r84 Inhibits VEGFR2 Figure 4. r84 therapy induces vascular changes within tumors. Frozen sections of A549, H460, H1299 tumors treated with control IgG (Ctrl), r84, or bevacizumab (bev) were analyzed by immunofluorescence for endothelial, pericyte (PC), and lymphatic markers, as well as for VEGFR2 expression. Number of positive-staining entities per high powered field was evaluated using Nikon Elements software. A, r84, bev treatment significantly decreases tumor MVD, shown by a reduction in tumor endomucin positive endothelial cells (red). r84, bev treatment induced a trend towards increased NG2 positive (green) PC coverage of vessels as compared to Ctrl. B, r84, bev treatment significantly reduces the number of VEGFR2 positive cells in H460, H1299 tumors as shown by RAFL-2 staining (red). Only r84 treatment significantly reduced VEGFR2 staining in A549 tumors. C, r84, bev treatment significantly decreased H460, H1299 tumor LVD, as indicated by a reduction in lyve1 positive cells (red). Only r84 treatment significantly reduced A549 tumor LVD. *p,0.05, **p,0.01, ***p,0.001, statistical differences compared to Ctrl, unless otherwise indicated. doi:10.1371/journal.pone.0012031.g004 disappointing, with modest increases in patient overall survival [27]. Therefore, there is still much to learn about angiogenic signaling and angiogenesis dependence within tumors, which can be aided through the development and use of new investigative tools. Here we describe r84, a fully human monoclonal antibody specific for VEGF, a key mediator of angiogenesis. r84 binds to human and mouse VEGF-A, but not other VEGF family members (VEGF-B, -C, -D, PlGF), and specifically blocks subsequent binding of VEGF to VEGFR2, leaving intact VEGF:VEGFR1 interaction. Through its unique VEGF binding properties, r84 blocked VEGFR2-mediated endothelial cell migration and signaling. In vivo, r84 controlled tumor growth in NOD/SCID mice similarly to bevacizumab. r84-treated tumors had reduced MVD, VEGFR2 expression, and LVD as compared to control- treated tumors, and showed a trend towards increased pericyte- associated blood vessels. Importantly, chronic exposure to r84 in tumor bearing and non-tumor bearing NOD/SCID mice and in a spontaneous, immunocompetent model of pancreatic cancer did not induce toxicity. The discriminating specificity of r84 in that it recognizes one ligand (VEGF) and inhibits binding only to VEGFR2 establishes r84 as a beneficial tool for elucidating VEGFR1 signaling pathways and functional contributions of VEGFR1 and VEGFR2 in vitro and in vivo. r84 binds both human and mouse VEGF (Figure 1A, B), and a mouse chimeric version of r84 (mcr84) has been developed, thereby obviating the need for complex mouse model systems genetically engineered to express human VEGF [12] to study contributions of host- and tumor-derived VEGF in human xenograft or syngeneic tumor models. Previous work has directly compared the efficacy of r84 with other anti-angiogenic agents in established human tumor xenografts and syngeneic tumor models [20–21]. In these studies, r84 has been shown to be more effective than bevacizumab, sunitinib, an anti-VEGFR2 antibody (RAFL-2), and a peptoid against VEGFR1 and VEGFR2 (GU81) in controlling tumor growth and infiltration of immune suppressor cell populations [19,21]. Functionally, r84 inhibits VEGFR2 activity by specifically blocking only VEGF. This distinguishes this r84 from anti-VEGFR2 antibodies such as DC101 that block the activity of all VEGFR2 ligands [28]. The importance of r84’s specificity is best observed through direct comparisons where r84 has been shown to outperform less specific anti-VEGFR2 strategies [21]. The present study supports the previous investigations, highlighting that selective inhibition of VEGFR2 with r84 can delay tumor take and control tumor growth similar to blockade of both VEGFR1 and VEGFR2 (Figure 3), bringing to question the function of VEGFR1 in tumor angiogenesis and in physiological homeostasis. A caveat to the specificity of r84 is that we have been unable to determine conclusively the effect of r84 on VEGF binding to neuropilin-1 or - 2, which might impact the biological effect of r84. Although the function and signaling pathways of VEGFR1 remain elusive, there is data supporting the concept that VEGFR1 is a negative regulator of VEGFR2 signaling. VEGFR1 deficient mice die in utero due to an over abundance of endothelial cells [29–30], whereas mice expressing only the extracellular domain of VEGFR1 are viable [31]. These studies established that VEGFR1 does not need to signal through its cytoplasmic domain and functions during development as a decoy receptor for VEGF, sequestering the ligand and regulating VEGFR2-mediated angiogenesis. Roberts et al., [32] demonstrated that the VEGFR1 mutant phenotype in embryonic stem cell-derived blood vessels could be rescued by incubation with small molecule inhibitors of VEGFR2. These data further supports that VEGFR1 controls blood vessel development by negatively regulating VEGFR2 signaling. In addition, work by Nozaki et al., [33] demonstrated that VEGF binding to VEGFR1 induced the activity of SHP-1 phosphatase that in turn reduced levels of VEGFR2 phosphory- lation. Therefore, active VEGF binding and signaling through VEGFR1 could potentially negatively regulate tumor angiogene- sis, an interesting concept that warrants further investigation. Hypertension is likely caused by decreased levels of nitric oxide (NO) resulting from blockade of VEGF signaling through VEGFR2 and VEGFR1 by current anti-angiogenic strategies. VEGF activation of VEGFR1 has been demonstrated to induce NO production [34–35]. Therefore, it is possible that hypertension may be reduced or eliminated following r84 therapy. Additionally, studies have demonstrated the importance of VEGFR1 function in tumor cell survival. Neutralizing antibodies against VEGFR1 [36–37] and PlGF [38], a VEGFR1 specific ligand, have successfully controlled tumor growth in preclinical models. Adding to the complexity of this pathway, PlGF over expression has also been shown to inhibit tumor growth and angiogenesis through increased levels of functionally inactive VEGF:PlGF heterodimers [39–40]. Further, Bais et al. recently demonstrated that although anti-PlGF antibodies were able to inhibit wound healing and cancer cell extravasation, these antibodies only inhibited tumor growth in tumors that over expressed VEGFR1 [41]. These papers question the importance of directly blocking PlGF or VEGFR1 therapeutically and highlight the potential benefit of anti-angiogenic agents such as r84 that allow for PlGF and VEGFR1 interactions. VEGFR1 has also been linked to tumor metastasis [42–43]. However, selective blockade of VEGFR2 in our models was sufficient to control tumor growth as compared to simultaneous inhibition of VEGFR1 and VEGFR2 (Figure 3A–D). Increased metastasis was not observed from tumor xenografts treated with r84 or the phenotypic precursor of r84, 2C3, in subcutaneous or orthotopic models [3,20]. Nevertheless, the effects of anti-angiogenic therapy on tumor progression and metastasis are still being elucidated [44–45] and could benefit from selective tools, such as r84, to delineate important pathways and mechanisms of action in these processes. In the present study, delayed tumor take through selective inhibition of VEGFR2 with r84 was associated with several histological changes. r84 reduced tumor MVD similar to bevacizumab treatment (Figure 4A). Consistent with the concept of anti-angiogenic therapies functioning by pruning nascent tumor vasculature, we observed a trend of increased pericyte association with endothelial cells in r84 and bevacizumab treated animals, though this only reached statistical significance in the H460 model PLoS ONE | www.plosone.org 9 August 2010 | Volume 5 | Issue 8 | e12031