binding and signaling through VEGFR2 without interrupting VEGFR1 signaling. The ability of r84 to bind human and mouse VEGF (Figure 1A, B) and block VEGF from binding and signaling through VEGFR2 (Figure 1C, Figure 2A–D, Figure S1A, B) makes r84 a unique tool for studying VEGF inhibition in tumor xenograft models, assessing possible toxicity induction and analyzing the importance of VEGFR1 signaling in these processes. r84 delays take of human xenograft tumors Previous studies in our lab have demonstrated the ability of r84 to control tumor growth and decrease tumor angiogenesis in established models of breast cancer [20–21]. The efficacy of r84 as a cancer therapeutic was further assessed in tumor xenograft models in NOD/SCID mice. Briefly, four- to six-week old female NOD/SCID mice were implanted subcutaneously (s.c.) with 2.5 million human non-small cell lung cancer (NSCLC) cell lines H460, H1299, or A549. Treatment began one day post tumor cell injection (TCI) and continued until the average tumor volume in control IgG treated tumors reached 1500 mm3, at which time all animals were sacrificed. Tumor-bearing animals were treated with 50 mg/kg/week r84 and 25 mg/kg/week bevacizumab and control IgG (palivizumab/SynagisH). r84 and bevacizumab similarly delayed tumor take, thereby controlling H460 and H1299 tumor growth compared to control IgG therapy by both tumor volume and final tumor weights at sacrifice (Figure 3A, B). In A549 xenografts, r84 delayed tumor take better than bevacizumab, and the mean final tumor weight at sacrifice of animals treated with r84 was significantly smaller than animals treated with bevacizumab (Figure 3C, p,0.05). Bevacizumab has a half life in mice of approximately two weeks [22]. Pharmacokinetic studies (data not shown) determined the half life of r84 in mice to be approximately five days. This difference, along with the fact that r84 binds both human and mouse VEGF and thus has more target to bind in tumor xenograft models than bevacizumab, led to the differences in antibody doses used in tumor studies. Consequently, this increase in dose could lead to better control of tumor growth as was seen in the A459 model (Figure 3C). To evaluate the effect of antibody dose, A549 tumor cells were implanted into mice as previously described. One day post TCI, animals began therapy, receiving 5, 15, or 50 mg/ kg/week of r84 or bevacizumab, or 15 mg/kg/week of a non- specific control human IgG. The different doses of bevacizumab had the same effect on tumor growth and final tumor weight (Figure 3D). In contrast, there was an observable titration of tumor growth and final tumor weight with r84 therapy, with tighter control seen at higher doses of antibody. In addition, treatment of A549 tumor-bearing animals with 15 and 50 mg/kg/week r84 resulted in smaller tumors as compared to the same dose of bevacizumab (Figure 3D, p,0.001 and p,0.01, respectively). Therefore, these results indicate that r84 may be more effective at controlling tumor take and growth than bevacizumab independent of dose in certain models. We propose that the appropriate therapeutic antibody dose should be determined independently for different tumor types to maximize therapeutic benefit with minimal induction of toxicity [23–25]. r84 effects the tumor microenvironment The phenotypic effects of r84 therapy within NSCLC tumors were assessed by immunohistochemistry (IHC). As was expected for anti-angiogenic therapies, treatment with r84 and bevacizu- mab resulted in a significant decrease in tumor MVD as demonstrated using two endothelial cell markers, MECA-32 (data not shown) and endomucin (Figure 4A). There was a trend towards an increase in the number of pericyte-(PC) associated r84 Inhibits VEGFR2 blood vessels in r84- and bevacizumab-treated tumors as compared to control IgG, although this increase only reached significance in the H460 model (Figure 4A). Treatment of H460 and H1299 xenograft tumors with r84 or bevacizumab also reduced the number of VEGFR2 positive cells, as analyzed by IHC (Figure 4B). Interestingly, VEGFR2 expression in A549 tumors was only decreased following r84 and not bevacizumab (Figure 4B, p,0.05) therapy, perhaps reflecting the difference in the efficacy of these two drugs in controlling tumor growth in this model. Additionally, inhibition of VEGF with r84 or bevacizumab decreased tumor LVD as compared to control IgG therapy in both H460 and H1299 models (Figure 4C). However, bevacizumab therapy failed to reduce LVD in the A549 model. These results suggest that in a model-dependent manner, r84 and bevacizumab may be able to disrupt lymphatic vessel-mediated tumor metastasis. Extended r84 therapy does not induce toxicity The use of bevacizumab and other anti-angiogenic therapies in the clinic is associated with a number of rare although serious toxicities. Toxicity associated with r84 can be evaluated in preclinical mouse xenograft models because of its ability of r84 to bind both human and mouse VEGF. To assess the potential of r84 to induce toxicities, NOD/SCID mice were injected with five million PANC-1 tumor cells (a slow-growing human pancreatic cancer line) s.c. Treatment began one day post TCI, with 50 mg/ kg/week r84 or a non-specific control IgG (palivizumab/ SynagisH). An equal number of non-tumor-bearing (NTB) animals received antibody treatment as well. Therapy continued for 12 weeks, at which point animals were sacrificed and tumor, organs and blood were collected for toxicity assessment. As was seen in the NSCLC models, r84 therapy significantly reduced PANC-1 tumor growth and final tumor weight, as compared to control (Figure S2A, p,0.05). In addition, r84 treatment resulted in decreased tumor MVD (Figure S2B, p,0.001). r84 did not induce histological changes (as assessed by a pathologist) within the kidney or liver of tumor-bearing (TB) or NTB mice as compared to age- matched na¨ıve animals (Figure 5A, na¨ıve and TB r84 displayed). Blood was collected from all animals at the time of sacrifice, and a serum analysis of 20 metabolic markers was performed at UT Southwestern Medical Center’s mouse metabolic phenotyping core (Table S1). There were no significant changes in any of these analytes between treated animals and age-matched na¨ıve animals. This analysis included no observable change in alanine amino- transferase, aspartate aminotransferase, and blood urea nitrogen levels (Figure 5B), markers of liver and kidney function, respectively. These three markers are elevated in correlation with toxicity in animals treated for 12 weeks with bevacizumab and high-affinity anti-VEGF antibodies [12]. It has been reported that anti-VEGF treatment can reduce pancreatic islet vascular density in adult mice, leaving the supporting pericytes behind [9]. In this study, the pancreatic islets of TB and NTB animals treated for 12 weeks with r84 showed a reduction in MVD as compared to control IgG-treated TB and NTB animals (p,0.01), but there was no significant change when compared to na¨ıve animals (Figure S3A). Additionally, there was no observable change in the percentage of pericytes without endothelial cell association (Figure S3A). Furthermore, there was no change in serum glucose levels, nor was there a change in insulin staining in pancreatic islets of experimental animals (Figure S3B, C). Taken together, long-term therapy with r84 produced no observable toxicity in TB or NTB animals. Since hypertension and proteinuria are among the most common toxicity-related side effects associated with anti-VEGF PLoS ONE | www.plosone.org 6 August 2010 | Volume 5 | Issue 8 | e12031