ONCOLOGY REPORTS 46: 132, 2021

An anti‑TROP2 monoclonal antibody TrMab‑6 exerts
antitumor activity in breast cancer mouse xenograft models
TOMOHIRO TANAKA1*, TOMOKAZU OHISHI2*, TEIZO ASANO1, JUNKO TAKEI1,3,
REN NANAMIYA1, HIDEKI HOSONO1, MASATO SANO1, HIROYUKI HARADA3,
MANABU KAWADA2, MIKA K. KANEKO1 and YUKINARI KATO1,4
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Aoba‑ku, Sendai,
Miyagi 980‑8575; 2Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
Numazu‑shi, Shizuoka 410‑0301; 3Department of Oral and Maxillofacial Surgery,
Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo‑ku, Tokyo 113‑8510;
4
New Industry Creation Hatchery Center, Tohoku University, Aoba‑ku, Sendai, Miyagi 980‑8575, Japan
Received February 27, 2021; Accepted April 21, 2021
DOI: 10.3892/or.2021.8083
Abstract. Trophoblast cell surface antigen 2 (TROP2),
reported to be overexpressed in several types of cancer, isinvolved in cell proliferation, invasion, metastasis, and poor
prognosis of many types of cancer. Previously, a highly sensi‑
tive anti‑TROP2 monoclonal antibody (clone TrMab‑6; mouse
IgG2b, κ) was developed using a Cell‑Based Immunizationand Screening (CBIS) method. TrMab‑6 was useful forinvestigations using flow cytometry, western blot, and immuno‑
histochemistry. The aim of the present study was to investigate
whether TrMab‑6 possesses in vitro antibody‑dependent
cellular cytotoxicity (ADCC) or complement‑dependent cyto‑
toxicity (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

Correspondence to: Professor Yukinari Kato, New Industry
Creation Hatchery Center, Tohoku University, 2‑1 Seiryo‑machi,
Aoba‑ku, Sendai, Miyagi 980‑8575, JapanE‑mail: yukinarikato@med.tohoku.ac.jp*

Contributed equally

Abbreviations: ADC, antibody‑drug conjugate; ADCC, antibodydependent cellular cytotoxicity; BSA, bovine serum albumin; CBIS,
Cell‑Based Immunization and Screening; CDC, complementdependent cytotoxicity; DMEM, Dulbecco's modified Eagle's
medium; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine
serum; mAb, monoclonal antibody; PBS, phosphate‑buffered saline;
PVDF, polyvinylidene difluoride; SEM, standard error of the mean
Key words: TROP2, monoclonal antibody, ADCC, CDC, antitumor
activity, breast cancer

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 xenografts
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 find‑ings suggest that TrMab‑6 is a promising treatment option for
TROP2‑expressing breast cancers.
Introduction
The loss of epithelial features in tumors, known as epithe‑lial‑mesenchymal transition (EMT), is significantly involved in
the malignant transformation of cancers, such as tumor initia‑
tion, migration, and metastasis (1,2). Previous findings haveidentified several molecules associated with the maintenanceof 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), anothermolecule of the TACSTD gene family, was identified as a cellsurface marker for invasive trophoblast cells (4). TROP2 is apromising therapeutic target (5), and its expression is associ‑ated 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 extracellulardomain (274 amino acids) with four N‑glycosylation sites, atransmembrane domain (23 amino acids), and a short intracel‑lular domain (26 amino acids). TROP2 possesses 49% identityand 67% similarity with EpCAM (4,5), and is expressed innormal tissues, such as skin, kidney, liver, breast, uretericbud, and renal tubules. TROP2 is highly expressed during thedevelopment 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,

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TANAKA et al: ANTITUMOR ACTIVITIES OF AN ANTI-TROP2 MAB

