Evaluation of drug mechanism and efficacy of a novel anti-angiogenic agent, TTAC-0001, using multi-modality bioimaging in a mouse breast cancer orthotopic model

Purpose Targeting of vascular endothelial growth factor receptors (VEGFRs) has potential anti-angiogenic effects because VEGFR-2 is the major signaling regulator of VEGF/VEGFR pathways. We aimed to elucidate the drug mechanism and anti-tumor efficacy of TTAC-0001, a novel, fully human anti-VEGFR-2/KDR monoclonal antibody, in mouse orthotopic breast cancer model using multi-modal bioimaging. Materials and methods We used orthotopic xenograft tumor model in which human breast cancer cells (MDA-MB-231) were injected into the right mammary fat pad of Balb/c nude mice. We investigated its biodistribution using serial fluorescence imaging after injecting fluorescent-labelled-drug and mode of action using Matrigel plug angiogenesis assays. The anti-tumor efficacy of drug was assessed using ultrasonography and bioluminescence imaging. Histopathologic analyses, including hematoxylin and eosin staining and immunohistochemistry with anti-CD31 and anti-Ki-67 antibodies, were performed. Each experiment had four groups: control, bevacizumab 10 mg/kg (BVZ-10 group), TTAC-0001 2 mg/kg (TTAC-2 group), and TTAC-0001 10 mg/kg (TTAC-10 group). Results The TTAC-10 group showed good tumor targeting that lasted for at least 6 days and had a good anti-angiogenic effect with decreased hemoglobin content and fewer CD31-positive cells in the Matrigel plug. Compared with BVZ-10 and TTAC-2 groups, the TTAC-10 group showed the strongest anti-tumor efficacy, inhibiting tumor growth as detected by ultrasonography and bioluminescence imaging. The TTAC-10 group also showed the lowest viable tumor and micro-vessel areas and the lowest Ki-67 index in histopathologic analyses. Conclusion We firstly demonstrated that TTAC-0001 effectively inhibited tumor growth and neovascularization in mouse orthotopic breast cancer model. It may provide a future treatment option for breast cancer.


Results
The TTAC-10 group showed good tumor targeting that lasted for at least 6 days and had a good anti-angiogenic effect with decreased hemoglobin content and fewer CD31-positive cells in the Matrigel plug. Compared with BVZ-10 and TTAC-2 groups, the TTAC-10 group PLOS

Introduction
Tumor angiogenesis is a potential target for anti-cancer therapy, as it plays an essential role in oxygen and nutrient supply [1,2]. Antibodies against either vascular endothelial growth factors (VEGFs) or their receptors have been developed to target tumor angiogenesis [3,4]. Bevacizumab, the first approved anti-angiogenic agent to target VEGF itself, achieved notable success as a novel targeted drug to treat several cancers, including colon, renal, and non-small cell lung cancer. Although its therapeutic efficacy is limited, it is generally used as part of a combination treatment regimen. Targeting VEGF receptors (VEGFRs) is an alternative approach to inhibit angiogenesis in tumors. In particular, inhibition of the VEGFR-2/kinase insert domain receptor (KDR) has potential anti-angiogenic effects because VEGFR-2 is the major signaling regulator of VEGF/ VEGFR pathways [5]. From this perspective, TTAC-0001, a human anti-VEGFR-2/KDR monoclonal antibody, was developed. TTAC-0001 binds to the VEGF-binding domain of VEGFR-2 and neutralizes the biological activity of VEGFR-2 by blocking the binding of VEGF [6]. Preclinical research revealed potential anti-tumor activity of TTAC-0001 in colorectal, non-small-cell lung cancer and glioblastoma tumor models [6][7][8][9][10]. Recently, a phase I clinical trial of TTAC-0001 was completed, and a phase IIa trial is ongoing. However, there have been no previous studies of TTAC-0001 for breast cancer.
In terms of chemotherapy for breast cancer, the major challenge is to develop an effective regimen for triple-negative breast cancer [11]. Bevacizumab had been incorporated as a second-line chemotherapy regimen for metastatic triple-negative breast cancer, but was revoked by the FDA due to inadequate therapeutic effect, suggesting that targeting the VEGF ligand itself may not be the best strategy [12,13]. Therefore, an alternative approach, inhibition of VEGFR-2/KDR, is worthy of investigation for treatment of triple-negative breast cancer.
Bioimaging plays important roles in anti-cancer drug research [14,15]. Multiple modalities such as magnetic resonance imaging, positron emission tomography have been applied for the oncologic drug development and each modality showed its own values to facilitate the development steps. Particularly, size measurement with ultrasonography (US) provides more accurate values of regressed tumor volumes [16,17] when the drugs are treated and the utilization of optical imaging (fluorescence and bioluminescence) enables the studies of mode of action mechanisms and biodistribution of the drugs [18].
The validation of TTAC-0001 in triple-negative breast cancer has not been performed yet. Therefore, we aimed to investigate the drug mechanism and anti-tumor efficacy of TTAC-0001, a novel anti-angiogenic agent, in a mouse orthotopic breast cancer model using multimodal bioimaging.

