ONCOLOGY REPORTS 44: 2517-2526, 2020

Anti‑EpCAM monoclonal antibody exerts antitumor
activity against oral squamous cell carcinomas
MIKA K. KANEKO1*, TOMOKAZU OHISHI2*, JUNKO TAKEI1,3*, MASATO SANO1,
TAKURO NAKAMURA1, HIDEKI HOSONO1, MIYUKI YANAKA1, TEIZO ASANO1,YUSUKE SAYAMA1, HIROYUKI HARADA3, MANABU KAWADA2 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 July 14, 2020; Accepted August 28, 2020
DOI: 10.3892/or.2020.7808
Abstract. The epithelial cell adhesion molecule (EpCAM)
is a calcium‑independent, homophilic, intercellular adhesion
factor classified as a transmembrane glycoprotein. In addition
to cell adhesion, EpCAM also contributes to cell signaling,differentiation, proliferation, and migration. EpCAM is an
essential factor in the carcinogenesis of numerous humancancers. In the present study, we developed and validated ananti‑EpCAM monoclonal antibody (mAb), EpMab‑16 (IgG2a,kappa), by immunizing mice with EpCAM‑overexpressing
CHO‑K1 cells. EpMab‑16 specifically reacted with endogenous EpCAM in oral squamous cell carcinoma (OSCC) celllines in flow cytometry and Western blot analyses. It exhibiteda plasma membrane‑like stain pattern in OSCC tissues upon
immunohistochemical analysis. The KD for EpMab‑16 in SAS
and HSC‑2 OSCC cells were assessed via flow cytometry at
1.1x10 ‑8 and 1.9x10 ‑8 M, respectively, suggesting moderatebinding affinity of EpMab‑16 for EpCAM. We then assessed

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: ADCC, antibody‑dependent cellular cytotoxicity;
BSA, bovine serum albumin; CBIS, Cell‑Based Immunizationand Screening; CDC, complement‑dependent cytotoxicity;
DMEM, Dulbecco's modified Eagle's medium; EDTA,
ethylenediaminetetraacetic acid; FBS, fetal bovine serum; mAb,
monoclonal antibody; OSCC, oral squamous cell carcinoma; PBS,
phosphate‑buffered saline
Key words: EpCAM, monoclonal antibody, ADCC, CDC,
antitumor activity, oral cancer

whether the EpMab‑16 induced antibody‑dependent cellularcytotoxicity (ADCC) and complement‑dependent cytotoxicity(CDC) against OSCC cell lines, and antitumor capacity in amurine xenograft model. In vitro experiments revealed strong
ADCC and CDC inducement against OSCC cells treated with
EpMab‑16. In vivo experiments on OSCC xenografts revealed
that EpMab‑16 treatment significantly reduced tumor growthcompared with the control mouse IgG. These data indicatedthat EpMab‑16 could be a promising treatment option for
EpCAM‑expressing OSCCs.
Introduction
Cellular junctions are critical for maintaining cellular architecture and are comprised of several different cell adhesion
molecules (CAM) (1). Thus, the four major CAM families are
selectins, cadherins, integrins, and the immunoglobulin CAM
superfamily (2). The extracellular domain of selectins consistsof a calcium‑dependent lectin domain, an epidermal growth
factor (EGF)‑like domain, a domain homologous to EGF, andtwo to nine consensus repeats. Selectins also contain a hydrophobic transmembrane domain and a short cytoplasmic tail. The
cadherins are calcium‑dependent glycoproteins that have an extracellular domain CAM with three to five internal repeats, alongwith a single‑span transmembrane domain and an intracellulardomain (2). Integrins are composed of two or more noncovalently
associated membrane‑spanning subunits labeled α and β (3). The
specific combination of α and β subunits confers specificity for
different extracellular ligands and their concomitant intracellular signaling events, and each represents a significant receptor
family within the context of interaction with the extracellularmatrix (3). The Ig‑CAMs are calcium‑independent, with an
extracellular domain comprising a ligand‑binding region of fourto six Ig‑like repeats, one to five fibronectin‑like repeats, a transmembrane domain, and an intracellular component (1). Whilethese families predominate, numerous CAMs do not share any
structural similarities with them, one of which is epithelial celladhesion molecule (EpCAM) (4).

