Humanized Anti-hepatocyte Growth Factor Monoclonal Antibody (YYB-101) Inhibits Ovarian Cancer Progression

Current chemotherapy regimens have certain limitations in improving the survival rates of patients with advanced ovarian cancer. Hepatocyte growth factor (HGF) is important in ovarian cancer cell migration and invasion. This study assessed the effects of YYB-101, a humanized monoclonal anti-HGF antibody, on the growth and metastasis of ovarian cancer cells. YYB-101 suppressed the phosphorylation of the HGF receptor c-MET and inhibited the migration and invasion of SKOV3 and A2780 ovarian cancer cells. Moreover, the combination of YYB-101 and paclitaxel synergistically inhibited tumor growth in an in vivo ovarian cancer mouse xenograft model and significantly increased the overall survival (OS) rate compared with either paclitaxel or YYB-101 alone. Taken together, these findings suggest that YYB-101 has therapeutic potential in ovarian cancer when combined with conventional chemotherapy agents.


INTRODUCTION
Ovarian cancer has the highest mortality rate among gynecological diseases and is the fifth leading cause of cancer deaths among women in the US. The American Cancer Society estimated that in 2018, 22,240 women in the US were diagnosed with ovarian cancer and 14,070 died of this disease. The 5 year overall survival (OS) rate of patients with stage III or higher ovarian cancer is <29% (1). Because ovarian cancer progresses without symptoms, it is likely to have reached an advanced stage, with metastases to the peritoneal cavity, at initial diagnosis (2). The metastatic process of ovarian carcinoma is believed to involve the attachment of cancer cells shed by ovarian tumors to the surface of the peritoneum or to organs inside the peritoneum (3). Cancer cell seeding of the peritoneal cavity is associated with ascites, which ultimately progresses to high-grade carcinomas (4).
The standard treatment for ovarian cancer is cytoreductive surgery, followed by combination platinum and taxane-based chemotherapy (5). The most widely used combination is paclitaxel and carboplatin (6,7). However, chemotherapy does not significantly improve the OS rate of patients diagnosed with advanced stage disease, mainly because of tumor resistance to chemotherapeutic agents (8). Efforts to improve clinical outcomes include co-treatment with targeted and chemotherapeutic agents. For example, the addition of bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), or cetuximab, a monoclonal antibody against epidermal growth factor receptor (EGFR), was found to increase survival rates in patients with ovarian cancer in chemotherapy (9,10). These findings suggest that targeted therapeutic agents may be effective and improve survival rates in patients with ovarian cancer.
Hepatocyte growth factor (HGF) is a scatter factor that promotes cell proliferation, migration, and invasion (11,12). HGF binds to the HGF receptor c-MET, inducing several biological activities involved in cancer progression. The HGF/c-MET axis especially affects the migration of cancer cells from the primary site to other organs by promoting epithelialmesenchymal transition (EMT), which initiates cancer cell metastasis (13,14). HGF also stimulates the proliferation and inhibits the apoptosis of ovarian cancer cells, thereby enhancing cell survival (15,16). Moreover, HGF concentrations were shown to be elevated in the ascitic fluid of ovarian cancer patients, suggesting that HGF enhances ovarian cancer cell migration and peritoneal dissemination (17,18). In addition, c-MET inhibitors such as PF-2341066, foretinib and DCC-2701 have shown effective antitumor activities in ovarian cancer xenograft models (19)(20)(21).
YYB-101 is a humanized monoclonal antibody against HGF and a potential cancer treatment. Treatment of a mouse xenograft model of human colorectal cancer with a combination of YYB-101 and irinotecan, a chemotherapeutic agent used to treat colorectal cancer, effectively inhibited tumor progression (22). Treatment of a mouse xenograft model of human glioblastoma with YYB-101 and temozolomide (TMZ) resulted in a 2-fold higher survival rate than treatment with TMZ alone did (23). These findings suggested that combination therapy with YYB-101 and chemotherapeutic agents may inhibit tumor progression, including in ovarian cancer.
Therefore, in the present study, we assessed the effects of YYB-101 on ovarian cancer cells and a mouse xenograft model of ovarian cancer. The results of this study suggested that a combination of YYB-101 and conventional chemotherapeutic agents may be effective in treating patients with ovarian cancer by effectively inhibiting tumor metastasis and growth.
A2780 cells overexpressing firefly luciferase were generated as previously described (24). Briefly, A2780 cells were transfected with the firefly luciferase reporter plasmid pGL 4.51 (luc2/CMV/Neo; Promega, Madison, WI, USA) using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). The cells were cultured in medium containing 100 µg/ml G418 to select positive clones. The expression of luciferase was determined using a Dual-Luciferase reporter assay system (Promega) and luminescence was measured using a Victor luminometer (Perkin Elmer, Waltham, MA, USA).
For drug combination experiments, synergistic effects were evaluated by the combination index (CI), which was calculated using CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA). CI values were interpreted as follows: CI < 1 indicated synergism, CI > 1 antagonism, and CI = 1 additive effect.

