In this study, we evaluated the anti-angiogenic drug TRC105, and its combination with bevacizumab, in a series of HUVEC functional assays. Initially, individual drugs were tested over a broad range of dosing levels under each assay condition. In every assay system, doses that induced moderate effects (typically around 25–30 % inhibition) were chosen for these analyses in order to detect potential additive or synergistic effects with both drugs. For most assays, the dose of TRC105 was empirically determined to be 100 μg/ml, as lower doses exhibited little effect on HUVEC function. This dose is clinically relevant as pharmacokinetic analyses revealed that the serum concentration of TRC105 following dosing in advanced cancer patients at the recommended phase 2 dose ranged from 200 to 600 μg/ml . For bevacizumab, the dose tested in most assays was empirically determined to be 100 ng/ml.
Despite the fact that TRC105 and bevacizumab are both monoclonal antibodies that block angiogenesis, the targets of each drug are different and utilize distinct mechanisms. Endoglin is an integral cell membrane receptor located on proliferating endothelial cells , whereas VEGF is a soluble angiogenic factor that is primarily released by tumor cells and tumor-associated stromal cells [21, 22]. Therefore, combining TRC105 and bevacizumab has the potential to block complementary pathways leading to improved efficacy.
We observed that individually, both TRC105 and bevacizumab blocked HUVEC tube formation, and the most robust inhibition was achieved when both drugs were combined (Fig. 1). The tube formation assay is a powerful tool to monitor ECs during vascular network formation, representing an end-point evaluation of the complicated interplay among many processes, including proliferation, differentiation, migration, apoptosis, etc. To further interrogate these processes, we next investigated specific EC functional biology in more defined assay systems.
When given at 100–1,000 μg/ml levels, TRC105 and its isotype control (IgG) similarly inhibited HUVEC viability, in both regular and nutrient-limited medium (Fig. 2). In contrast, although bevacizumab elicited little effect on HUVEC viability in regular medium, it showed dose-dependent, drug-specific inhibition in limited medium (Fig. 2b). While TRC105 had little effect on HUVEC viability, several observations are noteworthy. First, contrary to bevacizumab, TRC105 elicited no inhibitory effect on HUVEC growth in nutrient-limited medium. Second, given that the steady state plasma concentration of TRC105 in patients is in μg/ml range, the potential non-specific effect of IgG on EC growth should be considered. Third, Anderberg et al., reported that genetic knockdown of endoglin sensitizes tumors to VEGF inhibition , advocating for the potential benefit of co-administration of these drugs. The underlying mechanism is unlikely to be an effect on EC growth/viability, as suggested by our MTS data and suggests that the inhibition of HUVEC tube formation is not due to the impact of these agents on cell viability.
The effect of endoglin on cell motility is controversial. While endoglin is considered to be an inhibitor of cell migration through its extracellular RGD domain that binds to intercellular matrix proteins , evidence exists that endoglin promotes ALK1 signaling, leading to increased cell mobility . Additionally, endoglin has been shown to antagonize the inhibitory effect of TGF-β1 on HUVEC migration, suggesting a positive role of endoglin in HUVEC motility . Our scratch assay data support the latter hypothesis (Fig. 3). TRC105 moderately decreased HUVEC migration, as quantified by a reduced percentage of cells that migrated into the gap created by the scratch. Bevacizumab exhibited similar inhibitory effects, consistent with VEGF’s motility-promoting role . The combination of both drugs exhibited an additive inhibition when compared to either drug alone in the scratch filling assay.
EC apoptosis provided another opportunity to evaluate TRC105 and bevacizumab in both regular and limited medium. In the presence of multiple growth factors, TRC105 and bevacizumab exhibited similar potency in inducing HUVEC apoptosis (Fig. 4a). In limited medium, where VEGF is essentially the only growth factor, TRC105 was also active and exhibited dose-dependent induction of HUVEC apoptosis (Fig. 4b). These findings are in agreement with previous observations that SN6j, the parent antibody of TRC105, also led to increased HUVEC apoptosis in vitro . However, there were no additive effects by combining both drugs.