and poor prognosis in many cancer types (9‑12). It has beenreported that membrane‑localized TROP2 becomes an unfa‑
vorable target of prognosis, while the intracellular retentionof TROP2 is associated with less frequent tumor relapse andbetter survival in breast cancer patients (13). A high expressionof TROP2 and low expression of E‑cadherin are associatedwith 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 promo‑tion of EMT (14). Furthermore, TROP2 has been reported to
be involved in the chemotherapeutic resistance against lungcancer (15).
Previously, we developed a highly sensitive anti‑TROP2
monoclonal antibody (mAb; clone TrMab‑6; mouse IgG2b,
kappa) (16) using a Cell‑Based Immunization and Screening(CBIS) method (17). TrMab‑6 was useful for investigations
using flow cytometry, western blot, and immunohistochem‑
istry (16). The aim of this study was to investigate whether
TrMab‑6 possesses in vitro antibody‑dependent cellularcytotoxicity (ADCC) or complement‑dependent cytotoxicity(CDC) activities and in vivo antitumor activities using breast
cancer models.
Materials and methods
Cell lines. CHO‑K1 and the breast cancer cell lines,
MDA‑MB‑231 and MDA‑MB‑468 were obtained from theAmerican Type Culture Collection. The breast cancer cellline MCF7 was obtained from the Cell Resource Center for
Biomedical Research Institute of Development, Aging andCancer, at Tohoku University, Japan. C‑terminal PA‑tagged
TROP2‑overexpressed CHO‑K1 (CHO/TROP2) was previ‑
ously established by transfection of pCAG/TROP2‑PA to
CHO‑K1 cells using Lipofectamine LTX Reagent (Thermo
Fisher Scientific, Inc.) (16). The TROP2 gene‑knockout cellline, MCF7/TROP2‑KO (BINDS‑29), was previously generated
by transfection of CRISPR/Cas9 plasmids targeting TROP2(http://www.med‑tohoku‑antibody.com/topics/001_paper_cell.
htm), using the Neon Transfection System (Thermo Fisher
Scientific, Inc.). Stable transfectants were established by cellsorting using SH800 (Sony Biotechnology Corp.) (16). CHO‑K1,
CHO/TROP2, MCF7, and BINDS‑29 were cultured in Roswell
Park Memorial Institute (RPMI)‑1640 medium (Nacalai
Tesque, Inc.). MDA‑MB‑231 and MDA‑MB‑468 were cultured
in Dulbecco's modified Eagle's medium (DMEM; Nacalai
Tesque, Inc.). RPMI‑1640 and DMEM were supplemented with
10% heat‑inactivated fetal bovine serum (FBS; Thermo Fisher
Scientific Inc.), 100 U/ml of penicillin (Nacalai Tesque, Inc.),
100 µg/ml streptomycin (Nacalai Tesque, Inc.), and 0.25 µg/ml
amphotericin B (Nacalai Tesque, Inc.), and incubated at 37˚C ina humidified atmosphere containing 5% CO2.
Primary antibodies. Purified mouse IgG (cat. no. I8765)
and mouse IgG2b (cat. no. M1395) were purchased from
Sigma‑Aldrich; Merck KGaA. An anti‑TROP2 mAb was puri‑
fied using Protein G‑Sepharose (GE Healthcare Biosciences).
Western blot analysis. Cell pellets were resuspended inphosphate‑buffered saline (PBS; Nacalai Tesque, Inc.) with
1% Triton X‑100 (cat. no. 168‑11805; FUJIFILM Wako Pure

Chemical Corporation) and 50 µg/ml aprotinin (productno. 03346‑84; Nacalai Tesque, Inc.). Cell debris was removed bycentrifugation at 21,880 x g for 10 min at 4˚C. Protein concen‑
tration was determined by BCA method. Cell lysates wereboiled in sodium dodecyl sulfate sample buffer with a reducing
reagent (Nacalai Tesque, Inc.). These proteins (10 µg) were
electrophoresed on 5‑20% polyacrylamide gels (FUJIFILM
Wako Pure Chemical Corporation) and transferred onto polyvi‑
nylidene difluoride (PVDF) membranes (Merck KGaA). After
blocking with 4% skim milk (Nacalai Tesque, Inc.) at room
temperature for 30 min, the membranes were incubated withprimary antibodies, such as 1 µg/ml of TrMab‑6 or anti‑β‑actin
for control (clone AC‑15; Sigma‑Aldrich; Merck KGaA) at room
temperature for 30 min, followed by incubation with secondary
peroxidase‑conjugated anti‑mouse immunoglobulins (1:1,000;
cat. no. P044701‑2; Agilent Technologies Inc.) at room
temperature for 30 min. Finally, the proteins were visualizedwith ImmunoStar LD (cat. no. 290‑69904; FUJIFILM Wako
Pure Chemical Corporation) or Pierce™ ECL Plus Western
Blotting Substrate (cat. no. 32132; Thermo Fisher Scientific,Inc.), and were detected using the Sayaca‑Imager (DRC Co.
Ltd.). Qcapture Pro software (DRC Co. Ltd) was used for thedensitometry.
Flow cytometry. Cells (2x105 cells/ml) were harvested after
brief exposure to 0.25% trypsin in 1 mM ethylenediamine‑
tetraacetic acid (EDTA; Nacalai Tesque, Inc.). After washingwith 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 specificpathogen‑free conditions on an 11‑h light/13‑h dark cycle at
a temperature of 23±2˚C and 55±5% humidity with food andwater supplied ad libitum during the experimental periods.
After euthanasia by cervical dislocation, spleens were removed
aseptically, and single‑cell suspensions were obtained byforcing spleen tissues through a sterile cell strainer (productno. 352360; Corning, Inc.) with a syringe. Erythrocytes werelysed 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 designatedas effector cells. The target tumor cells were labeled with
10 µg/ml Calcein‑AM (Thermo Fisher Scientific, Inc.) andresuspended in the same medium. The target cells were trans‑ferred to 96‑well plates, at 2x104 cells/well, and mixed witheffector cells at an effector‑to‑target ratio of 100:1, along with
100 µg/ml of an anti‑TROP2 mAb or control mouse IgG2b.
After a 5‑h incubation at 37˚C, the Calcein‑AM release into
the supernatant was measured for each well. Fluorescenceintensity 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

