Eradication of growth of HER2-positive ovarian cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate in mouse xenograft model.

ORIGINAL STUDY

Eradication of Growth of HER2-Positive Ovarian Cancer With Trastuzumab-DM1, an Antibody-Cytotoxic Drug Conjugate in Mouse Xenograft Model Lin Yu, PhD,*Þ Yuxi Wang,* Yuqin Yao, PhD,* Wenting Li,* Qinhuai Lai,* Jun Li,* Yongjun Zhou,* Tairan Kang, PhD,* Yongmei Xie, PhD,* Yangping Wu,* Xiangzhen Chen,* Cheng Yi, PhD,*Þ Lantu Gou, PhD,*Þ and Jinliang Yang, PhD*Þ Objective: Ovarian cancer is 1 kind of a highly malignant gynecologic tumor, and current treatments have not achieved satisfactory effects. Human epidermal growth factor receptor 2 (HER2)Ytargeted therapies including trastuzumab and trastuzumab-DM1 (T-DM1) (antibody-cytotoxic drug conjugates) have been applied to treat HER2-overexpressing breast cancers in clinic. In the present study, we explored whether T-DM1 could effectively treat HER2-positive human ovarian carcinoma in vitro and in vivo. Methods: HER2 expressions of 6 ovarian cancer cell lines and 2 breast carcinoma cell lines were validated, and the binding capacity of T-DM1 to HER2-positive ovarian cancer SKOV3 cells were analyzed by flow cytometry. Nude mice bearing intraperitoneal and subcutaneous SKOV3 xenografts were used to investigate the antitumor effect of T-DM1. Results: High HER2 expressions in SKOV3 cell lines were detected. The binding capacity of T-DM1 to HER2-positive SKOV3 cells was in a similar manner comparing with trastuzumab. In vitro, T-DM1 showed strong growth inhibitory on SKOV3 cells, with IC50 values of 0.15 nmol/L. Nude mice bearing intraperitoneal and subcutaneous SKOV3 xenografts were used to investigate the antitumor effects of T-DM1 in vivo. In subcutaneous xenografts model, T-DM1 (30 mg/kg and 10 mg/kg) indicated significant anticancer effects. It is noteworthy that tumors were completely eradicated in the T-DM1 (30 mg/kg) group, and no regrowth was observed in a long time after the termination of the treatment. In the peritoneal xenograft model, tumor nodules in 3 of 7 mice were hardly observed in the abdominal cavity of mice after intraperitoneal injection of T-DM1 (30 mg/kg). At the same time, tumor nodules from the other 4 mice weighed on the average of only 0.07 g versus 1.77 g in control group. Conclusions: Our data showed that T-DM1 possessed promising antitumor effects on HER2-overexpressing ovarian cancer in mouse model, which provided valuable references for the future clinical trials. Key Words: HER2, Ovarian cancer, Antibody drug conjugates, Trastuzumab-DM1 Received November 22, 2014, and in revised form April 6, 2014. Accepted for publication April 29, 2014. (Int J Gynecol Cancer 2014;24: 1158Y1164)

*State Key Laboratory of Biotherapy, and †Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, China. Address correspondence and reprint requests to Lantu Gou and Jinliang Yang, State Key Laboratory of Biotherapy and Cancer Copyright * 2014 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0000000000000179

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Center, West China Hospital, West China Medical School, Sichuan University, People’s South Rd 17, Chengdu 610041, China. E-mail: [email protected]; [email protected]. This study was supported by the National Natural Science Foundation of China (no. 30872742, no. 81071818, and no. 81372822) and the National Science and Technology Major Project of China (2012ZX09103301-030). The authors declare no conflicts of interest.

International Journal of Gynecological Cancer

& Volume 24, Number 7, September 2014

Copyright © 2014 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.



