Cell lines were confirmed to be pathogen free and were authenticated to confirm origin prior to use. Subcutaneous xenograft therapy study. 2.5 million H460, H1299, A549 NSCLC cells (provided by Dr. John Minna) were injected (in PBS) subcutaneously into the right flank of NOD/ SCID mice. Mice were treated with 50 mg/kg/week r84 and 25 mg/kg/week bevacizumab/AvastinH and palivizumab/ SynagisH (anti-respiratory syncytial virus) via intraperitoneal (IP) injection starting one day post tumor cell injection (TCI) (n = 8–9/ group). Mice were monitored twice a week, recording weights, taking perpendicular tumor measurements, and observing for signs of distress such as weight loss and inactivity. Therapy continued until average control-treated tumor volume reached 1500 mm3 or until day 60 post TCI, at which point animals were sacrificed. Toxicity studies. 5 million PANC-1 human pancreatic cancer cells (ATCC, Manassas, VA) (in PBS) were injected subcutaneously into the right flank of NOD/SCID mice. An equal number of NOD/SCID mice were not injected with tumor cells. Therapy began one day post TCI. Tumor bearing (TB) and non- tumor bearing (NTB) mice were treated with 50 mg/kg/week r84 and palivizumab via IP injection. Each group consisted of five mice. Mice were monitored as previously described. All mice were sacrificed following 12 weeks of continuous therapy and evaluated for r84-induced toxicity. Blood was collected from animals at sacrifice; serum was isolated following centrifugation and analyzed by the mouse metabolic phenotyping core at the University of Texas Southwestern Medical Center. A second toxicity study was performed in immunocompetent mice harbouring spontaneous pancreatic cancer (p48cre/KrasG12D/INK4a) [17]. Mice were treated with saline (n = 4) or 25 mg/kg/week mouse chimeric r84 (mcr84, n = 3) via IP injection or with 50 mg/kg/week sunitinib (n = 4) by daily oral gavage five days per week. Sunitinib was purchased from LC laboratories (Woburn, MA). Therapy began when mice reached eight weeks old. Mice were monitored for weight gain as previously described. At weeks two and seven of therapy, tail vein cuff blood pressures of all mice were measured using the Visitech Systems BP-2000 Series II Blood Pressure Analysis SystemTM through the O’Brien Kidney Research Core Center at the University of Texas Southwestern Medical Center. To familiarize mice to the procedure, tail cuff blood pressures were measured for five consecutive days, with data collection on the fifth day. Average systolic pressures were calculated from data collected on the last day of measurement (day five). At week six of therapy metabolic cages obtained through the O’Brien Kidney Research Core Center at the University of Texas Southwestern Medical Center were used to collect urine from all animals over a 24-hour collection period. Fresh urine samples were then submitted to the University of Texas Southwestern Medical Center mouse metabolic phenotyping core for analysis of total levels of urine protein and creatine. All mice were sacrificed following eight weeks of continuous therapy and evaluated for mcr84- and sunitinib-induced toxicity. Blood was collected from animals at sacrifice; serum was isolated following centrifugation and analyzed by the mouse metabolic phenotyping core at the University of Texas Southwestern Medical Center. Therapy dose titration. 2.5 million A549 NSCLC cells (in PBS) were injected subcutaneously into the right flank of NOD/ SCID mice. Mice were treated with 5, 15, or 50 mg/kg/week r84 and bevacizumab and 15 mg/kg/week control IgG (Peregrine Pharmaceuticals, Inc.) via IP injection starting one day post tumor cell injection (TCI). Each group consisted of eight mice and were monitored as above. Therapy continued until average control- treated tumor volume reached 1200 mm3, at which point animals were sacrificed. r84 Inhibits VEGFR2 Histology and Immunohistochemical Studies Formalin-fixed, paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin by the molecular pathology core laboratory at the University of Texas Southwestern Medical Center. Snap frozen tumors were sectioned, blocked with 20% Aquablock (East Coast Biologics, North Berwick, ME) and stained for markers of interest. Primary antibodies used include MECA-32 (DSHB; University of Iowa), endomucin (Santa Cruz BiotechnologyH, Inc., Santa Cruz, CA), NG2 (MilliporeH, Billerica, MA), smooth muscle actin (NeoMarkers, Fremont, CA), Lyve1, VEGFR2 (55B11) (Cell Signaling TechnologyH, Danvers, MA), rabbit anti-VEGFR2 T014 (purified in our laboratory) [4,18], rat anti-VEGFR2 RAFL-2 [19], and insulin (Dako, Glostrup, Denmark). Statistics Data were analyzed using GraphPad software (GraphPad Prism version 5.00 for Windows, GraphPad Software). Results are expressed as mean 6 SE. Differences are analyzed by t test or ANOVA, and results are considered significant at a p value of ,0.05. Results Generation of a fully human monoclonal antibody against VEGF The success of 2C3 in preclinical models led to the development of a fully human monoclonal antibody that recognizes VEGF and retains many of the characteristics of 2C3. A number of anti- VEGF human single chain variable fragments (scFv) were screened for several characteristics such as a competition with 2C3 for binding to VEGF, the ability to block VEGF:VEGFR2 binding, and the ability of the scFv to bind to different VEGF isoforms such as VEGF165 and VEGF121. r84 binds human and mouse VEGF-A and specifically blocks VEGF from binding to VEGFR2 To determine the binding specificity of r84, a series of ELISAs were performed. A titration of r84 against recombinant human or mouse VEGF demonstrated that r84 binds with equal affinity to both species (Figure 1A). This established r84 as an important tool in evaluating the contribution of both tumor cell- and host-derived VEGF in tumor progression using xenograft models. The binding specificity of r84 differs from other anti-VEGF antibodies, such as bevacizumab and 2C3 that recognize only human VEGF. Next, the specificity of r84 within the VEGF family was determined. r84 only bound wells coated with recombinant human and mouse VEGF-A (Figure 1B). The effect of r84 on VEGF binding to VEGFR1 and VEGFR2 was determined using ligand-receptor ELISAs. 2C3 and r84 at increasing fold molar excess significantly reduced biotinylated- VEGF binding to VEGFR2, compared to binding of biotinylated- VEGF alone or in the presence of a non-specific control IgG (Figure 1C). In contrast, neither 2C3 nor r84 inhibited binding of biotinylated-VEGF to VEGFR1 (Figure 1D, left panel). However, at a 500 fold molar excess of antibody to biotinylated-VEGF, bevacizumab decreased VEGF binding to VEGFR1 by approx- imately 80% (Figure 1D, right panel). These blocking ELISAs demonstrate the precise binding of r84 to VEGF to selectively inhibit the VEGF:VEGFR2 interaction. r84 effects VEGFR2-mediated endothelial cell function The effect of r84 on endothelial cells was determined using several in vitro assays. First, a transwell assay was used to test the PLoS ONE | www.plosone.org 3 August 2010 | Volume 5 | Issue 8 | e12031