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KANEKO et al: ANTITUMOR ACTIVITIES OF AN anti-EpCAM mAB

EpCAM was discovered approximately 40 years ago
and was one of the first identified human tumor‑associatedbiomarkers (5). These days, EpCAM is also considered asa marker for tumor‑initiating cells (6). EpCAM is a transmembrane, calcium‑independent, homophilic, intercellularadhesion glycoprotein. It has three distinct domains, extracellular, transmembrane, and intracellular. In addition tocell adhesion, EpCAM functions include cell signaling,differentiation, proliferation, and migration (4). EpCAM has
been implicated in carcinogenesis and is expressed robustly innumerous human epithelial cancers, including cancers of thelung, colon, breast, ovary, cervix, and oral cavity, making it apromising target for cancer diagnosis and therapy (7‑9).
Oral cancers account for approximately 2% of all cancer
cases worldwide (10). More than 350,000 individuals are
diagnosed with oral cancer each year, and it is ultimately fatalin nearly half of all cases (11,12). Of the defined histologicaltypes of oral cancers, >90% of patients develop oral squamous cell carcinoma (OSCC) on the lips or within the oralcavity (13). The most effective current treatments for OSCC
vary. Stage‑I and ‑II OSCCs are treated with surgery or radiotherapy; advanced stage‑III and ‑IV disease are treated witha combination of excision, radiation, and chemotherapy (14).
Typically, chemotherapeutic regimens include cisplatin asa first‑line agent; it is often combined with docetaxel or
5‑fluorouracil (15,16). Other anticancer drugs, such as paclitaxel, methotrexate, and carboplatin, have also been used for
OSCCs (17). Effective molecular‑targeting drugs, however,including antibody therapies, remain lacking.
In the present study, we developed a set of new anti‑EpCAM
monoclonal antibodies (mAbs), using Cell‑Based Immunizationand Screening (CBIS) methods (18). We then tested whether
these anti‑EpCAM mAbs induced antibody‑dependent cellularcytotoxicity (ADCC), complement‑dependent cytotoxicity(CDC), or other antitumor activity against oral cancers in amurine xenograft model.
Materials and methods
Plasmids. The Genome Network Project clone IRAK021G03(EpCAM) was provided by the RIKEN BioResource ResearchCenter through the National BioResource Project of the MEXT
and AMED agencies of Japan (19‑22). EpCAM DNA plus a
C‑terminal PA tag that is recognized by the anti‑PA tag mAb(NZ‑1), was subcloned into a pCAG‑Ble vector (FUJIFILM
Wako Pure Chemical Corporation).
Cell lines. P3X63Ag8U.1 (P3U1) and CHO‑K1 cells wereobtained from the American Type Culture Collection(ATCC). CHO/EpCAM was established by transfecting
pCAG/EpCAM‑PA into CHO‑K1 cells using the Neon
Transfection System (Thermo Fisher Scientific, Inc.). A few
days after transfection, cells positive for anti‑EpCAM mAb(clone 9C4; cat. no. 324202; BioLegend, Inc.) were sorted
using a cell sorter (SH800; Sony Biotecnology Corp.), andstable transfectants were cultured at 37˚C for 14 days on media
containing 0.5 mg/ml zeocin (InvivoGen). OSCC cell lines,
including SAS (tongue) and HSC‑2 (oral cavity), were obtained
from the Japanese Collection of Research Bioresources Cell
Bank. P3U1, CHO‑K1 and CHO/EpCAM were cultured in