Cells and drugs
Human breast cancer cells (MDA-MB-231) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and were used in cell culture and animal experiments. For the establishment of MDA-MB-231+luc cells, MDA-MB-231 cells were transfected with a lentiviral vector containing the firefly luciferase reporter gene. Both MDA-MB-231 and MDA-MB-231+luc cells were cultured in Dulbecco's modified Eagle's medium (Welgene, Seoul, Korea) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (GIBCO, Seoul, Korea).

Animal experiments
All animal experiments were performed according to our Institutional Animal Care and Use Committee approved protocol. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Asan Medical Center (IACUC Number 2015-12-066). Female Balb/c nude mice (n = 91), 6 weeks old and weighing 20-25 g, were used. In our experiment, we used an orthotopic xenograft tumor model in which suspended cells were injected into the right mammary fat pad of the animals. The mice were treated with therapeutic agents approximately 2 weeks after tumor implantation, when the tumor diameter had reached approximately 5-6 mm in diameter (70-130 mm 3 in volume). All therapeutic agents were dissolved in saline (0.1 mL) and injected intraperitoneally. Detailed information regarding the number of animals and experimental groups is presented in Fig 1. Our study was composed of three experimental parts as follows: 1. Biodistribution of the drug, focusing on tumor targeting, by serial fluorescence imaging after injecting fluorescently labeled drug.
2. Use of Matrigel plug angiogenesis assays to evaluate the mode of action of the anti-angiogenic effect.
3. Monitoring of anti-tumor efficacy of the drugs by ultrasonography and bioluminescence imaging.

Biodistribution for tumor targeting
Experimental groups. For the biodistribution assay, the animals were divided into three groups: a control group administered immunoglobulin G (n = 5), a TTAC Ã -2 group treated TTAC-0001 is a fully human monoclonal antibody derived from a fully human single chain variable fragment phargelibrary. The TTAC-0001 neutralizes the vascular endothelial growth factor receptor2/vascular endothelial growth factor axis, which is a major mediator of tumor-angiogenesis, and therefore blocks angiogenesis and inhibits tumor growth and metastasis.
Labeling of TTAC-0001. To perform fluorescent labeling of TTAC-0001, we used a commercial kit (Alexa Fluor1 647 Antibody Labelling Kit, Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's protocol. Briefly, we induced a conjugation reaction between the Alexa Fluor1 647 reactive dye and TTAC-0001, and the purified labeled TTAC-0001 was collected. To determine the degree of labeling, the concentration of the dye was calculated based on absorbance at 280 and 650 nm. The collected labeled drug was stabilized at room temperature for 1 h and then intravenously injected into the mice.
Serial fluorescence imaging. Biodistribution of Alexa Fluor1 647-labeled TTAC-0001 was performed using the IVIS Lumina II machine (PerkinElmer, Waltham, MA, USA). Mice were anesthetized with 1% isoflurane (Forane1, Choongwae, Korea), and Alexa Fluor1 647-labeled TTAC-0001 was intravenously injected at doses of 2 and 10 mg/kg. Serial fluorescence images were then acquired at baseline and at 1, 2, 4, and 8 h and 1, 2, 4, and 6 days after drug injection. Imaging was performed with an exposure time of 1 s and an f/stop of 1. Using Living Image1 4.2 software (Caliper Life Sciences, Hopkinton, MA, USA), the peak total signal was measured by placing regions of interest (ROIs) at the tumors, which reflected the accumulated amount of Alexa Fluor1 647-labeled drug.
Matrigel plug angiogenesis assay. We used Matrigel plug angiogenesis assay (Corning, NY, USA) to evaluate the in vivo anti-angiogenic effect of TTAC-0001. It is based on the fact that VEGF secreted from Matrigel tumor cells exerts pro-angiogenic effects on the surrounding tissue, thus promoting neovascular development that is visualized in the Matrigel. If a drug inhibits the effect of VEGF by inhibiting either VEGF or VEGFR, then the decreased levels of neovascular development in the Matrigel can be evaluated. Matrigel (0.5 mL) was premixed with 5 × 10 6 MDA-MB-231 cells and then engrafted into the right mammary fat pad of Balb/c nude mice (n = 5). A single 2 or 10 mg/kg treatment of TTAC-0001 or saline was administered to the respective groups by intravenous injection after implantation. After 10 days, Matrigel plugs were removed and frozen for immunofluorescence analysis. To measure the hemoglobin (Hb) content, excised plugs (n = 5 plugs/group) were cut into small pieces and placed in 500 uL of cold, distilled water at 4˚C overnight to liquefy the Matrigel. Specimens were centrifuged at 1500 rpm for 20 mins, and the supernatant was collected. Hb content was quantified using a Hb assay kit (Sigma-Aldrich) and spectrophotometry.
The immunofluorescence images were analyzed using NIH-Image J software (National Institutes of Health, Bethesda, Maryland, USA). A free-hand ROI was drawn around the tumor, and CD31-positive cells were selected using the global histogram-derived thresholding method [19]. The percentage area of CD31-positive cells was calculated by dividing the area of the CD31-positive cells by the area of the tumor ROI.