Intraperitoneal Xenograft Mouse Model of Ovarian Cancer
All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC, NCC-16-342) of the National Cancer Center, Republic of Korea. Luciferase-overexpressing A2780/luc cells (1 × 10 7 in 200 µL phosphate-buffered saline [PBS]) were intraperitoneally injected into 7 week-old female BALB/c-nude mice (Orient bio, Korea). Three days later, the mice were intravenously injected with 40 mg/kg YYB-101 twice weekly and with 10 mg/kg paclitaxel once weekly. Ovarian cancer progression was monitored using bioluminescence imaging using the in vivo Imaging System (IVIS; Caliper Life Science, Waltham, MA, USA), and bioluminescence was quantified using Living Images software with identical standardized square regions of interest (ROI).

Orthotopic Mouse Xenograft Model of Ovarian Cancer
Female BALB/C-nude mice were anesthetized with isoflurane and the right lateral sides of their abdomens were incised. The ovarian bursa was ejected, A2780/luc cells (1 × 10 5 cells in 10 µL) were injected into the ovarian bursa, and the incision site was closed. One week later, the entire ovary of each mouse was excised and the mice were randomly grouped (n = 4-6 per group). Mice were injected intraperitoneally with 10 mg/kg paclitaxel once weekly and intravenously with 15 or 30 mg/kg YYB-101 twice weekly. Mice were monitored using the IVIS bioluminescence imaging device once weekly for 15 weeks.

Statistical Analysis
All statistical analyses were performed using the GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). The survival rate of xenografts was calculated using the Kaplan-Meier plots and compared using the log-rank test. Differences were analyzed with the Student's t-test or one-way analysis of variance (ANOVA) and a p < 0.05 was considered significant.
Migration and invasion assays were performed to determine whether HGF affects the movement of ovarian cancer cells. Using a Transwell system, we found that the migration ( Figure 1A) and invasion ( Figure 1C) of A2780/luc cells were maximal after treatment with 80 ng/mL HGF. The migration of SKOV3 cells was also maximal following treatment with 80 ng/mL HGF (Figure 1B), whereas invasiveness was HGF concentrationdependent ( Figure 1D). In another experiment, we measured the HGF concentration of A2780/luc cells of the upper and lower chambers in the migration assay system without adding exogenous HGF to the lower chamber. As a result, we found that the concentration of HGF was gradually increased in the upper chamber after 8 h. However, the concentration of HGF was not detected in the lower chamber (data not shown). These data suggest that A2780/luc cells secrete HGF; however, the concentration and secretion period are not sufficient to affect the migration and invasion of the cells in this assay system. It is also possible that the initial concentration in the lower chamber is important to induce A2780/luc migration and invasion in this assay system.

YYB-101 Inhibits Metastasis of Ovarian Cancer Cells in vitro and Suppresses Phosphorylation of C-MET
We next tested the effect of YYB-101 on the migration and invasion of ovarian cancer cells. Cells were treated with 80 ng/mL HGF to induce migration, followed by treatment with 0.3 or 3 µM YYB-101 (Figure 2). The migration of A2780/luc and SKOV3 cells was reduced following treatment with 0.1 µM crizotinib, a small molecule inhibitor of lymphoma kinase (ALK), the c-ros oncogene (ROS1), and c-MET and was dosedependently inhibited by YYB-101. The effect of YYB-101 on ovarian cancer cell invasiveness was tested using a Transwell system coated with Matrigel. Similar to its effects on migration, YYB-101 reduced the invasiveness of A2780/luc, SKOV3, Caov-3 and JHOC-5 ovarian cancer cells (Figure 3). Because paclitaxel is used as a first-line chemotherapeutics for patients with advanced ovarian cancer, we thought that co-treatment with YYB-101 and paclitaxel might have a synergistic effect on inhibition of invasion and migration of ovarian cancer cells. When A2780 cells were co-treated with paclitaxel and YYB-101, the migration, and invasion rates were effectively inhibited (Figure 4). These findings suggested that the HGF-induced migration and invasion of ovarian cancer cells was effectively inhibited by YYB-101.
The expression level of c-MET, the only known receptor for HGF, was assessed in SKOV3 cells using western blot analysis. In untreated and hIgG-treated SKOV3 cells, c-MET was phosphorylated on Tyr 1234/1235 ; however, phosphorylated c-MET was not detected in YYB-101-treated SKOV3 cells ( Figure S2A). In addition, treatment with YYB-101 reduced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, which occurs downstream of c-MET, compared with that in untreated or hIgG-treated cells. Furthermore, we found that c-MET phosphorylation was reduced in YYB-101-treated Caov-3 cells, as well as in SKOV3 cells (Figure S2B). Similarly, crizotinib treatment decrease c-MET activation at Tyr 1234/1235 while PTX treatment did not.
Furthermore, some studies showed that HGF does not increase the viability of ovarian cancer cells (26,27). We confirmed these results by WST-1 assay in SKOV3 and A2780 cells (Figure S3). Therefore, our results suggested that HGF-induced migration and invasion were not influenced by cell growth.