RPMI‑1640 media (Nacalai Tesque, Inc.). SAS and HSC‑2 were
cultured in Dulbecco's modified Eagle's medium (DMEM;
Nacalai Tesque, Inc.). The media 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
for 14 days in a humidified atmosphere containing 5% CO2.
Antibodies. Purified mouse IgG (cat. no. I8765) and mouse
IgG2a (cat. no. M7769) were purchased from Sigma‑Aldrich;
Merck KGaA. Anti‑EpCAM mAbs were purified using Protein
G‑Sepharose (GE Healthcare Bio‑Sciences).
Animals. Thirty‑eight 5‑week‑old female BALB/c nude mice(mean weight, 15±3 g) were purchased from Charles River
Laboratories, Inc. 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. 2019‑066) approved theanimal studies for ADCC and antitumor activity. Mice weremaintained in a specific 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 healthand weight every 1 or 4 days. Experiments on mice wereconducted in three or fewer weeks. Weight loss exceeding
25% or tumor size exceeding 3,000 mm3 were identified as
humane endpoints for euthanasia. At humane and experimental
endpoints, mice were euthanized by cervical dislocation, anddeath was verified by validating respiratory and cardiac arrest.
Hybridoma production. We used a CBIS method (18) todevelop mAbs against EpCAM. Briefly, one BALB/c mouse
was intraperitoneally (i.p.) immunized with CHO/EpCAM
cells (1x108) along with Imject Alum adjuvant (Thermo Fisher
Scientific, Inc.). The procedure included three additional immunizations, followed by a final booster injection administered i.p.
two days before spleen cell harvesting. Spleen cells were thenfused with P3U1 cells using PEG1500 (Roche Diagnostics).
Hybridomas were grown at 37˚C for 10 days in RPMI‑1640
media with HAT Supplement (50x) (cat. no. 21060017; Thermo
Fisher Scientific, Inc.) for selection. Culture supernatants werescreened using flow cytometry.
Flow cytometry. Cells (2x105 cells/ml) were harvested after
brief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). After washingwith 0.1% bovine serum albumin (BSA; Nacalai Tesque, Inc.)in phosphate‑buffered saline (PBS; Nacalai Tesque, Inc.), cells
were treated with 1 µg/ml of anti‑EpCAM mAbs 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 an EC800 Cell
Analyzer (Sony Biotechnology Corp.).
Western blot analyses. Cell pellets were resuspended in
PBS with 1% Triton X‑100 (cat. no. 168‑11805; FUJIFILM
Wako Pure Chemical Corporation) and 50 µg/ml aprotinin

ONCOLOGY REPORTS 44: 2517-2526, 2020

(product no. 03346‑84; Nacalai Tesque, Inc.). Protein concentration was determined by BCA method. Cell lysates wereboiled in sodium dodecyl sulfate sample buffer (Nacalai
Tesque, Inc.). The samples (10 µg/lane) were then electrophoresed on 5‑20% polyacrylamide gels (Nacalai Tesque, Inc.) andtransferred onto polyvinylidene difluoride (PVDF) membranes(Merck KGaA). After blocking with 4% skim milk (Nacalai
Tesque, Inc.) for 1 h, the membrane was incubated with ananti‑EpCAM mAb (5 µg/ml) or anti‑β‑actin (1 µg/ml; clone
AC‑15; cat. no. A5441; Sigma‑Aldrich; Merck KGaA) for
1 h, followed by incubation with HRP‑conjugated anti‑mouse
immunoglobulins (cat. no. P0260; Agilent Technologies, Inc.)or anti‑rat IgG (cat. no. A9542; Sigma‑Aldrich; Merck KGaA)at a 1:2,000 dilution for 1 h. The membrane was developed
using the ImmunoStar LD Chemiluminescence Reagent(FUJIFILM Wako Pure Chemical Corporation) and a
Sayaca‑Imager (DRC Co., Ltd.). All western blot procedures
were performed at room temperature.
Immunohistochemical analyses. Histologic sections
4‑µm thick of an oral cancer tissue array (cat. no. OR481;
US Biomax, Inc.) were autoclaved directly in EnVision FLEX
Target Retrieval Solution, High pH (Agilent Technologies, Inc.)for 20 min. Sections were then incubated with 10 µg/ml of ananti‑EpCAM mAb for 1 h at room temperature and treatedusing an Envision+ kit (Agilent Technologies, Inc.) for 30 min
according to the manufacturer's instructions. The color wasdeveloped using 3, 3'‑diaminobenzidine tetrahydrochloride(DAB; Agilent Technologies Inc.) for 2 min at room temperature, and sections were then counterstained with hematoxylin(FUJIFILM Wako Pure Chemical Corporation) at room
temperature for 5 min. Hematoxylin and eosin (H&E) staining(FUJIFILM Wako Pure Chemical Corporation) was performed
using consecutive tissue sections at room temperature for5 min. Leica DMD108 (Leica Microsystems GmbH) was used
to examine the sections and obtain images (x100 and x400).
Determination of the binding affinity. Cells (2x105 cells/ml)
were suspended in 100 µl of serially diluted anti‑EpCAM
mAb (6 ng/ml‑100 µg/ml), followed by the addition of
Alexa Fluor 488‑conjugated anti‑mouse IgG (1:200;
cat. no. 4408; Cell Signaling Technology, Inc.). Fluorescencedata were collected using an EC800 Cell Analyzer (Sony
Biotechnology Corp.). The dissociation constant (K D)
was calculated by fitting binding isotherms to built‑in,
one‑site binding models in GraphPad Prism 6 (GraphPad
Software, Inc.).
ADCC. ADCC inducement by EpCAM was assayed as follows.
Six female five‑week‑old BALB/c nude mice (mean weight,
15±3 g) were purchased from Charles River Laboratories,
Inc. After euthanasia by cervical dislocation, spleens were
removed aseptically, and single‑cell suspensions wereobtained by forcing spleen tissues through a sterile cell strainer(product no. 352360; BD Falcon; Corning, Inc.) with a syringe.
Erythrocytes were lysed with a 10‑sec exposure to ice‑cold
distilled water. The splenocytes were washed with DMEM andresuspended in DMEM with 10% FBS; this preparation was
designated as effector cells. The target tumor cells were labeledwith 10 µg/ml Calcein‑AM (Thermo Fisher Scientific, Inc.)2519