Anti-tumor efficacy
To evaluate the anti-tumor efficacy of the drugs, we used two types of cells and imaging methods as follows: (1) MDA-MB-231 cells for ultrasonography (US) evaluation and (2) MDA-MB-231+luc cells for bioluminescence imaging (BLI). The reason why we used imaging for tumor monitoring is that the orthotopic tumors in the mammary fat pad may be barely visible, thus making it very difficult to measure tumor size if the tumor decreases during treatment. In addition, utilization of both US and BLI modalities allows a more accurate evaluation of the tumor volume and viability, respectively.
Mice were monitored daily for up to 30 days after treatment for tumor volume, body weight, and general body condition, such as appearance, food/water intake, respiration, and ambulation. Animals were euthanized when they showed signs of distress, when the tumors were >2 cm in diameter, when the weight loss was >15% of body weight, or when the tumor interfered with the ability to eat or drink.
Ultrasonography. All US examinations were performed by a board-certified radiologist (K.W.K) using an iU22 unit (Philips Healthcare, Bothell, WA, USA) with a 20 MHz linear transducer. To assess tumor volume, we measured the longest diameter of the tumor on the axial and coronal axes and the height of the tumor every 3 days during the 1 month follow-up. We calculated the tumor volume according to the following formula: 4.19 × axial longest diameter × coronal longest diameter × height.
Bioluminescence imaging. BLI is an established in vivo imaging techniques assessing assessing angiogenesis and evaluating the efficacy of angiogenesis-directed therapies [20,21]. BLI was performed using the IVIS Lumina II machine (PerkinElmer, Waltham, MA, USA). Mice were anesthetized with 1% isoflurane (Forane1, Choongwae, Korea), then D-Luciferin (Caliper Life Sciences, Hopkinton, MA, USA) dissolved in phosphate-buffered saline (PBS; 1.5 mg luciferin/100 μL PBS) was injected intraperitoneally at a dose of 150 mg luciferin/kg, and serial images were acquired with an exposure time of 10 s, an f/stop of 1, and pixel binning of 8, over 20 min. The BLI was performed at baseline (i.e., 2 h before injecting therapeutic agents) and after treatment (every 5 days during the 1 month follow-up). Using Living Image1 4.2 software (Caliper Life Sciences, Hopkinton, MA, USA), we measured the total flux (photons/s) of the tumor bioluminescence signal by placing an ROI at the tumor.
Histopathologic study. All animals were euthanized at day 30 after treatment. The extracted tumors were perfused with PBS and fixed with 4% paraformaldehyde in PBS. Tumors were then embedded in paraffin and sectioned at a thickness of 5 μm at the largest tumor area.
To evaluate the tumor morphology and extent of viable tumor, hematoxylin and eosin (H&E) staining was performed. In representative sections of the tumor, H&E images were analyzed using NIH-Image J software. The viable tumor cells were regarded as cells stained with H&E in both the nucleus and cytoplasm, whereas necrotic/apoptotic areas were regarded as cells stained with eosin only or no stain. After drawing ROIs around the whole tumor, viable cells were selected based on the hematoxylin staining of the nuclei using the global histogramderived thresholding method [19]. The percentage of viable tumor area was calculated by dividing the area of viable cells by the whole tumor area.
To evaluate the microvessel areas, immunohistochemical staining for a vascular endothelial antigen, CD31, was performed using a primary rabbit anti-mouse CD31 antibody (BD Pharmingen, San Diego, CA, USA) and a secondary goat anti-rabbit antibody (1:1000, Molecular Probes, Eugene, OR, USA). The images of immunohistochemical stains were analyzed using NIH-Image J software to calculate microvessel area. In the tumor ROI, microvessels were selected based on areas of CD31-positive cells [19]. The percentage microvessel area was calculated by dividing the area of CD31-positive cells by the area of the tumor ROI.
To evaluate tumor cell proliferation (i.e., the Ki-67 index), immunohistochemical stating for Ki-67 was performed using an anti-mouse Ki-67 antibody (1:100, Novus Biologicals, CO, USA). Detection was performed by incubating with the Dako EnVision + System HRP-labeled polyclonal anti-rabbit antibody (Agilent Inc., Palo Alto, CA, USA) for 30 min, followed by DAB chromogen (Agilent Inc.). In the tumor ROI, the Ki-67-positive cells were selected based on brownish nuclear staining using the global histogram-derived thresholding method [19]. We set the threshold to select the hematoxylin-stained nuclei and count the number of whole tumor cells. Then we reset the color threshold to select the brownish nuclei and count the number of Ki-67-stained tumor cells. The Ki-67 index was calculated by dividing the number of Ki-67-positive cells by the number of whole tumor cells.

