An anti-TROP2 monoclonal antibody TrMab-6 exerts antitumor activity in breast cancer mouse xenograft models

Trophoblast cell surface antigen 2 (TROP2), reported to be overexpressed in several types of cancer, is involved in cell proliferation, invasion, metastasis, and poor prognosis of many types of cancer. Previously, a highly sensitive anti-TROP2 monoclonal antibody (clone TrMab-6; mouse IgG2b, κ) was developed using a Cell-Based Immunization and Screening (CBIS) method. TrMab-6 was useful for investigations using flow cytometry, western blot, and immunohistochemistry. The aim of the present study was to investigate whether TrMab-6 possesses in vitro antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activities or in vivo antitumor activities using mouse xenograft models of TROP2-overexpressed CHO-K1 (CHO/TROP2) and breast cancer cell lines, including MCF7, MDA-MB-231, and MDA-MB-468. In vitro experiments revealed that TrMab-6 strongly induced ADCC and CDC activities against CHO/TROP2 and the three breast cancer cell lines, whereas it did not show those activities against parental CHO-K1 and MCF7/TROP2-knockout cells. Furthermore, in vivo experiments on CHO/TROP2 and MCF7 ×enografts revealed that TrMab-6 significantly reduced tumor growth, whereas it did not show antitumor activities against parental CHO-K1 and MCF7/TROP2-knockout xenografts. The findings suggest that TrMab-6 is a promising treatment option for TROP2-expressing breast cancers.


Introduction
The loss of epithelial features in tumors, known as epithelial-mesenchymal transition (EMT), is significantly involved in the malignant transformation of cancers, such as tumor initiation, migration, and metastasis (1,2). Previous findings have identified several molecules associated with the maintenance of the epithelial features of cells (3). Epithelial cell adhesion molecule (EpCAM) is a cell adhesion transmembrane molecule, which is overexpressed in tumors. EpCAM is also known as trophoblast cell surface antigen 1 (TROP1) and it is encoded by the tumor-associated calcium signal transducer 1 (TACSTD1) gene (3). Trophoblast cell surface antigen 2 (TROP2), another molecule of the TACSTD gene family, was identified as a cell surface marker for invasive trophoblast cells (4). TROP2 is a promising therapeutic target (5), and its expression is associated with cancer malignancy in various solid tumors including breast cancers (6).
TROP2 is a 46-kDa type I transmembrane glycoprotein (323 amino acids), which consists of a large extracellular domain (274 amino acids) with four N-glycosylation sites, a transmembrane domain (23 amino acids), and a short intracellular domain (26 amino acids). TROP2 possesses 49% identity and 67% similarity with EpCAM (4,5), and is expressed in normal tissues, such as skin, kidney, liver, breast, ureteric bud, and renal tubules. TROP2 is highly expressed during the development of mammalian embryos and fetus (4,7,8).
TROP2 has been reported to be overexpressed in cancers, and is involved in cell proliferation, invasion, metastasis, and poor prognosis in many cancer types (9)(10)(11)(12). It has been reported that membrane-localized TROP2 becomes an unfavorable target of prognosis, while the intracellular retention of TROP2 is associated with less frequent tumor relapse and better survival in breast cancer patients (13). A high expression of TROP2 and low expression of E-cadherin are associated with lymph node status, metastasis, tumor/node/metastasis (TNM) stage, and ER/PR/HER2 expression, indicating that TROP2 is considered to have a potential role in the promotion of EMT (14). Furthermore, TROP2 has been reported to be involved in the chemotherapeutic resistance against lung cancer (15).
Previously, we developed a highly sensitive anti-TROP2 monoclonal antibody (mAb; clone TrMab-6; mouse IgG 2b , kappa) (16) using a Cell-Based Immunization and Screening (CBIS) method (17). TrMab-6 was useful for investigations using flow cytometry, western blot, and immunohistochemistry (16). The aim of this study was to investigate whether TrMab-6 possesses in vitro antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activities and in vivo antitumor activities using breast cancer models.