and resuspended in the same medium. The target cells weretransferred to 96‑well plates, at 2x104 cells/well, and mixed
with effector cells at an effector‑to‑target ratio of 50:1, alongwith 100 µg/ml of anti‑EpCAM antibodies or control mouse
IgG2a. After a 4‑h incubation at 37˚C, 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 wavelengthof 485 nm and an emission wavelength of 538 nm. Cytolytic
activity was measured as a percentage of lysis and calculatedusing the equation: Percentage of lysis (%) = (E‑S)/(M‑S) x100,
where E is the fluorescence measured in combined cultures oftarget and effector cells, S is the spontaneous fluorescence ofthe 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.
CDC. CDC inducement by EpCAM 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 2x104 cells/well,and 10% rabbit complement (Low‑Tox‑M rabbit complement;
Cedarlane Laboratories), 100 µg/ml of anti‑EpCAM antibodies, or control IgG (mouse IgG2a) was added to each well.
After 4 h of incubation at 37˚C, Calcein‑AM release into thesupernatant was measured for each well. Fluorescence intensity was calculated as described in the ADCC section above.
Antitumor activity of anti‑EpCAM mAbs in xenografts oforal cancers. Thirty‑two five‑week‑old female BALB/c nude
mice were purchased from Charles River Laboratories Japan,
Inc. After a two‑week acclimation period, these mice wereused in experiments at seven weeks of age (mean weight,
16±2 g). HSC‑2 and SAS cells (0.3 ml of 1.33x108 cells/ml in
DMEM) were mixed with 0.5 ml BD Matrigel Matrix Growth
Factor Reduced (BD Biosciences), and 100 µl of this suspension (5x106 cells) was injected subcutaneously into the left
flank of each animal. On day 1 post‑inoculation, 100 µg ofan anti‑EpCAM mAb or control mouse IgG in 100 µl PBS
was injected i.p. Additional antibody inoculations were
performed on days 8 and 14. Seventeen days or eighteen days
after cell implantation, all mice were euthanized by cervicaldislocation, and tumor diameters and volumes were measured
and recorded.
Statistical analyses. All data are expressed as mean ± SEM.
Statistical analysis was conducted with one-way ANOVA
and Tukey's multiple comparisons tests for ADCC and CDC,
one-way ANOVA and Sidak's multiple comparisons tests fortumor volume and mouse weight, and Welch's t‑test for tumor
weight. All calculations were performed with GraphPad Prism 7(GraphPad Software, Inc.). A P‑value of <0.05 was considered
to indicate a statistically significant difference.
Results
Development and characterization of anti‑EpCAM mAbs.
Anti‑EpCAM mAbs were developed by immunizing a single
mouse with CHO/EpCAM cells. Hybridomas were cultured,and supernatants positive for CHO/EpCAM and negative for2520

KANEKO et al: ANTITUMOR ACTIVITIES OF AN anti-EpCAM mAB

Figure 1. Recognition of EpCAM by an anti‑EpCAM mAb. CHO/EpCAM, CHO‑K1, SAS, and HSC‑2 cells were treated with EpMab‑16 (1 µg/ml) and buffer
control, followed by secondary antibodies. EpCAM, epithelial cell adhesion molecule.