ONCOLOGY REPORTS 46: 132, 2021

(as % lysis) was measured using the formula: Percentage
of lysis (%)=(E‑S)/(M‑S) x100, where E is the fluorescencereleased in combined cultures of target cells and effector
cells, S is the spontaneous fluorescence released in culturesof only target cells, and M is the maximum fluorescencemeasured 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 wereapproved 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 asfollows. 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 2x104 cells/well,and 10% rabbit complement (Low‑Tox‑M rabbit complement;
Cedarlane Laboratories) and 100 μg/ml of an anti‑TROP2 mAbor control IgG (mouse IgG2b) were added to each well. After 5 hof incubation at 37˚C, the Calcein‑AM release into the super‑
natant was measured for each well. Fluorescence intensity wascalculated as described in the ADCC section above.
Antitumor activity of an anti‑TROP2 mAb in a mouse xeno‑
graft 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, andiv) BINDS‑29‑bearing mice. On day 7, each group was subdi‑
vided 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 performedin accordance with institutional guidelines and regulations
to minimize animal suffering and distress in the laboratory.
The Institutional Committee for experiments of the Instituteof Microbial Chemistry (permit no. 2020‑015) approved theanimal 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˚Cand 55±5% humidity, with food and water supplied ad libitum
throughout the experiments. Mice were monitored for healthand weight every three or four days. Experiments on mice
were conducted in four weeks. Weight loss >25% or tumor
volume >3,000 mm3 was identified as humane endpoints foreuthanasia. At humane and experimental endpoints, mice wereeuthanized by cervical dislocation, and death was verified byvalidating 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 1.33x108 cells/ml in DMEM) weremixed with 0.5 ml BD Matrigel Matrix Growth Factor
Reduced (BD Biosciences). A total of 100 µl of this suspen‑sion (5x106 cells) was injected subcutaneously into the left
flank of each animal. On day 7 post‑inoculation, 100 µg ofan anti‑TROP2 mAb or control mouse IgG in 100 µl PBS was
injected intraperitoneally (i.p.). Additional antibody inocula‑tions were performed on days 14 and 21. Twenty‑four days
after cell implantation, all mice were euthanized by cervicaldislocation, and tumor diameters and volumes were measured
and recorded.