cancer is 1 of the 3 major gynecologic maligO varian nancies and causes the first death rate in tumors in the

female reproductive system.1 Reports showed that the 5-year survival rate of all patients with ovarian carcinoma was less than 40%.2 So far, satisfactory treatments have not been found; therefore it is an urgent need to develop new drugs to improve the survival rate of patients. Human epidermal growth factor receptor 2 (HER2), also known EGFR2, has been demonstrated to highly express in many tumors, and thus HER2 has been used as therapy target in many solid tumors such as breast cancer, gastric cancer, ovarian cancer, nonYsmall cell lung cancer, prostate cancer, glioma, and bladder cancer.3Y9 Trastuzumab (Herceptin), a humanized monoclonal antibody against HER2, has been used as a treatment in combination with chemotherapeutic agents in patients with HER2-positive breast cancer and gastric cancer.10,11 It was reported that percentage of HER2positive cases of overexpression in invasive epithelial ovarian cancer varied from 1.8% to 76%.12 Studies about HER2targeted therapy in human ovarian cancer have been explored in recent years, and there are potential values of antibodytargeted therapy for ovarian cancer.13 The combination of monoclonal antibody and chemotherapy had increased effects on tumors with reducing the doses of chemotherapy drugs.14 As a new emerging class of antibody-based drugs, antibody-cytotoxic drug conjugates (ADCs) have been developed and paid more attentions for tumor-targeting therapy. The conjugates have been endowed with a degree of tumor selectivity and impressive anticancer activity, and thus enhance their therapeutic index even at lower doses.15,16 Trastuzumab-DM1 (T-DM1), trastuzumab conjugated with highly cytotoxic molecule DM1 (derivative of maytansine 1), has been approved by the Food and Drug Administration in 2013 to use as treatment for patients with HER2-positive metastatic breast cancer.17 In this study, we explored whether T-DM1 could effectively treat HER2-positive human ovarian carcinoma cells in vitro and in vivo. The results showed that T-DM1 possessed promising antitumor effects on HER2-overexpressing ovarian cancer in mouse model, which provided valuable references for the future clinical trials.

MATERIALS AND METHODS Cell Lines Tumor cell lines (ovarian carcinoma cell lines SKOV3, ES-2, A2780s, A2780cp, PA-1, and HO8910 and breast carcinoma cell lines SKBR-3 and MCF-7) were obtained from the American Type Culture Collection. Cells (SKBR-3, MCF-7, SKOV3, ES-2, A2780s, A2780cp, and PA-1) were cultured in high-glucose DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 Hg/mL). HO8910 cells were cultured in RPMI-1640 supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 Hg/mL). All cells were maintained in a humidified 5% CO2 atmosphere at 37-C.18

Trastuzumab-DM1 in Mouse Xenograft Model

Herceptin and T-DM1 Trastuzumab (Herceptin) was purchased from Roche. The T-DM1, an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent DM1 via the free amino group of lysine residue of antibody and maleimide moiety of DM1-MCC, was prepared in our laboratory. Briefly, the antibody trastuzumab and DM1MCC were mixed in a molar ratio of 1:10 in conjugation buffer 50 mM (NaH2PO4/Na2HPO4)/50 mM NaCl/2 mM EDTA, pH 6.5. and stirred overnight at 25-C and centrifuged for 5 minutes at 17,000g. The supernatant was purified by size-exclusion chromatography (Thermo Pierce Zeba Spin Desalting Columns and Devices, 7K MWCO; 4.5 mmol/L, histidine buffer [pH 6.0]) to yield T-DM1 conjugates, and the drug/antibody ratio of T-DM1 was kept around 3.2, which was calculated through the differential absorption of 252 nm and 280 nm measured by the ultraviolet spectrophotometer.19,20

Flow Cytometric Analysis

For flow cytometric analysis, trypsinized cells (1 106) were centrifuged and resuspended in 100 KL PBS (pH 7.2). Then the cells were incubated with a concentration (10 Kg/mL) of trastuzumab, T-DM1, or PBS for 30 minutes at 4-C, respectively. After incubation, cells were washed 3 times with PBS and then labeled with FITC-conjugated AffiniPure goat antiYhuman IgG (Jackson ImmunoResearch) about 20 minutes at 4-C. After washing 3 times with PBS, the fluorescence intensity of FITC was determined using flow cytometer (BD FACSCalibur).21 Three independent experiments of flow cytometric analysis were conducted.