YYB-101 Effectively Reduces the Progression of Ovarian Cancer in vivo
The effect of YYB-101 in vivo was examined using mouse xenograft models established by injecting A2780/luc cells intraperitoneally or implanting them orthotopically into the ovaries. In the intraperitoneal model, mice were treated with paclitaxel and YYB-101, starting 3 days after intraperitoneal injection, and bioluminescence imaging was performed weekly (Figure 5A). Following implantation of ovarian cancer cells, bioluminescence emission increased throughout the abdomens of control mice and those treated with YYB-101 or paclitaxel alone ( Figure 5B). In contrast, bioluminescence emission was significantly reduced throughout the abdomens of mice cotreated with paclitaxel and YYB-101 (p < 0.01). Total bioluminescence (ROI) was also significantly lower in mice treated with paclitaxel and YYB-101 than in control mice ( Figure 5C). Survival rates for up to 100 days after A2780/luc cell implantation were improved in mice treated with paclitaxel,  YYB-101, or both compared with the control mice, with the OS rate being significantly higher in mice treated with paclitaxel and YYB-101 than in control mice ( Figure 5D). The survival rate of the co-treatment group analyzed using the Log-Rank test was significantly (p < 0.001) different from that of the control group (Figure 5E). Change in body weight did not differ between mice treated with YYB-101 and control mice ( Figure 5F). Statistical analyses of body weight changes showed no significant differences between each experimental group and the control group.
Another in vivo mouse model, the orthotopic xenograft model, mimicked the treatment paradigm used to treat patients with ovarian cancer, consisting of cytoreductive surgery followed by paclitaxel treatment. The orthotopic xenograft model was designed to confirm the effect of paclitaxel and YYB-101 on cancer cells disseminated into the peritoneal cavity after cytoreductive surgery. We assessed the efficacy of YYB-101 in the orthotopic mouse xenograft model ( Figure 6A) and co-treatment with YYB-101 and paclitaxel reduced the bioluminescence emission in the abdomen, whereas paclitaxel alone did not ( Figure 6B). Mice treated with paclitaxel and YYB-101 had a significantly higher OS rate than mice treated with paclitaxel alone or control mice did (Figure 6C). We assessed whether cotreatment had a synergistic effect compared to single treatment, and used the Kaplan-Meier and Log-Rank test to analyze the statistically significant correlations with OS time. The  In the lower chamber, 1 pM paclitaxel, 0.3 µM YYB-101, or paclitaxel plus YYB-101 was added to the serum free culture media with 80 ng/mL hepatocyte growth factor (HGF). After 48 h culture, migrated cells were observed using light microscopy. (B) A2780/luc (2 × 10 5 ) cells were seeded onto Matrigel-coated upper chambers. In the lower chamber, 1 pM paclitaxel, 0.3 µM YYB-101, or paclitaxel plus YYB-101 was added to serum free culture medium with 80 ng/mL HGF. After 72 h culture, migrated cells were observed using light microscopy. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control, one-way ANOVA, followed by Tukey's multiple comparison test.
result showed that the survival rate of the co-treatment group was prolonged more than that of the single treatment group (Figure 6D). These findings indicate that, when combined with a chemotherapeutic agent, YYB-101 can inhibit ovarian cancer progression in vivo.

