Angiogenesis is a crucial process during embryonic development and normal physiology, and during tumor development, growth, and progression 
. Anti-angiogenic therapy therefore presents an exciting and rational approach for tumor therapy. However, the clinical efficacy of anti-angiogenic therapies have been mostly disappointing, with modest increases in patient overall survival 
. 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 (), 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 
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 
. 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 
. 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 
. The importance of r84's specificity is best observed through direct comparisons where r84 has been shown to outperform less specific anti-VEGFR2 strategies 
. 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 (), 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 
, whereas mice expressing only the extracellular domain of VEGFR1 are viable 
. 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.
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.
demonstrated that VEGF binding to VEGFR1 induced the activity of SHP-1 phosphatase that in turn reduced levels of VEGFR2 phosphorylation. Therefore, active VEGF binding and signaling through VEGFR1 could potentially negatively regulate tumor angiogenesis, 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 
. 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 
and PlGF 
, 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 
. 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 
. 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 
. However, selective blockade of VEGFR2 in our models was sufficient to control tumor growth as compared to simultaneous inhibition of VEGFR1 and VEGFR2 (). Increased metastasis was not observed from tumor xenografts treated with r84 or the phenotypic precursor of r84, 2C3, in subcutaneous or orthotopic models 
. Nevertheless, the effects of anti-angiogenic therapy on tumor progression and metastasis are still being elucidated 
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 (). 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 (). NSCLC tumors treated with r84 and bevacizumab showed a reduction in VEGFR2 staining (), 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 
. 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 
. In a corneal injury model, Cursiefen et al.
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 
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 hepatic 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 
. 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 
. Our current work with r84 in the A549 xenograft model () 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 (, 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 
. 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 (, Table S2
) and acute increases in systolic blood pressure were resolved over time without cessation of therapy (). 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.