3

Statistical analysis. Data are expressed as mean ± standard
error of the mean (SEM). Statistical analysis was conductedwith Welch's t‑test for ADCC and CDC, ANOVA and Sidak's
multiple comparisons tests for tumor volume and mouseweight, 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.
Results
Western blot analysis. We performed western blot analysis
using TrMab‑6. TrMab‑6 detected TROP2 with a 40‑kDa
band in CHO/TROP2 (16), MCF7, MDA‑MB‑231, andMDA‑MB‑468 cells; however, it did not detect any proteinsin CHO‑K1 and BINDS‑29 cells (Fig. 1A), indicatingthat TrMab‑6 is specific for TROP2. As TROP2 is overexpresed in CHO/TROP2, the band in the CHO/TROP2cell was broader than that of the other cells, such as MCF7,
MDA‑MB‑231, and MDA‑MB‑468. We used β ‑actin as aninternal control.
Flow cytometry. We investigated whether TrMab‑6 can reactwith CHO‑K1, CHO/TROP2 (16), MCF7, BINDS‑29 (MCF7/
TROP2‑KO), MDA‑MB‑231, and MDA‑MB‑468 by flow
cytometry. We used a blocking buffer as negative control.
TrMab‑6 recognized the CHO/TROP2 cells, but not theparental CHO‑K1 cells (Fig. 1B). TrMab‑6 also recognized theendogenous TROP2 in MCF7 breast cancer cells (Fig. 1B). By
contrast, the reaction of TrMab‑6 to BINDS‑29 was lost after
the knockout of TROP2 in MCF7 cells (Fig. 1B), indicatingthat TrMab‑6 is specific for TROP2. TrMab‑6 also detected
TROP2 of MDA‑MB‑231 and MDA‑MB‑468 (Fig. 1B).
ADCC and CDC activities of TrMab‑6 in TROP2‑expressing
cell lines. The effect of TrMab‑6 (mouse IgG2b) in the ADCC
and CDC activity in TROP2‑expressing cells, such as CHO/
TROP2 (16) or MCF7, MDA‑MB‑231, and MDA‑MB‑468
breast cancer cell lines, was analyzed. First, TrMab‑6
exhibited higher ADCC (63.2% cytotoxicity) in CHO/
TROP2 cells than that of the control mouse IgG2b (40.9%
cytotoxicity; P<0.05) (Fig. 2). By contrast, TrMab‑6 did notshow any ADCC activity in CHO‑K1 cells compared with therespective control (Fig. 2). Additionally, TrMab‑6 exhibited
higher ADCC (53.3% cytotoxicity) in MCF7 cells that in thecontrol mouse IgG2b (22.7% cytotoxicity; P<0.05); however,no ADCC activity was observed in BINDS‑29 cells (Fig. 2).
TrMab‑6 also exhibited higher ADCC (34.2% cytotox‑
icity) in MDA‑MB‑231 cells than that of the control mouse
IgG2b (16.4% cytotoxicity; P<0.05) (Fig. 2). Furthermore,
TrMab‑6 exhibited higher ADCC (40.2% cytotoxicity) in
MDA‑MB‑468 cells than that of the control mouse IgG2b(18.7% cytotoxicity; P<0.01) (Fig. 2).
TrMab‑6 was also associated with more robust CDC activity(67.7% cytotoxicity) in CHO/TROP2 cells compared to control
mouse IgG2b (33.9% cytotoxicity; P<0.05), in contrast to its
CDC activity in CHO‑K1 cells (Fig. 3). Furthermore, while
TrMab‑6 exhibited higher CDC (51.6% cytotoxicity) in MCF7
cells compared to the control (30.2% cytotoxicity; P<0.05),
this was not evident in BINDS‑29 cells (Fig. 3). TrMab‑6 also
exhibited higher CDC (36.0% cytotoxicity) in MDA‑MB‑231

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TANAKA et al: ANTITUMOR ACTIVITIES OF AN ANTI-TROP2 MAB

Figure 1. (A) Detection of TROP2 by TrMab‑6 by western blot analysis. Cell lysates of CHO/TROP2, CHO‑K1, MCF7, BINDS‑29, MDA‑MB‑231, andMDA‑MB‑468 cells were electrophoresed and transferred onto PVDF membranes. These membranes were treated with TrMab‑6 (left panel) or anti‑ β‑actin(right panel), followed by incubation with peroxidase‑conjugated anti‑mouse immunoglobulin. (B) Flow cytometry using TrMab‑6. CHO/TROP2, CHO‑K1,
MCF7, BINDS‑29, MDA‑MB‑231, and MDA‑MB‑468 cells were treated with 1 µg/ml of TrMab‑6, followed by a treatment with Alexa Fluor 488‑conjugatedanti‑mouse IgG. Black line, negative control (blocking buffer).