MTT Assay The growth inhibition of the T-DM1 conjugates and trastuzumab on ovarian cancer cells and breast cancer cells were determined by an MTT (Amresco) assay. Cells (1.6Y4.5 103/ well) were seeded in a 100-KL medium per well in 96-well plates. After 24 hours of culture, cells were treated with trastuzumab or T-DM1 of various concentrations in culture medium, and the culture medium was used as control. After 72 hours posttreatment, a volume of 20 KL sterile MTT (5 mg/mL) solution was added to each well. After 2 to 3 hours of incubation, the medium was aspirated from the wells as completely as possible without disturbing the formazan crystals and cells on the plastic surface. A 150-KL volume of DMSO was added to each well. Plates were agitated on a shaker for 5 minutes, and the absorbance at 570 nm was measured by enzyme-linked immunosorbent assay reader (Thermo, Multiskcan MK3). All experiments were conducted independently in triplicate. The effects of each agent on the proliferation of ovarian cancer cell lines were expressed as the percentage of cell growth inhibition using the following formula: %inhibition = [(A570 of control j A570 of treated cells)/A570 of control] 100%.22 In the statistical analysis, the significant difference of T-DM1Ytreated and trastuzumabtreated cells was statistically analyzed by paired Student t test, and the results were considered statistically significant when P G 0.05.

* 2014 IGCS and ESGO


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In Vivo Antitumor Activity Female Balb/c nude mice (age, 5Y6 weeks) were purchased from Beijing HFK Bioscience and were acclimated for 1 week before the experiment. Animal experiments were approved by the Institutional Animal Care and Treatment Committee of the State Key Laboratory of Biotherapy in Sichuan University. All in vivo experiments in the study of the antitumor effect of T-DM1 have been independently repeated twice. To establish subcutaneous xenograft model, the mice were given a single subcutaneous injection with 1 107 SKOV3 cells suspended in a 100-HL cell culture medium (DMEM). One week later, the mice were divided into 5 groups, and each group has 5 mice. T-DM1 (3, 10, and 30 mg/kg), trastuzumab (30 mg/kg), and a control (vehicle) were administered via tail vein injection to mice every 3 days with a total of 3 times. For the subcutaneous xenograft model, tumor volume and body weight were monitored every 3 days throughout the treatment. When the subcutaneous xenografts were more than 2000 mm3, the mice were euthanized, and the experiment was terminated. To establish intraperitoneal xenograft, the mice were given peritoneal injections supplemented with Matrigel Basement Membrane Matrix (BD Bioscience) with 1 107 SKOV3 cells suspended in a 100-HL cell culture medium (DMEM). One week later, the mice were also divided into 5 groups, and each group has 7 mice. T-DM1 (3, 10, and 30 mg/kg), trastuzumab (30 mg/kg), and a control (vehicle) were administered via intraperitoneal injection to mice every 3 days with a total of 3 times. For the intraperitoneal xenograft model, mice were only weighed every 3 days and were killed when the mean normal body weight from control mice decreased to 14 g, and tumor nodules in the abdominal cavity were collected to be photographed and weighed.23


RESULTS HER2 Expression in Ovarian Cancer Cell Lines The expression of HER2 in ovarian cancer can be tested by immunohistochemistry, fluorescence in situ hybridization, chromogenic in situ hybridization, and enzyme-linked immunosorbent assay.12 In this study, the expressions of HER2 in 6 kinds of ovarian cancer cell lines and 2 kinds of breast cancer cell lines were analyzed by flow cytometric. High HER2 expressions in SKOV3 and SKBR-3 cells were detected, and the expression of HER2 in A2780s, HO8910, A2780cp, PA-1, and MCF-7 cells were low. However, there was almost no expression of HER2 protein in the ES-2 ovarian cancer cell line (Fig. 1). The repeated experiments showed similar results.

Binding Capacity of T-DM1 and Trastuzumab to HER2-Positive Ovarian Cancer Cell Line To investigate the influence on the binding capacity of antibody after conjugating DM1-MCC, ovarian cancer HER2-positive SKOV3 cells and breast cancer HER2-positive SKBR-3 cells were incubated with T-DM1 or trastuzumab. The 3 independent results all showed that T-DM1 and trastuzumab bound to HER2-positive SKOV3 and SKBR-3 cells in a similar manner, which indicated conjugating trastuzumab with DM1, did not influence the binding capacity to HER2-positive cells (Fig. 2).

Potent Cytotoxicity of T-DM1 to HER2-Positive Cell Lines In Vitro The T-DM1 conjugates were endowed with both tumor targeting from trastuzumab and stronger tumor killing from

FIGURE 1. HER2 expression of human ovarian and breast cancer cell lines. Ovarian cancer cell lines (A) SKOV3, (B) ES-2, (C) PA-1, (D) A2780s, (E) HO8910, and (F) A2780cp and breast cancer cell lines (G) SKBR-3 and (H) MCF-7 were incubated with PBS (purple) and trastuzumab (green), respectively. After labeling by FITC-conjugated AffiniPure goat antiYhuman IgG, fluorescence intensities were determined by flow cytometric to analyze HER2 expression. Three independent experiments were conducted, and similar results were obtained.