Statistical analyses
For the Matrigel plug angiogenesis assay, Hb content and percentage area of CD31-positive cells in the four different treatment groups were compared using one-way ANOVA with a post-hoc t-test with least significant difference significance (LSD).
For US and BLI analyses, the tumor volume on US and signal intensity on BLI over time (every 3 days on USI and every 5 days on BLI) using a repeated measures analysis of variance and post-hoc comparison tests with LSD were used. In addition, we compared the mean values of the tumor volume on US and signal intensity on BLI at 30 days in the four different treatment groups using one-way ANOVA with a post-hoc t-test with LSD.
For histopathologic analysis, we compared the percentage viable tumor area, percentage microvessel area, and Ki-67 index at 30 days in the four different treatment groups using oneway ANOVA with a post-hoc t-test with LSD.
Statistical analysis was performed using a computer software package (SPSS, version 21.0; SPSS; Chicago, IL, USA).

Biodistribution for tumor targeting
After injecting Alexa Fluor1 647-labeled TTAC-0001, a focal signal appeared in the right flank area after 24 h and was detected in both TTAC-2 and TTAC-10 groups, but not in the control group. These results are suggestive of tumor targeting of TTAC-0001 to the right mammary fat pad of the mouse. The TTAC-10 group demonstrated higher fluorescence accumulation at the tumor implantation site and a longer fluorescence accumulation of up to 6 days than TTAC-2 group (Fig 2), indicating a higher and longer level of tumor targeting in the high-dose TTAC-10 group.

