r84 Inhibits VEGFR2 saline, 25 mg/kg/week mcr84, or 50 mg/kg/week sunitinib. After 2 weeks of therapy, mcr84 and sunitinib significantly increased mean systolic blood pressure (C, left panel, ***p,0.001), but this effect was lost by week 7 of continuous therapy (C, right panel). D, Urine samples collected during week 6 of therapy and assayed for total levels of urine protein and creatine (Upro/Creatine ratio displayed) showed no significant difference between treated animals as compared to control, indicating that extended therapy with mcr84 and sunitinib did not induce kidney damage in this model. doi:10.1371/journal.pone.0012031.g005 (Figure 4A). NSCLC tumors treated with r84 and bevacizumab showed a reduction in VEGFR2 staining (Figure 4B), with the exception of A549 tumors where bevacizumab had no effect, suggesting specific inhibition of VEGF:VEGFR2 binding by r84 can down regulate receptor expression. As VEGFR2 is considered the predominant angiogenic signaling receptor, decreasing its expression within tumors could promote the anti-angiogenic effects of r84. Tumor LVD was also decreased in mice treated with r84 and bevacizumab, with the exception of A549 tumors where bevacizumab had no effect. In several tumor types, including lung cancer, lymphatic vasculature participates in tumor metastasis [46]. Although predominately mediated by VEGF-C and -D interaction with VEGFR3, recent data demonstrated elevated expression of tumor-derived VEGF-A contributes to pathological lymphangiogenesis [47–48]. In a corneal injury model, Cursiefen et al., [47] demonstrated that elevated levels of VEGF-A recruits macrophages and inflammatory cells secreting VEGF-C and -D to the site of injury, thereby inducing lymphangiogenesis. This mechanism may explain the decrease in LVD seen in treated tumors in our studies. Therefore, reduced LVD observed with r84 and bevacizumab therapy is perhaps mechanistically similar to the reduction in LVD observed in 2C3- treated breast cancer xenografts, which correlated with a VEGFR2-mediated down regulation of VEGFR3 in lymphatic endothelial cells and a decrease in Ang-2 expression in endothelial cells and tumor cells [49]. Extended therapy with r84 in tumor bearing and non-tumor bearing mice did not induce toxicity, as measured by weight maintenance, blood pressure levels, proteinuria analysis, and preservation of renal, hepatic, and pancreatic structure and function. Previous studies assessing the safety of anti-VEGF antibodies, including bevacizumab, demonstrated increased he- patic and renal damage with antibodies of increasing affinity to VEGF. Hepatic and renal toxicity produced elevated serum levels of ALT, AST, and BUN as well as glomerulosclerosis and loss of structural integrity seen by H&E staining [12]. These toxicity- inducing antibodies were first characterized in 2006 as cross- reactive antibodies that recognized human and mouse VEGF and highlighted the importance of blocking stromal-derived VEGF in some tumor models [50]. Our current work with r84 in the A549 xenograft model (Figure 3C–D) highlights the importance of host VEGF in the progression of some tumors. However, in our studies, long-term therapy with r84 does not induce the renal or hepatic toxicities (Figure 5, Table S1). This separates r84 from previously developed cross-reactive antibodies as a unique therapeutic tool with the potential to answer key questions on the function of stromal VEGF in tumor progression and the importance of VEGFR1 activity in avoiding anti-VEGF induced toxicity. The endocrine pancreas is especially sensitive to VEGF inhibition [9,51]. However, extended therapy with r84 did not result in changes in pancreatic islet structure or function (Figure S3A-C). In an immunocompetent model of spontaneous pancreatic cancer, extended therapy with mcr84 did not induce renal or hepatic toxicities as indicated by urine analysis and serum metabolic markers (Figure 5D, Table S2) and acute increases in systolic blood pressure were resolved over time without cessation of therapy (Figure 5C). Thus, we conclude that r84 and mcr84 do not induce significant toxicities in mice perhaps due to the lower affinity of r84 for VEGF as compared to other anti-VEGF antibodies or from a protective function of VEGFR1. Overall, the in vitro and in vivo characteristics of r84 establish this antibody as an important tool to further elucidate the importance of VEGF signaling through VEGFR1 and VEGFR2 within tumors and during normal physiology and as a potential adjuvant therapy. At the present time the production of clinical grade r84 is being evaluated and we anticipate that initial safety trials in humans will begin in the near future. Supporting Information Table S1 Extended r84 therapy does not induce significant changes in blood serum chemistry. NOD/SCID mice bearing subcutaneous PANC-1 tumors received long-term 12-week therapy with 50 mg/kg/week r84 or a control IgG. Blood chemistry analysis of serum samples collected from mice at sacrifice indicated that extended r84 treatment does not induce changes in serum levels of 20 different markers, as compared to control-treated (Ctrl) or Na¨ıve animals. Found at: doi:10.1371/journal.pone.0012031.s001 (0.03 MB DOC) Table S2 Extended mcr84 therapy does not induce significant changes in blood serum chemistry. Immunocompetent mice heterozygous for a spontaneous model of pancreatic cancer received extended 8-week therapy with saline, 25 mg/kg/week mouse chimeric r84 (mcr84), or 50 mg/kg/week sunitinib. Blood chemistry analysis of serum samples collected from mice at sacrifice indicated that extended mcr84 and sunitinib treatment does not induce changes in serum levels of 18 different markers, as compared to saline-treated animals in this model. Found at: doi:10.1371/journal.pone.0012031.s002 (0.03 MB DOC) Figure S1 r84 reduces mouse VEGF-induced endothelial cell migration and signaling in vitro. A, A modified Boyden chamber migration assay was used to assess the effect of r84, bevacizumab (bev) on mouse VEGF-induced endothelial cell (EC) migration. 20,000 PAE-KDR cells were plated on 8.0 mm cell culture inserts and allowed to migrate overnight towards SFM or mouse VEGF (100 ng/mL)+/2500-fold molar excess antibody (bev, r84, control IgG). Only r84 blocks mouse VEGF-induced migration of VEGFR2-expressing PAE-KDR ECs. B, Western blots of mouse VEGF-induced signaling in PAE-KDR lysates following stimula- tion of cells with 50 ng/mL mouse VEGF+/2500-fold molar excess antibody (bev, r84, control IgG). Only r84 blocks p- VEGFR2 and downstream phosphorylation (PLC-c, ERK1/2) in mouse VEGF-stimulated cells. ***p,0.001, statistical differences in A compared to mouse VEGF alone, unless otherwise indicated. Found at: doi:10.1371/journal.pone.0012031.s003 (0.68 MB TIF) Figure S2 Efficacy of long-term anti-VEGF therapy. r84 and mcr84 were able to control tumor growth in two extended therapy models. A–B, NOD/SCID mice bearing subcutaneous PANC-1 tumors received long-term 12-week therapy with 50 mg/kg/week r84 or a control IgG. r84 therapy significantly controls tumor growth and final tumor weight compared to control IgG (A, *p,0.05). B, r84 significantly decreases PANC-1 tumor micro- vessel density as compared to control IgG (Ctrl) treatment as PLoS ONE | www.plosone.org 11 August 2010 | Volume 5 | Issue 8 | e12031