DISCUSSION
The standard treatment regimen for patients diagnosed with ovarian cancer consists of surgery followed by chemotherapy. Conventional chemotherapy for ovarian cancer consists of combinations of carboplatin with taxane-based agents such as paclitaxel (6,7,28). Adjuvant chemotherapy was found to improve OS and recurrence rate in patients with early stage (I to IIA) ovarian cancer (29). However, 60-70% of patients are diagnosed with stage III or IV ovarian cancer or abdominal metastasis, and the recurrence rate in patients with advanced stage ovarian cancer is over 70% (7,30). Therefore, it is important to provide new treatment options, such as new targeted therapy agents, for patients with advanced stage ovarian cancer.
Several co-treatments, including chemotherapeutic and targeted therapy agents, may overcome drug resistance and improve the efficacy of chemotherapy. Clinical trials have shown that bevacizumab, a monoclonal anti-VEGF antibody, administered alone or in combination with chemotherapy, was effective in patients with recurrent ovarian cancer (10).  Because EGFR is overexpressed in up to 70% of ovarian cancers and is associated with poor prognosis, monoclonal anti-EGFR antibodies such as cetuximab have been added to paclitaxel and platinum-based agents in patients with recurrent ovarian cancer (9,30). However, clinical trials of cetuximab in combination with paclitaxel and cisplatin showed only modest efficacy, including an 18 month progression free survival (PFS) rate of only 38.8% (9). The EGFR kinase inhibitor erlotinib and antagonistic antibody panitumumab have also shown limited success, improving PFS by 12.7 and 2.7 months, respectively (31,32). Although several targeted agents, such as those against poly ADP ribose polymerase (PARP) and insulin-like growth factor receptor (IGFR), have been tested in clinical trials, they did not significantly improve PFS or OS (33,34). New targeted agents are needed because current targeted agents have shown only limited success.
The results of the present study suggest that HGF has promise as a target molecule for the treatment of patients with ovarian cancer. HGF activates the c-MET signaling pathway, which stimulates the invasive and metastatic potential of various tumor cells (13,14). The HGF/c-MET axis has recently become a therapeutic target for the treatment of various types of cancer. In several studies, c-MET inhibitors such as crizotinib and foretinib effectively inhibited the development and metastasis of ovarian cancer in animal models (19,20,35). HGF is expressed in normal ovarian epithelial cells and benign ovarian tumor cells, but to a higher degree in ovarian cancer cells (36,37). Our results demonstrated that high levels of HGF were secreted by various ovarian cancer cell lines and HGF was involved in the increased migration and invasiveness of ovarian cancer cells.
YYB-101 was developed as a humanized neutralizing monoclonal antibody against HGF. YYB-101 was shown to inhibit HGF-induced scattering in MDCK-2 cells and block the phosphorylation of ERK, a downstream signaling molecule of c-MET that affects cell proliferation (23). YYB-101 was also found to be effective in various cancer models, including mouse xenograft models of colorectal cancer and glioblastoma, when coadministered with a chemotherapeutic agent (22,23).
The present study verified the efficacy of YYB-101 in two mouse xenograft models of ovarian cancer, a widely used intraperitoneal model and a model where the ovaries were surgically removed after orthotopic xenografting. Although the efficacy of YYB-101 alone was similar to that of paclitaxel alone, their combination was highly effective, inhibiting the peritoneal progression of cancer cells and enhancing OS. These results suggested that co-treatment with YYB-101 and paclitaxel may significantly improve the survival rate by inhibiting the progression of ovarian cancer. paclitaxel induces the formation dysfunctional mitotic spindles, resulting in cell death through an apoptosis pathway. Despite of its activity, ∼60% of patients with advanced stage ovarian cancer receiving standard treatment, consisting of cytoreductive surgery followed by chemotherapy with paclitaxel, experienced recurrence or drug resistance within 6 months, reducing their survival rate (38). However, we found that YYB-101 increased survival rate when combined with standard treatment, suggesting that adding YYB-101 to the standard treatment may improve outcomes.
In conclusion, the present study demonstrated that HGF was blocked by YYB-101, inhibiting the growth of ovarian cancer cells through the signaling pathway mediated by c-MET, the target receptor for HGF. However, although YYB-101 alone did not significantly inhibit metastasis of ovarian cancer, it showed synergistic effects with paclitaxel by inhibiting ovarian cancer progression in vivo. Further studies would be needed to elucidate the mechanism of the synergistic effect of combination therapy with YYB-101 and paclitaxel and to confirm the efficacy of YYB-101 in the xenograft model using various ovarian cancer cell types. Based on these results, co-treatment with YYB-101 and chemotherapeutic agents may overcome the limitations of chemotherapeutic agents alone, enhancing the treatment of advanced ovarian cancer. Currently, YYB-101 is undergoing Phase I (NCT02499224) clinical trials for solid cancers. The combination regimen of YYB-101 plus chemotherapy may benefit ovarian cancer patients who receive chemotherapy after cytoreductive surgery, which should be confirmed in Phase II clinical trials.

DATA AVAILABILITY
All datasets generated for this study are included in the manuscript and/or the Supplementary Files.