubated with primary human EphB4 specific MAb265 at final concentration 0.5 μg/ml for 1 hour. Membrane was washed three times for 10 minutes each and incubated with secondary HRP-labeled anti-mouse antibody for 40 minutes. Finally, the membrane was washed three times with TBST and HRP signal was detected using chemiluminescent substrate.17Kumar SR Singh J Xia G Krasnoperov V Hassanieh L Ley EJ Scehnet J Kumar NG Hawes D Press MF Weaver FA Gill PS Receptor tyrosine kinase EphB4 is a survival factor in breast cancer.Am J Pathol. 2006; 169: 279-293Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Frozen sections of tumors (5 μm) were fixed in 4% paraformaldehyde and washed in PBS. Endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 10 minutes. SuperBlock blocking buffer from Pierce (Rockford, IL) was used to prevent nonspecific binding. Sections were then incubated with primary antibody overnight at 4°C, followed by corresponding secondary antibody for 30 minutes at room temperature. Antibody binding was localized with ABC staining kit from Vector Laboratories (Burlingame, CA).17Kumar SR Singh J Xia G Krasnoperov V Hassanieh L Ley EJ Scehnet J Kumar NG Hawes D Press MF Weaver FA Gill PS Receptor tyrosine kinase EphB4 is a survival factor in breast cancer.Am J Pathol. 2006; 169: 279-293Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar In immunofluorescence assays, biotinylated secondary antibodies were used and the signal was detected with FITC-streptavidin. Nuclei were counterstained with 6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), and images were obtained with Olympus AX70 fluorescence microscope and Spot v2.2.2 digital imaging system.17Kumar SR Singh J Xia G Krasnoperov V Hassanieh L Ley EJ Scehnet J Kumar NG Hawes D Press MF Weaver FA Gill PS Receptor tyrosine kinase EphB4 is a survival factor in breast cancer.Am J Pathol. 2006; 169: 279-293Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Antibody mediated receptor endocytosis was examined on HT29 tumor cells cultured on BD Falcon 4-well culture slide in RPMI1640 with 10% FBS. Cells were incubate with Cy5 labeled IgG, MAb47, or MAb131 (2 μg/ml) for 30 minutes on ice or 37°C. Cells were washed in PBS and fixed with 4% PFA for 20 minutes at room temperature, washed in PBS and mounted with VectaShield® mounting medium with DAPI (Vector Laboratories). Images were taken using Zeiss LSM510 confocal microscope. Growth factor–reduced Matrigel from BD Biosciences (Palo Alto, CA) was kept liquid on ice and reconstituted with either VEGF alone (100 ng/ml), VEGF (100 ng/ml) plus MAbs (10 μg/ml), or VEGF (100 ng/ml) plus sEphB4 (soluble extracellular domain of EphB4, 10 μg/ml) as positive control for inhibition of angiogenesis.16Kertesz N Krasnoperov V Reddy R Leshanski L Kumar SR Zozulya S Gill PS The soluble extracellular domain of EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and inhibits tumor growth.Blood. 2006; 107: 2330-2338Crossref PubMed Scopus (134) Google Scholar Matrigel (0.5 ml per injection) from each treatment group was injected in female Balb/C nu/nu mice (Charles River Laboratories) subcutaneously on each side of the midline of abdominal wall. On day 7, plugs were excised under anesthesia, fixed in 10% formalin, embedded in paraffin, and sectioned (5-μm section) for histochemical analysis. Vascular identity of the infiltrating cells was established with CD31 immunostaining. The experiment was repeated three times. The vascularized area in each section was calculated using ImageJ software (National Institutes of Health, Bethesda, MD).17Kumar SR Singh J Xia G Krasnoperov V Hassanieh L Ley EJ Scehnet J Kumar NG Hawes D Press MF Weaver FA Gill PS Receptor tyrosine kinase EphB4 is a survival factor in breast cancer.Am J Pathol. 2006; 169: 279-293Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar EphB4-positive tumor cell lines PC3M (a metastatic clone of PC3, a prostate carcinoma cell line, 1 × 106 cells), Hey (ovarian cancer cell line, 1 × 106 cells), HT29 (colorectal cancer cell line, 1 × 106 cells), SCC15 (head and neck cancer cell line, 5 × 106 cells) and MCF-7 (breast cancer cell line, 1 × 107 cells), and EphB4-negative KS-SLK (Kaposi sarcoma cell line, 2 × 106 cells) were injected subcutaneously on day 0 bilaterally in the flanks of 8-week-old Balb/C nu/nu mice (Charles River Laboratories). Beginning day 4, tumor volumes were calculated as 0.52 × a × b2, where "a" and "b" are the largest and smallest lengths of the visible tumor. Animals were distributed into treatment and control groups (n = 10 tumors per group) such that the mean tumor volume of each group was comparable and the standard error (SE) between groups was minimal. Each group was treated by i.p. injection of antibody three times a week at a dose of 10 mg/kg once tumors were about 150 mm3. Treatment was continued until the end of the experiment when mice were sacrificed for tissue analysis. All procedures were approved by Institutional Animal Care and Use Committee and performed in accordance with the Animal Welfare Act regulations. Rhodamine Ricinus communis agglutinin I (RCA) from Vector Laboratories (Burlingame, CA; 0.5 mg in 100 μl) was injected into the tail vein and allowed to circulate for 7 minutes before the mice were euthanized. The tumors were harvested, frozen on dry ice, and stored at −80°C until analysis.18Kumar SR Scehnet JS Ley EJ Singh J Krasnoperov V Liu R Manchanda PK Ladner RD Hawes D Weaver FA Beart RW Singh G Nguyen C Kahn M Gill PS Preferential induction of EphB4 over EphB2 and its implication in colorectal cancer progression.Cancer Res. 2009; 69: 3736-3745Crossref PubMed Scopus (108) Google Scholar Male Balb/C nu/nu mice (6 to 7 weeks old) were anesthetized, the spleen was exposed via a left flank incision, and 1 × 107 HT29 cells were then slowly injected into the lower half of the splenic pulp. After 2 minutes, the hilum was ligated, splenectomy was performed, and the incision was closed.32Heijstek MW Kranenburg O Borel Rinkes IH Mouse models of colorectal cancer and liver metastases.Dig Surg. 2005; 22: 16-25Crossref PubMed Scopus (96) Google Scholar The animals were randomly assigned to four treatment groups (five mice per group): hAb47, hAb131, combination of both hAbs (at half of dose each), and PBS. Treatment was given three times a week i.p. starting from day 0. After 39 days, mice were sacrificed and livers were evaluated for tumor metastasis. Anti-human EphB4 antibodies were generated in mice immunized with the extracellular domain of human EphB4. Despite the very high sequence homology between human and mouse EphB4 (89% identity and 94% similarity) within extracellular domain, we identified nearly 100 hybridomas producing distinct monoclonal antibodies to EphB4. Antibodies were screened for their ability to bind native protein - hEphB4-ECD by immunoprecipitation. Selected monoclonal antibodies were characterized in vitro and in vivo, epitopes were mapped, and affinity and isotypes were determined. Two antibodies were selected for further investigations. The specificity of MAb47 and MAb131 was determined using extracellular domains of EphB receptors