cells than that of the control mouse IgG2b (14.7% cytotoxicity;
P<0.05) (Fig. 3). Furthermore, TrMab‑6 exhibited higher CDC(47.0% cytotoxicity) in MDA‑MB‑468 cells than that of thecontrol mouse IgG2b (20.4% cytotoxicity; P<0.01) (Fig. 3).
These favorable ADCC/CDC activities indicated that
TrMab‑6 may induce strong antitumor activity against breast
cancer cells in vivo.
Antitumor effect of TrMab‑ 6 in mouse xenografts of
TROP2‑expressed CHO/TROP2 cells. CHO/TROP2 wasdeveloped in our previous study (16). Tumor formation of 16
CHO/TROP2‑bearing mice was observed on day 7. Then,these 16 CHO/TROP2‑bearing mice were divided into a
TrMab‑6‑treated group and a control group. On days 7,
14 and 21 after CHO/TROP2 cell injections into the mice,
TrMab‑6 (100 µg) or control mouse IgG (100 µg) were
injected i.p. to the mice. Tumor volume was measured ondays 7, 10, 14, 17, 21 and 24 after CHO/TROP2 cell injection.

TrMab‑6‑treated mice exhibited significantly less tumor
growth on days 14 (P<0.05), 17 (P<0.01), 21 (P<0.01), and 24(P<0.01) compared with IgG‑treated control mice (Fig. 4A,
upper panel). On day 24, there was a reduction of the tumor
volume of 61.9% in TrMab‑6‑treated mice (Fig. 4A, upper
panel). Tumors from TrMab‑6‑treated mice weighed signifi‑
cantly less than tumors from IgG‑treated control mice on
day 24 (52.9% reduction, P<0.05; Fig. 4A, middle panels).
These results indicated that TrMab‑6 reduced the growth of
CHO/TROP2 xenografts, but without full elimination. Total
body weights did not significantly differ between the treatment
and control groups (Fig. 4A, lower panel).
Similarly, tumor formation of 16 CHO‑K1‑bearing
mice was observed on day 7, before they were divided intoa TrMab‑6‑treated group and a control group. On days 7,
14 and 21 after CHO‑K1 cell injections, TrMab‑6 (100 µg) orcontrol mouse IgG (100 µg) were injected i.p. into the mice.
Tumor volume was measured on days 7, 10, 14, 17, 21 and 24

ONCOLOGY REPORTS 46: 132, 2021

5

Figure 2. Evaluation of ADCC activities by TrMab‑6. ADCC activities by TrMab‑6 and control mouse IgG2b in CHO/TROP2, CHO‑K1, MCF7, BINDS‑29,
MDA‑MB‑231, and MDA‑MB‑468 cells. The cells were incubated with splenocytes from female nude mice in the presence of the indicated antibodies
at 100 µg/ml for 5 h. Cytotoxicity (%) was calculated as described in Materials and methods. Values are mean ± SEM. Asterisk indicates statistical significance(**P<0.01, *P<0.05, n.s., not significant, Welch's t‑test).

after CHO‑K1 cell injection. Both TrMab‑6‑treated andcontrol 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).
Antitumor effect of TrMab‑ 6 in mouse xenografts of
TROP2‑expressing MCF7 breast cancer cell lines. The
tumor formation of 16 MCF7‑bearing mice was observed on
day 7 before mice were divided into a TrMab‑6‑treated group
and a control group. On days 7, 14 and 21 after MCF7 cell
injections into the mice, either 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 MCF7 cell injection. TrMab‑6‑treated mice exhibited
significantly less tumor growth on days 10 (P<0.01), 14(P<0.01), 17 (P<0.01), 21 (P<0.01), and 24 (P<0.01) comparedwith IgG‑treated control mice (Fig. 5A, upper panel). On
day 24, a reduction of the tumor volume of 46.6% was seenin TrMab‑6‑treated mice (Fig. 5A, upper panel). Tumors from
TrMab‑6‑treated mice weighed significantly less than tumors
from IgG‑treated control mice on day 24 (37.8% reduction,
P<0.05; Fig. 5A, middle panels). These results indicated that
TrMab‑6 reduced the growth of MCF7 xenografts, but did notcontribute towards their total elimination. Total body weights
did not significantly differ between the treatment and control
groups (Fig. 5A, lower panel).
Similarly, the tumor formation of 16 BINDS‑29‑bearing
mice was observed on day 7, before the 16 BINDS‑29‑bearing

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TANAKA et al: ANTITUMOR ACTIVITIES OF AN ANTI-TROP2 MAB

Figure 3. Evaluation of CDC activities by TrMab‑6. CDC activities by TrMab‑6 and control mouse IgG2b in CHO/TROP2, CHO‑K1, MCF7, BINDS‑29,
MDA‑MB‑231, and MDA‑MB‑468 cells. The cells were incubated with 10% rabbit complement in the presence of the indicated antibodies for 5 h.
Cytotoxicity (%) was calculated as described in Materials and methods. Values are mean ± SEM. Asterisk indicates statistical significance (**P<0.01, *P<0.05,
n.s., not significant, Welch's t‑test).