Mode of action of the anti-angiogenic effect
Matrigel plugs from TTAC-0001-treated groups were pale white in appearance, but those from the control group were bright red (Fig 3), indicating a reduction in new blood vessel formation in the TTAC-0001-treated groups. The Hb content in the Matrigel plugs was the highest in the control group, followed by BVZ-10, TTAC-2, and TTAC-10 groups. A significant difference was noted between both TTAC-0001-treated groups (TTAC-2 and TTAC-10 groups) and the BVZ-10 group (P = 0.004 and P = 0.004, Fig 3). However, there was no significant difference between the TTAC-2 and TTAC-10 Matrigel plugs (P = 0.971, Fig 3).
The percentage area of CD31-positive cells showed a similar tendency in that the TTAC-2 and TTAC-10 groups had a significantly reduced area of CD31-positive cells compared with the control group and the BVZ-10 group (P < 0.05, Fig 3). These results consistently indicated that the mode of action of TTAC-0001 was an anti-angiogenetic effect, which was the strongest in the TTAC-10 group, followed by the TTAC-2 and BVZ-10 groups.

Anti-tumor efficacy
Of the 56 mice involved in the anti-tumor efficacy evaluation, none were found dead during the 30-day treatment period. No significant differences in weights were seen between the four groups in the US and BLI experiments (S1 Fig and S2 Fig). Signs of drug toxicity, such as ruffled fur, anorexia, cachexia, skin tenting, skin ulcerations, or toxic death [22], were not seen in any of the mice.
Serial US monitoring. Serial changes in the mean tumor volume in mice transplanted with MDA-MB-231 cells were measured every 3 days and are summarized in Table 2. The control group showed a continuous increase in tumor volume over time. In contrast, all treated groups (BVZ-10, TTAC-2, and TTAC-10) showed significantly smaller mean tumor volumes (P < 0.001, Fig 4). The efficacy of tumor growth inhibition was the strongest in the TTAC-10 group, followed by the TTAC-2 and BVZ-10 groups (P = 0.018), but the TTAC-10 group did not differ significantly from the TTAC-2 group (P = 0.182).
Serial BLI monitoring. The BLI signal intensities of MDA-MB-231+luc cell tumors showed different characteristics between the groups, as summarized in Table 3. In the control group, the signal intensity of tumors continuously increased until 30 days. In the BVZ-10 group, the signal intensity of the tumors fluctuated during the treatment period (Fig 5). In both TTAC-2 and TTAC-10 groups, the signal intensity of tumors showed a tendency to decrease. Notably, the TTAC-10 group showed a rapid drop in the signal intensity of tumors after 20 days (Fig 5). At the end of the treatment period (30 th day), the efficacy of tumor inhibition was the strongest in the TTAC-10 group, followed by the TTAC-2 and BVZ-10 groups (P = 0.040), but the TTAC-10 group did not differ from TTAC-2 group (P = 0.831).
Histopathologic findings. H&E staining demonstrated different characteristics of tumor apoptosis/necrosis between the four groups (Fig 6). Tumor cells with active mitosis were abundant in the control group, whereas they were sparse in the treatment groups. The percentage of viable tumor areas significantly differed between groups (P < 0.001, one-way ANOVA) ( Fig  6). The post-hoc test showed that the percentage of viable tumor area was the lowest in the TTAC-10 group, followed by the TTAC-2 and BVZ-10 groups, with a significant difference in each pair (P 0.016).  Immunohistochemical staining for CD31 showed that tumor vessels were abundant in the control group, but sparse in treated groups, with smaller microvessel areas (Fig 7). One-way ANOVA revealed that the percentage of microvessel area significantly differed between the four groups (P < 0.001). The post-hoc test revealed that the TTAC-10 group showed the lowest percentage of microvessel area, followed by the TTAC-2 (P = 0.001) and BVZ-10 groups. However, there was no significant difference between the TTAC-2 and TTAC-10 groups (P = 0.084). Table 3. Serial changes in the mean signal intensity from bioluminescence imaging of mice treated with bevacizumab or TTAC-0001.