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FIGURE 2. Binding capacities of T-DM1 and trastuzumab to human ovarian and breast cancer cell lines. Ovarian cancer cell lines (A) SKOV3 and (B) ES-2 and breast cancer cell lines (C) SKBR-3 and (D) MCF-7 were incubated with PBS (purple), trastuzumab (green), and T-DM1 (pink), respectively. Three independent flow cytometric analyses showed that T-DM1 possessed good binding to HER2-positive SKOV3 and SKBR-3 cells but poor binding to HER2-negative MCF-7 and ES-2 cells, presenting similar binding capacity comparable with trastuzumab. DM1 in HER2-positive ovarian cancer cells. So the antiproliferative activities of T-DM1 were compared with trastuzumab

in HER2-positive cells (SKOV3 and SKBR-3) and HER2negative (ES-2 and MCF-7) cells using an MTT assay. The

FIGURE 3. In vitro cytotoxicity of T-DM1 and trastuzumab. Ovarian cancer cell lines (A) SKOV3 and (B) ES-2 and breast cancer cell lines (C) SKBR-3 and (D) MCF-7 were treated with various concentrations of T-DM1 (circle) and trastuzumab (cross) for 72 hours. Untreated cells were used as controls. Cell viability was determined by 3 independent experiments of MTT assay and calculated as the absorbance ratio of treated to control groups. Data were presented as mean T SE,*P G 0.05. * 2014 IGCS and ESGO


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results showed that T-DM1 exerted strong inhibitory effects in SKOV3 and SKBR-3 cells, with IC50 values of 0.15 nmol/L and 0.22 nmol/L, respectively (Figs. 3A, C). In contrast, the inhibitory effects of T-DM1 to HER2-negative cells (ES-2 and MCF-7) were less effective with IC50 values of more than 1200 nmol/L and 720 nmol/L, respectively (Fig. 3B, D). These independently repeated results indicated that the antitumor effect of trastuzumab after conjugation with DM1 had been increased under the same concentration in vitro. Data were presented as mean T SE (P G 0.05).

Eradication of Growth of HER2-Positive SKOV3 Xenograft Tumors by T-DM1 Subcutaneous and intraperitoneal HER2-positive SKOV3 xenograft models were used for comparing the efficacy of T-DM1 with that of trastuzumab in vivo, and all tumor progression experiments have been independently repeated twice; 2 independent experiments have obtained similar results, and a typical result of the subcutaneous and intraperitoneal xenograft models had been shown (Fig. 4). In the subcutaneous SKOV3 xenograft model, complete tumor regression was observed in mice receiving 30 mg/kg T-DM1, and no regrowth was observed in a long time after termination of the treatment. However, for the T-DM1 (10 mg/kg) treatment group, 3 of 5 mice showed complete remission without regrowth, whereas trastuzumab (30 mg/kg) and T-DM1 (3 mg/kg) showed similar


effect and no significant decrease in tumor size (Fig. 4A). Besides, the body weight of the mice from the T-DM1 (30 mg/kg and 10 mg/kg) group kept increasing after cessation of treatment, which showed low or no toxic effect (Fig. 4B). The activity of T-DM1 was also investigated in intraperitoneal xenograft model. Under the treatment with T-DM1 (10 mg/kg and 30 mg/kg), potent inhibition of tumor growth was observed. Especially in the T-DM1 (30 mg/kg) group, tumor nodules in 3 of 7 mice were hardly observed in the abdominal cavity of mice (Fig. 4C). At the same time, tumor nodules from the other 4 mice weighed on the average of only 0.07 g versus 1.77 g in control group (Fig. 4D). In the T-DM1 (3 mg/kg) group, tumor nodules were not regressed, and TDM1 (3 mg/kg) showed no significant antitumor effect. So these data indicated excellent anticancer activities of T-DM1 including in the subcutaneous and intraperitoneal xenograft models.