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 varietyof 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 tobe associated with poor overall and disease‑free survival
across several types of solid tumors (20). Furthermore, theknockdown of TROP2 decreased cell proliferation and migra‑
tion (21). Altogether, these results suggest that TROP2 is apotential target for antitumor treatments.

ONCOLOGY REPORTS 46: 132, 2021

7

Figure 4. Evaluation of antitumor activity of TrMab‑6 in CHO/TROP2 or CHO‑K1 xenografts. (A, upper panel) CHO/TROP2 cells (5x106 cells) were injectedsubcutaneously into the left flank. After day 7, 100 µg of TrMab‑6 and control mouse IgG in 100 µl PBS were injected i.p. into treated and control mice, respec‑tively. Additional antibodies were then injected on days 14 and 21. The tumor volume was measured on days 7, 10, 14, 17, 21 and 24. Values are mean ± SEM.
Asterisk indicates statistical significance (**P<0.01, *P<0.05, n.s., not significant, ANOVA and Sidak's multiple comparisons test). (A, middle panels) Tumors of
CHO/TROP2 xenografts were resected from TrMab‑6 and control mouse IgG groups. Tumor weight on day 24 was measured from excised xenografts. Values
are mean ± SEM. Asterisk indicates statistical significance (*P<0.05, Welch's t‑test). Resected tumors of CHO/TROP2 xenografts from control mouse IgG and
TrMab‑6 groups on day 24. Scale bar, 1 cm. (A, lower panel) Body weights of the mice implanted with CHO/TROP2 xenografts were recorded on days 7, 10, 14,
17, 21 and 24 (n.s., not significant). (B, upper panel) CHO‑K1 cells (5x106 cells) were injected subcutaneously into the left flank. After day 7, 100 µg of TrMab‑6and control mouse IgG in 100 µl PBS were injected i.p. into treated and control mice, respectively. Additional antibodies were then injected on days 14 and 21.
Tumor volume was measured on days 7, 10, 14, 17, 21 and 24. Values are mean ± SEM. n.s., not significant. (B, middle panels) Tumors of CHO‑K1 xenografts
were resected from TrMab‑6 and control mouse IgG groups. Tumor weight on day 24 was measured from excised xenografts. Values are mean ± SEM. n.s., notsignificant. Resected tumors of CHO‑K1 xenografts from control mouse IgG and TrMab‑6 groups on day 24. Scale bar, 1 cm. (B, lower panel) Body weights ofthe mice implanted with CHO‑K1 xenografts were recorded on days 7, 10, 14, 17, 21 and 24 (n.s., not significant).

Antibody‑based therapy is a rapidly emerging field for treat‑ment of several diseases, including cancer. The development ofantibody 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 mainmodality of antibody drugs (22). Recently, the first anti‑TROP2
ADC, sacituzumab govitecan, which is a humanized IgG1
conjugated to irinotecan metabolite (SN‑38), has been approvedby the US Food and Drug Administration against metastatic

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TANAKA et al: ANTITUMOR ACTIVITIES OF AN ANTI-TROP2 MAB