Lastly, we explored the molecular mechanisms underlying the ability of TRC105 to block HUVEC function. Since endoglin is a type III receptor for TGF-β and BMP, we investigated the effects of TRC105 on Smad signaling, pathways known to play pivotal roles in EC proliferation and viability. In assessing Smad phosphorylation (Fig. 5), three doses of TRC105 were chosen: 0.2, 100, and 1,000 μg/ml. A dose of 0.2 μg/ml is the target concentration predicted to saturate endoglin on cell surface . Doses of 100 and 1,000 μg/ml fall within or close to the range of TRC105 plasma concentrations (200–600 μg/ml) achieved in cancer patients following dosing at the recommended phase 2 dose .
While endoglin’s role in canonical TGF-β signaling is well documented , recent data indicate that BMP-9 and −10 are the important endoglin ligands that mediate Smad 1/5/8 phosphorylation needed for activation of primary EC [10, 29]. Our data corroborate that BMP-9 effectively stimulates Smad 1/5/8 phosphorylation (more than 30 fold induction), while TGF-β1 only modestly stimulates Smad 2/3 phosphorylation (approximately 5 fold induction). Our observation is consistent with the model suggesting that the BMP9-ALK1-Smad1/5/8 and the TGFβ-ALK5-Smad2/3 axes co-exist in parallel in primary human EC .
When HUVEC were pre-treated with TRC105, BMP-9-induced Smad 1/5/8 phosphorylation was inhibited by 50 %, even at the lowest dose of 0.2 μg/ml (Fig. 5b). In contrast, TGF-β1-induced Smad 2/3 phosphorylation was less affected (<20 % inhibition). It is well established that Smad 1/5/8 signaling promotes EC activation. Therefore, the inhibition of this pathway exerted by TRC105 would lead to EC deactivation, contributing to the anti-angiogenic, anti-tumor function of TRC105.
In addition to blocking BMP9/Smad1/5/8 signaling, other mechanisms may be responsible for TRC105 anti-angiogenic function. For example, biomarker analyses from cancer patients treated with escalating doses of TRC105 revealed significant increases of soluble endoglin (sEnd) after TRC105 administration . Kumar et al. proposed two mechanisms as to how TRC105 could induce sEnd shedding . First, TRC105 could stabilize endoglin/MMP-14 complexes on the cell surface; second, TRC105 could induce MMP-14 gene expression to facilitate enzymatic cleavage of endoglin. In either event, sEnd would be released and serve as a trap for its ligand BMP-9, further diminishing the BMP9/Smad 1/5/8 pathway . Another mechanism may involve the intracellular domain of endoglin. This domain contains important structural elements that serve as docking sites for multiple adaptor proteins, such as zyxin, zyxin-related protein 1 , β-arrestin2 , and GIPC . It remains unclear how TRC105 binding to endoglin would affect these proteins and what role (s) the intracellular domain of endoglin plays in TRC105 function.
Clinically relevant doses of TRC105 were tested in these in vitro cell-based assays and could facilitate our understanding of the efficacy in vivo. Based on the superior effects of TRC105 and bevacizumab in HUVEC tube formation assays, the combination of both drugs has the potential to increase drug efficacy and reduce resistance. Currently, developing rationale-based combinations of multiple anti-angiogenic agents is a favored strategy to overcome resistance in the clinic . Bevacizumab has been successfully added to chemo- and radiotherapy regimens and has significantly improved patient outcomes . In the case of TRC105 and bevacizumab, with each drug targeting an independent pathway, more effective blockage of angiogenesis is expected. However, caution needs to be taken when determining drug dosing, relative ratio, frequency of administration, etc., to prevent the possible convergence of downstream effectors becoming exhausted or saturated.
In summary, we have demonstrated that the combination of TRC105 and bevacizumab led to greater inhibition in HUVEC functional assays, such as tube formation and migration, than either drug alone. We also explored Smad signaling and confirmed that diminished BMP9/Smad 1/5/8 signaling is a mechanism contributing to TRC105’s anti-angiogenic, anti-tumor effect. The superior potency demonstrated by the drug combination advocates their co-administration in vivo as a therapeutic strategy.