monotherapy for glioblastoma, and in combination with interferon for renal cell carcinoma [9–11]. Treatment with bevacizumab in these cancer types results in a delay of tumor progression and increases in patient survival [2,9]. However, treatment with bevacizumab, sorafenib, and sunitinib, is also associated with a number of rare although serious toxicities including gastro- intestinal perforations, bleeding, proteinurea, and glomeruloscle- rosis [9,12–13]. IMC-1121b is a high affinity, fully human IgG1 monoclonal antibody that recognizes VEGFR2. IMC-1121b binding to VEGFR2 inhibits ligand-induced activation of the receptor. There are several on-going phase I, II, and III clinical trials evaluating the efficacy of IMC-1121b in a number of tumor types [6]. VEGF-Trap is comprised of the second and third extracellular immunoglobulin domains of VEGFR1 and VEGFR2, respective- ly, joined by an IgG1 Fc region. The resulting fusion protein traps with high affinity multiple VEGF family members including VEGF and placental growth factor (PlGF) [14]. Currently, VEGF- Trap is being tested in phase III clinical trials in a number of tumor types [6]. 2C3 is a murine, monoclonal antibody against VEGF that specifically blocks human VEGF binding to VEGFR2 [4]. The selective inhibition of VEGF:VEGFR2 signaling by 2C3 reduces vascular permeability, decreases endothelial cell growth, and decreases tumor growth in murine xenograft models. Additionally, 2C3 reduces tumor microvessel density (MVD) and macrophage infiltration and down-regulates VEGFR2 expression on the tumor vasculature [3–5,15]. The desirable anti-angiogenic effects of 2C3 lead to the development of a human antibody that retains 2C3 specificity. Here we describe a fully human monoclonal antibody, r84 (AT001, Affitech AS) that binds to mouse and human VEGF and specifically inhibits VEGF binding to VEGFR2, while leaving intact VEGF interaction with VEGFR1. Through blockade of VEGFR2 signaling, r84 inhibits the migration of VEGFR2 positive endothelial cells, and blocks VEGFR2 phosphorylation and downstream signaling. In addition, treatment of mice bearing tumor xenografts with r84 delays tumor take resulting in tumor vascular changes, including reductions in tumor MVD and in tumor lymphatic vessel density (LVD). Furthermore, extended treatment with r84 does not induce significant systemic toxicity in mice. Materials and Methods Construction of human IgG anti-VEGF antibodies Human anti-VEGF single chain variable fragments (scFvs) were created by Affitech AS (Olso, Norway) and Peregrine Pharma- ceuticals, Inc. (Tustin, CA) and screened for specific VEGF binding characteristics. The most desirable scFvs were cloned into full length antibody expression vectors containing the glutamine synthetase gene, transfected into CHO K1SV cells, and selected in a glutamine free cell culture media. The cells were plated into flat bottom 96 well culture plates, and wells with antibody production were diluted and the cells were subcloned. Once subcloned, the high production cells were grown to 500 mL cultures and the antibody was purified by Protein-A affinity chromatography and size-exclusion chromatography for purities of greater than 90% monomer. ELISA analysis of r84 To evaluate the binding specificities of r84, a series of ELISAs were performed. r84 Inhibits VEGFR2 Determination of r84 specificity. Relative binding affinity of r84 for mouse and human VEGF was determined by ELISA. Recombinant human VEGF (R&D SystemsH, Minneapolis, MN) or mouse VEGF (Sigma-AldrichH, St. Louis, MO) was coated onto the bottom of 96-well plates at 0.5 mg/mL. Wells were blocked and then incubated with r84 starting at 2 mg/mL with a serial dilution factor of four. Antibody bound to the wells was detected by incubation with anti-human Fc HRP-conjugated antibody followed by development with HRP substrate. r84 specificity within VEGF family. Human VEGF-A, mouse VEGF-A, human VEGF-B, human VEGF-C, human VEGF-D, and human PlGF (R&D SystemsH) were coated onto 96-well ELISA plates at 0.5 mg/mL. Wells were blocked and then incubated with human r84 at 1 mg/mL. Antibody bound to the wells was detected as described above. r84 receptor blocking ELISAs. Recombinant human VEGFR1/Fc or VEGFR2/Fc (R&D SystemsH) was coated onto the bottom of 96-well plates at 1 mg/mL. Wells were blocked and then incubated with 2.38 nM or 4.76 nM biotinylated VEGF for VEGFR1 and VEGFR2, respectively, +/2 fold the indicated molar excess of antibody. Labelled VEGF bound to the wells was detected by incubation with strepavidin HRP-conjugated antibody, developed as described above, and displayed as a percentage of VEGF binding alone in the absence of antibody. Endothelial cell in vitro assays The effect of r84 on endothelial cell function and signaling in vitro was assessed using HDMEC (ScienCellTM Research Labora- tories, Carlsbad, CA), PAE-KDR [16], PAE-Flt-1 [16] endothelial cell lines. Migration assays. A modified Boyden chamber assay was used. 20,000 endothelial cells (ECs) (HDMEC (ScienCellTM Research Laboratories, Carlsbad, CA), PAE-KDR [16], PAE- Flt-1 [16]) were plated in serum free media on 8.0 mm pore size cell culture inserts (BD FalconTM, San Jose, CA) and allowed to migrate overnight at 37uC. Recombinant human or mouse VEGF (Sigma-AldrichH) was used as a chemo-attractant at 100 ng/mL, with antibodies added at a 500-fold molar excess. Insert membranes were isolated following migration and stained with DAPI to allow for quantification of migrated cells (total magnification, 1006). Stimulation assays. HDMEC and PAE-KDR, -Flt-1 cell lines were maintained in 100 mm2 tissue culture dishes in MCDB 131 (GibcoH, Carlsbad, CA) media supplemented with 0.4 mg/mL ECGF and 20% fetal bovine serum (FBS). Following 24 hour serum starvation, cells were stimulated for two minutes with 100 ng recombinant human VEGF or mouse (R&D SystemsH) +/ 2 500-fold molar excess antibody. Cell lysates of stimulated cells were prepared and analyzed by Western blot using commercially- available antibodies specific for targets of interest ( total and phospho- VEGFR2, p38, PLCc, Erk1/2 (Cell Signaling TechnologyH, Danvers, MA), and VEGFR1 (AbcamH, Cambridge, MA)). Animal studies 4–6-week-old NOD/SCID mice were purchased from the breeding core at the University of Texas Southwestern Medical Center. Animals were housed in a pathogen-free facility and all procedures were performed in accordance with a protocol (APN 0974-07-05-1) approved by the IACUC of the University of Texas Southwestern Medical Center. Tumor models and treatment. All tumor cells (H460, H1299, A549, PANC-1) were grown in culture in RPMI-1640 medium (HyCloneH, Waltham, MA) supplemented with 5% FBS. PLoS ONE | www.plosone.org 2 August 2010 | Volume 5 | Issue 8 | e12031