DISCUSSION Because of its tumor selectivity and impressive anticancer activity, ADCs have achieved hopeful progress in recent years. In this study, we prepared T-DM1, and this ADC was endowed with both tumor targeting from trastuzumab and stronger tumor killing from DM1 in HER2-positive ovarian cancer in vivo. Our experiments showed that T-DM1 possessed binding capacity to HER2-positive SKOV3 and SKBR-3 cells

FIGURE 4. Therapeutic effects of T-DM1 and trastuzumab against SKOV3 ovarian cancer xenografts. Subcutaneous or intraperitoneal tumorYbearing mice were treated with PBS, trastuzumab, and T-DM1 at indicated doses. A, T-DM1 showed more powerful inhibition of subcutaneous xenografts growth compared with trastuzumab, even at lower dose. B, Body weight of mice did not show significant changes during the treatment of subcutaneous xenografts model. C, T-DM1 showed better efficacy versus trastuzumab for treatment of intraperitoneal xenografts. D, Tumor nodules in abdominal cavity were weighed and statistically analyzed. One of 2 independent tumor progression experiments of the subcutaneous and intraperitoneal xenograft models had been shown. Data were presented as mean T SE.

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in a similar manner with that of trastuzumab. At the same time, the cytotoxic potencies of T-DM1 to all HER2-positive cell lines in vitro were both in nanomolar levels, which was similar to free DM1 but more potent than trastuzumab. Ovarian carcinoma is prone to occurring widespread abdominal metastasis, which makes it a kind of highly malignant gynecologic tumor. So the efficacies of T-DM1 were studied both in subcutaneous and intraperitoneal xenograft models. When the dose of T-DM1 was the same with that of trastuzumab (30 mg/kg), tumors were completely regressed in subcutaneous and intraperitoneal xenograft model. In the subcutaneous xenograft model, the dose of T-DM1 was only a third of the dose of trastuzumab (30 mg/kg); tumors were almost complete regression. When the dose of T-DM1 was only a third of the dose of trastuzumab (30 mg/kg), tumors were almost complete regression. However, T-DM1 (3 mg/kg) produced similar antitumor effect with trastuzumab, whereas the dose of trastuzumab was 10-fold that of T-DM1. Based on our observations, there were no influences on body weight of the mice treated with T-DM1 even at a high dose, which showed that T-DM1 has good drug tolerance to mice. As a result, T-DM1 still showed promising antitumor effect in subcutaneous and intraperitoneal xenograft models. Therefore, T-DM1 may inhibit not only primary ovarian tumor growth but also metastatic spread in the future clinical trials. Compared with chemotherapeutic drugs, antibodytargeted therapeutics with target selectivity and a substantial reduction of toxicity are of great significance for cancer therapy. In addition, studies have shed light on the mechanisms of action of antibodies, and the main recognized antitumor functions of antibody were perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), and induction of adaptive immunity.24 In addition, ADCC plays a key role in the in vivo antitumor effect of trastuzumab.25 In this study, the ADC conjugate T-DM1 showed that the cytotoxic potencies to all HER2-positive ovarian cancer cell lines in vitro were both in nanometer levels, which was similar to free DM1 but more potent than trastuzumab. The results indicated that the T-DM1 conjugates were endowed with both tumor selectivity from trastuzumab and impressive anticancer activity from DM1. The following reasons could account for why trastuzumab were less effective both in vitro and in vivo than T-DM1. First, trastuzumab can only disturb tumor cell signaling to indirectly kill tumor cells in vitro and cannot mediate the effect of ADCC to kill tumor cells without peripheral blood mononuclear cells in vitro. Second, the incubation time after drug treatment in this study was much shorter, and the method of detecting the growth inhibition of trastuzumab on HER2-positive cells was different from the initial study.21,26 Third, clinical studies about the effects of trastuzumab showed that patients should continue to be given administration of trastuzumab with an initial dose and followed by another dose. However, for the subcutaneous and intraperitoneal xenograft model created in this study, trastuzumab was respectively administered only via tail vein injection and intraperitoneal injection to mice every 3 days with a total of 3 times. Besides, clinical studies about trastuzumab showed that patients should continue to be given administration of trastuzumab with an


initial dose and followed by another dose.27 However, for the subcutaneous and intraperitoneal xenograft model created in this study, trastuzumab was respectively administered only via tail vein injection and intraperitoneal injection to mice every 3 days with a total of 3 times. So T-DM1 was more effective than trastuzumab both in vitro and in vivo. In summary, T-DM1 is shown to be highly effective in HER2-positive ovarian cancer models, indicating that T-DM1 represents an important new approach for treating HER2positive human ovarian cancer. These promising results provide valuable references for the future clinical studies to assess the safety and efficacy of T-DM1 in patients through HER2targeted therapies.

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