Figure 5. Evaluation of antitumor activity of TrMab‑6 in MCF7 or BINDS‑29 xenografts. (A, upper panel) MCF7 cells (5x106 cells) were injected subcutane‑
ously into the left flank. After day 7, 100 µg of TrMab‑6 and control mouse IgG in 100 µl PBS were injected i.p. into treated and control mice, respectively.
Additional antibodies were then injected on days 14 and 21. Tumor volume was measured on days 7, 10, 14, 17, 21 and 24. Values are mean ± SEM. Asterisk
indicates statistical significance (**P<0.01, ANOVA and Sidak's multiple comparisons test). (A, middle panels) Tumors of MCF7 xenografts were resectedfrom TrMab‑6 and control mouse IgG groups. Tumor weight on day 24 was measured from excised xenografts. Values are mean ± SEM. Asterisk indicatesstatistical significance (*P<0.05, Welch's t‑test). Resected tumors of MCF7 xenografts from control mouse IgG and TrMab‑6 groups on day 24. Scale bar, 1 cm.
(A, lower panel) Body weights of the mice implanted with MCF7 xenografts were recorded on days 7, 10, 14, 17, 21 and 24 (n.s., not significant). (B, upper
panel) BINDS‑29 cells (5x106 cells) were injected subcutaneously into the left flank. After day 7, 100 µg of TrMab‑6 and control mouse IgG in 100 µl PBS
were injected i.p. into treated and control mice, respectively. Additional antibodies were then injected on days 14 and 21. Tumor volume was measured ondays 7, 10, 14, 17, 21 and 24. Values are mean ± SEM. n.s., not significant. (B, middle panels) Tumors of BINDS‑29 xenografts were resected from TrMab‑6and control mouse IgG groups. Tumor weight on day 24 was measured from excised xenografts. Values are mean ± SEM. n.s., not significant. Resected tumorsof BINDS‑29 xenografts from control mouse IgG and TrMab‑6 groups on day 24. Scale bar, 1 cm. (B, lower panel) Body weights of the mice implanted with
BINDS‑29 xenografts were recorded on days 7, 10, 14, 17, 21 and 24 (n.s., not significant).

triple‑negative breast cancers (23). ADCs have also beendeveloped 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 thepatients trialed and had manageable side effects (24). The
combination of these ADCs with immune checkpoint inhibi‑
tors 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).

ONCOLOGY REPORTS 46: 132, 2021

Development of new antibodies and improvement ofantibody‑based drugs are required to overcome therapeutic
resistance and reduce the possibility of identifying suitable
candidates for clinical application (27). In this study, wedemonstrated 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‑resistantsolid 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 clinicaldiagnosis for biopsies and resected specimens. TrMab‑6
may be used to ascertain patients who should receive theanti‑TROP2‑targeted therapy.
The CBIS method, which uses antigen‑expressing celllines for both immunization and screening, can help toeffectively develop mAbs which may be useful as antitumor
agents. We have recently succeeded in developing numerous
mAbs that target membrane proteins, including CD19 (28),CD20 (29), CD44 (30), CD133 (17), EpCAM (31), andTROP2 (16). Of these, CMab‑43 (mouse IgG2a) for CD133
showed significant ADCC/CDC activities against coloncancer cells and antitumor activity against colon cancer
xenograft models (32). EpMab‑16 (mouse IgG2a) for EpCAM
also demonstrated significant antitumor activity againstcolon cancer xenograft models (31), and oral squamous cell
carcinomas (33). Furthermore, 5‑mG2a‑f (a defucosylatedmouse IgG2a‑type of clone C44Mab‑5) for CD44 exerted anti‑
tumor effects in mouse xenograft models of oral squamouscell carcinomas (34).
I n t he present st udy, we investigated whet her
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), demonstratingthat 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.
Acknowledgments
We would like to thank Mr. Takuro Nakamura, Ms. Miyuki
Yanaka, Ms. Saori Handa, and Mr. Yu Komatsu (Departmentof Antibody Drug Development, Tohoku University Graduate
School of Medicine) for technical assistance of in vitro experi‑ments, and Ms. Akiko Harakawa and Mr. Shun‑ichi Ohba
[Institute of Microbial Chemistry (BIKAKEN), Numazu,
Microbial Chemistry Research Foundation] for technicalassistance of animal experiments.

9

Funding
This research was supported in part by the Japan Agency for
Medical Research and Development (AMED) under grantnos. JP21am0401013 (to YK) and JP21am0101078 (to YK),
and by the Japan Society for the Promotion of Science (JSPS)
Grants‑in‑Aid for Scientific Research (KAKENHI) grantnos. 21K15523 (to TA), 21K07168 (to MKK), 19K07705(to YK) and 20K16322 (to MS).
Availability of data and materials
The datasets used and/or analyzed during the study are avail‑able from the corresponding author on reasonable request.
Authors' contributions
TT, TO, TA, RN, HHo, and MS performed the experiments. JT
and MKK analyzed the experimental data. HHa, MK, and YK
designed the present study. TT, TO, and YK wrote the manu‑
script. All the authors read and approved the final manuscript.
Ethics approval and consent to participate
Animal studies for ADCC and the antitumor activity wereapproved by the Institutional Committee for experiments of
the Institute of Microbial Chemistry (permit no. 2020‑015).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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