Flow cytometry. Cells (2x10 5 cells/ml) were harvested after brief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin (BSA, Nacalai Tesque, Inc.) in PBS the cells were treated with 1 µg/ml of an anti-TROP2 mAb or control (1% BSA in PBS; blocking buffer) for 30 min at 4˚C, and then with Alexa Fluor 488-conjugated anti-mouse IgG (1:1,000; product no. 4408; Cell Signaling Technology, Inc.). Fluorescence data were collected using flow cytometer: SA3800 Cell Analyzer (Sony Biotechnology Corp.). ADCC. ADCC stimulation by an anti-TROP2 mAb was assayed as follows. Five female five-week-old BALB/c nude mice (mean weight, 15±3 g) were purchased from Charles River Laboratories, Inc. Mice were kept under specific pathogen-free conditions on an 11-h light/13-h dark cycle at a temperature of 23±2˚C and 55±5% humidity with food and water supplied ad libitum during the experimental periods. After euthanasia by cervical dislocation, spleens were removed aseptically, and single-cell suspensions were obtained by forcing spleen tissues through a sterile cell strainer (product no. 352360; Corning, Inc.) with a syringe. Erythrocytes were lysed with 10-sec exposure to ice-cold distilled water. The splenocytes were then washed with DMEM and resuspended in DMEM with 10% FBS; this preparation was designated as effector cells. The target tumor cells were labeled with 10 µg/ml Calcein-AM (Thermo Fisher Scientific, Inc.) and resuspended in the same medium. The target cells were transferred to 96-well plates, at 2x10 4 cells/well, and mixed with effector cells at an effector-to-target ratio of 100:1, along with 100 µg/ml of an anti-TROP2 mAb or control mouse IgG 2b . After a 5-h incubation at 37˚C, the Calcein-AM release into the supernatant was measured for each well. Fluorescence intensity was assessed using a microplate reader (Power Scan HT; BioTek Instruments, Inc.) with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Cytotoxicity (as % lysis) was measured using the formula: Percentage of lysis (%)=(E-S)/(M-S) x100, where E is the fluorescence released in combined cultures of target cells and effector cells, S is the spontaneous fluorescence released in cultures of only target cells, and M is the maximum fluorescence measured after lysis of all cells with buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4), and 10 mM EDTA. Animal studies for ADCC and the antitumor activity were approved by the Institutional Committee for experiments of the Institute of Microbial Chemistry (permit no. 2020-015).
CDC. CDC stimulation by an anti-TROP2 mAb was assayed as follows. Target cells were labeled with 10 µg/ml Calcein-AM (Thermo Fisher Scientific, Inc.) and resuspended in medium. Target cells were plated in 96-well plates, at 2x10 4 cells/well, and 10% rabbit complement (Low-Tox-M rabbit complement; Cedarlane Laboratories) and 100 μg/ml of an anti-TROP2 mAb or control IgG (mouse IgG 2b ) were added to each well. After 5 h of incubation at 37˚C, the Calcein-AM release into the supernatant was measured for each well. Fluorescence intensity was calculated as described in the ADCC section above.
Antitumor activity of an anti-TROP2 mAb in a mouse xenograft model. Sixty-four five-week-old female BALB/c nude mice (mean weight, 15±3 g) were purchased from Charles River Laboratories, Inc., and were divided into the following four groups (n=16 in each group): i) CHO/TROP2-bearing mice, ii) CHO-K1-bearing mice, iii) MCF7-bearing mice, and iv) BINDS-29-bearing mice. On day 7, each group was subdivided into 2 groups (n=8 in each group) with equal mean tumor volume: A control mouse IgG-treated group or an anti-TROP2 mAb-treated group. All animal experiments were performed in accordance with institutional guidelines and regulations to minimize animal suffering and distress in the laboratory. The Institutional Committee for experiments of the Institute of Microbial Chemistry (permit no. 2020-015) approved the animal studies for antitumor activity.
Mice were maintained in a pathogen-free environment, on an 11-h light/13-h dark cycle at a temperature of 23±2˚C and 55±5% humidity, with food and water supplied ad libitum throughout the experiments. Mice were monitored for health and weight every three or four days. Experiments on mice were conducted in four weeks. Weight loss >25% or tumor volume >3,000 mm 3 was identified as humane endpoints for euthanasia. At humane and experimental endpoints, mice were euthanized by cervical dislocation, and death was verified by validating respiratory and cardiac arrest.
After an acclimation period of one week, these mice were used in experiments at six weeks of age (mean weight, 16±2 g). Cells (0.3 ml of 1.33x10 8 cells/ml in DMEM) were mixed with 0.5 ml BD Matrigel Matrix Growth Factor Reduced (BD Biosciences). A total of 100 µl of this suspension (5x10 6 cells) was injected subcutaneously into the left flank of each animal. On day 7 post-inoculation, 100 µg of an anti-TROP2 mAb or control mouse IgG in 100 µl PBS was injected intraperitoneally (i.p.). Additional antibody inoculations were performed on days 14 and 21. Twenty-four days after cell implantation, all mice were euthanized by cervical dislocation, and tumor diameters and volumes were measured and recorded.
Statistical analysis. Data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was conducted with Welch's t-test for ADCC and CDC, ANOVA and Sidak's multiple comparisons tests for tumor volume and mouse weight, and Welch's t-test for tumor weight. All calculations were performed using GraphPad Prism 7 (GraphPad Software, Inc.). P<0.05 was considered statistically significant.
These favorable ADCC/CDC activities indicated that TrMab-6 may induce strong antitumor activity against breast cancer cells in vivo.