Discussion
In our study, TTAC-0001, a novel fully human anti-VEGFR-2/KDR monoclonal antibody that blocks VEGF/VEGFR-2 signaling, showed selective tumor targeting that lasted at least 6 days, an anti-angiogenic effect to inhibit neovascularization, and anti-tumor efficacy. Among the three treatment groups, TTAC-10 showed the highest level of tumor targeting, anti-angiogenic effects, and anti-tumor efficacy, compared with the TTAC-2 and the BVZ-10 groups. The antitumor efficacy for inhibition of tumor growth was similar between the TTAC-2 and BVZ-10 groups. In histopathologic examination, the TTAC-10 group showed the lowest viable tumor area, microvessel area, and cellular proliferation among the treatment groups. In addition, no significant toxicity or death was noted for all treated mice. Consistent with a previous report [8], our results indicate the validity of TTAC-0001 10 mg/kg as a potent anti-angiogenic agent.
Targeting VEGFR-2/KDR may be more effective than targeting the VEGF ligand itself. Considering the fact that other VEGF-like heparin-binding growth factors can prevent specific inhibition of the anti-VEGF antibody in hypoxic tumor microenvironments, targeting VEGFR-2/KDR is more effective for anti-angiogenesis [8, 23,24]. VEGFR-2/KDR is more easily targetable than VEGF because VEGFR-2/KDR is highly expressed on the surface of the activated endothelium in tumor tissues, in contrast to VEGF, which is mainly located in the interstitial space between cells. Therefore, the VEGFR-2 antibody can more effectively inhibit neo-angiogenesis [23,24]. Moreover, different acting mechanisms of anti-VEGFR-2 antibody which inhibits the binding of VEGFR2 and all of VEGF-A, VEGF-C, and VEGF-D compared to VEGF antagonist, which only inhibits VEGF-A activity may contribute the increased antiangiogenic efficacy of TTAC-0001 [25]. These findings support our results showing that TTAC-0001 10 mg/kg has a more potent anti-angiogenic effect and anti-tumor efficacy than bevacizumab 10 mg/kg.
Preclinical results do not guarantee the success of clinical trials. However, the more information obtained in the preclinical drug development stage, the greater the probability of success in clinical trials [26]. As the researchers recognized the values of information from the Drug mechanism and efficacy of TTAC-0001 in mouse breast cancer imaging experiments, utilization of various imaging modalities has become increased. In our study, we showed the anti-tumor efficacy by using an orthotopic tumor model and US/BLI imaging methods, through which we expect to increase the translatability of our results to clinical trials [27]. Moreover, validation of targeting capacity of TTAC-0001 confirmed by the biodistribution exams can be used as a reference for predicting the mode of action and therapeutic efficacy of TTAC-0001 in humans as the TTAC-0001 has cross-reactivity between human and murine species [7]. It is expected that TTAC-0001 can binds to the N-terminus of these domains in the extracellular region of either human or mice KDR [8]. Although bevacizumab has no cross-reactivity between human and murine species, previous studies demonstrated anti-angiogenic effect in human cancer cell line xenografts [28,29].
Although the anti-tumor activity of TTAC-0001 has been shown in colorectal, non-smallcell lung cancer [8-10], our study is the first study to demonstrate anti-tumor activity of TTAC-0001 in triple-negative breast cancer. In contrast to previous studies, as we used orthotopic xenograft tumor model, the assessment of anti-tumor activity is more reliable than other tumor models, as the orthotopic xenograft models have similar tumor microenvironment as the original tumor [30,31]. In addition, we first showed the drug mechanism and the antitumor efficacy of the TTAC-0001 using bioimaging methods.
In patients with triple-negative breast cancer, anti-angiogenic agents, such as bevacizumab and ramucirumab, have not been successful [32]. In particular, bevacizumab, the first approved anti-angiogenic agent to bind all isoforms of VEGF-A, was approved for metastatic, HER2-negative breast cancer in 2008, based on the E2100 trial in which the progression-free survival was significantly improved [13,33]. However, subsequent trials did not show that improvement of progression-free survival leads to an overall survival gain in patients with metastatic breast cancer [34,35]. Although data from basic and translational science and the clinical success in improving progression-free survival may imply the effectiveness of antiangiogenic therapy for inhibition of tumor growth, anti-angiogenic agents might be effective only in a proportion of breast cancer patients as the breast cancer is regarded as a heterogenous group of tumors [36]. The next step may include the development of a more potent anti-angiogenic agent covering more targets, and the selective use of anti-angiogenic agents in a selected population of breast cancer patients [11,32,36].

Conclusions
In summary, TTAC-0001, a novel fully human monoclonal antibody against VEGFR-2/KDR, was shown to have anti-angiogenic effects and good anti-tumor efficacy for inhibition of tumor growth and neovascularization in a mouse orthotopic triple-negative breast cancer model. These preclinical results may assist translation into clinical investigation of TTAC-0001 for breast cancer patients.