Similarly, tumor formation of 16 CHO-K1-bearing mice was observed on day 7, before they were divided into a TrMab-6-treated group and a control group. On days 7, 14 and 21 after CHO-K1 cell injections, TrMab-6 (100 µg) or control mouse IgG (100 µg) were injected i.p. into the mice. Tumor volume was measured on days 7, 10, 14, 17, 21 and 24 after CHO-K1 cell injection. Both TrMab-6-treated and control groups exhibited similar tumor growth on all days (Fig. 4B, upper panel) and no difference in the tumor weight was observed between the two groups on day 24 (Fig. 4B, middle panels). These results indicated that TrMab-6 did not reduce the growth of TROP2-negative CHO-K1 xenografts. Additionally, the total body weights did not significantly differ between the two study groups (Fig. 4B, lower panel).
Similarly, the tumor formation of 16 BINDS-29-bearing mice was observed on day 7, before the 16 BINDS-29-bearing mice were divided into a TrMab-6-treated group and a control group. On days 7, 14 and 21 after BINDS-29 cell injections into the mice, TrMab-6 (100 µg) or control mouse IgG (100 µg) was injected i.p. into the mice. The tumor volume was measured on days 7, 10, 14, 17, 21 and 24 after BINDS-29 cell injection. The TrMab-6-treated and control groups exhibited similar tumor growth on all days (not significant; Fig. 5B, upper panel) and no difference in the tumor weight was observed between the two groups, on day 24 (Fig. 5B, middle panels). These results indicated that TrMab-6 did not reduce the growth of TROP2-negative BINDS-29 xenografts. Total body weights did not significantly differ between the treatment and control groups (Fig. 5B, lower panel).

Discussion
TROP2 has been demonstrated to be overexpressed in a variety of tumors (18). A gene expression pattern analysis comparing gastric tumors and their normal counterparts revealed that TROP2 was not overexpressed in normal tissues (19). In addition, in a meta-analysis that included 16 studies involving 2,569 participants, TROP2 overexpression was found to be associated with poor overall and disease-free survival across several types of solid tumors (20). Furthermore, the knockdown of TROP2 decreased cell proliferation and migration (21). Altogether, these results suggest that TROP2 is a potential target for antitumor treatments. Antibody-based therapy is a rapidly emerging field for treatment of several diseases, including cancer. The development of antibody drugs for TROP2 has been accelerated in recent years due to identification of the extracellular domain of TROP2 as a potential prominent target for TROP2-positive cancers (5,6).
Among them, antibody-drug conjugates (ADCs) are the main modality of antibody drugs (22). Recently, the first anti-TROP2 ADC, sacituzumab govitecan, which is a humanized IgG 1 conjugated to irinotecan metabolite (SN-38), has been approved by the US Food and Drug Administration against metastatic triple-negative breast cancers (23). ADCs have also been developed against hormone receptor-positive breast cancers and HER2-negative metastatic breast cancers (23). Preliminary findings have demonstrated that datopotamab deruxtecan (DS-1062) is active in patients with advanced or metastatic non-small cell lung cancer (24). In phase I trials of datopotamab deruxtecan, this drug induced responses in almost 25% of the patients trialed and had manageable side effects (24). The combination of these ADCs with immune checkpoint inhibitors is also expected to be effective (5,22,23,25).
The adaptive or acquired resistance to targeted antibody cancer therapies is of importance for clinical outcomes (26). Development of new antibodies and improvement of antibody-based drugs are required to overcome therapeutic resistance and reduce the possibility of identifying suitable candidates for clinical application (27). In this study, we demonstrated the efficacy of a new anti-TROP2 antibody, TrMab-6. The development of anti-TROP2 ADC is a potential therapeutic option for cancer patients with therapy-resistant solid tumors by itself or in combination with other anticancer drugs. Furthermore, TrMab-6 can be used in flow cytometry, immunohistochemistry, and western blot analyses (16). In histopathology, immunohistochemistry is used for clinical diagnosis for biopsies and resected specimens. TrMab-6 may be used to ascertain patients who should receive the anti-TROP2-targeted therapy.
In the present study, we investigated whether TrMab-6 (16), developed using the CBIS method, could exhibit ADCC/CDC activities in vitro and antitumor activity in vivo against breast cancers. In vitro experiments revealed strong ADCC/CDC inducement against CHO/ TROP2, MCF7, MDA-MB-231, and MDA-MB-468 cells by TrMab-6 (Figs. 2 and 3). In vivo experiments on CHO/ TROP2 (Fig. 4) and MCF7 (Fig. 5) xenografts revealed that the TrMab-6 treatment significantly reduced tumor growth, compared with the control mouse IgG. By contrast, TrMab-6 did not demonstrate ADCC/CDC in vitro (Figs. 2 and 3) and antitumor activity in vivo against TROP2-negative CHO-K1 (Fig. 4) and BINDS-29 (Fig. 5), demonstrating that the toxicity of TrMab-6 is specific for TROP2. These data indicated that TrMab-6 is a promising treatment option for TROP2-expressing breast cancers. For future studies, several modalities, such as ADC or chimeric antigen receptor (CAR)-T of TrMab-6, should be developed to strengthen the antitumor activity against breast cancers.