CHO‑K1 were selected by flow cytometry. Further screening
using western blot and immunohistochemical assays were
performed for validation, resulting in EpMab‑16 (IgG2a, kappa)
identification and establishment.
Flow cytometry was then used to assess the sensitivity of
EpMab‑16 in CHO/EpCAM and OSCC cell lines (SAS and
HSC‑2). EpMab‑16 reacted with CHO/EpCAM cells but notwith CHO‑K1 cells (Fig. 1). EpMab‑16 also reacted with
SAS and HSC‑2 cells (Fig. 1). Flow cytometric data indicatedthat EpMab‑16 was highly sensitive and highly specific for
EpCAM.
Then a western blot assay was conducted to delineate thesensitivity of EpMab‑16 further. Lysates of CHO/EpCAM,
SAS, and HSC‑2 cells were probed, and the results revealed
that EpMab‑16 detected 35 kDa band of EpCAM strongly incell lysates from CHO/EpCAM, whereas it detected EpCAM
moderately in cell lysates from SAS, and faintly from HSC‑2
cells, indicating that EpMab‑16 could detect both exogenousand endogenous EpCAM (Fig. 2).
Immunohistochemical analysis results revealed that
EpMab‑16 detected membrane antigens in oral cancer tissues(Fig. 3A, B, E and F). Among 38 OSCC cases in the oral
cancer tissue array, 6 cases (16%) were stained by EpMab‑16.
In this staining, antigen retrieval using EnVision FLEX Target
Retrieval Solution High pH was performed. The signal was
lower for citrate buffer (pH 6.0) antigen retrieval (data notshown), such that antigen retrieval using high pH was deemed

Figure 2. Western blot analysis using an anti‑EpCAM mAb. Cell lysates(10 µg) of CHO‑K1, CHO/EpCAM, SAS, and HSC‑2 were electrophoresed
and transferred onto PVDF membranes. The membranes were incubatedwith 5 µg/ml of EpMab‑16 and 1 µg/ml of anti‑β‑actin, followed by peroxidase‑conjugated anti‑mouse immunoglobulins. The red arrow indicates
EpCAM. The blue arrows indicate non‑specific bands, which were detectedin both CHO‑K1 and CHO/EpCAM cells. EpCAM, epithelial cell adhesion
molecule; mAb, monoclonal antibody; PVDF, polyvinylidene difluoride.

ONCOLOGY REPORTS 44: 2517-2526, 2020

Figure 3. Immunohistochemical analyses of OSCC tissues using EpMab‑16.
(A and B) FFPE tissue sections of OSCC (case 1) were incubated with 10 µg/ml
of EpMab‑16. (C and D) H&E staining against consecutive OSCC tissues(case 1). (E and F) FFPE tissue sections of OSCC (case 2) were incubated with
10 µg/ml of EpMab‑16. (G and H) H&E staining against consecutive OSCC
tissues (case 2). Scale bar, 100 µm. OSCC, oral squamous cell carcinoma;
FFPE, formalin‑fixed paraffin‑embedded; H&E, hematoxylin and eosin.

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Figure 4. Determination of binding affinity of an anti‑EpCAM mAb for OSCC
cells. (A) SAS and (B) HSC‑2 cells were suspended in 100 µl of serially diluted
EpMab‑16, followed by the addition of Alexa Fluor 488‑conjugated anti‑mouse
IgG, and fluorescence data were collected. EpCAM, epithelial cell adhesion
molecule; mAb, monoclonal antibody; OSCC, oral squamous cell carcinoma.

essential for immunostaining using EpMab‑16. Hematoxylin
and eosin (H&E) staining was performed using consecutive OSCC tissues (Fig. 3C, D, G and H). Results indicatedthat EpMab‑16 detected EpCAM in immunohistochemical
analysis on formalin‑fixed paraffin‑embedded (FFPE) tissues
effectively.
Then a kinetic analysis of the interactions of EpMab‑16
with SAS and HSC‑2 oral cancer cell lines was then conductedusing flow cytometry. The KD for EpMab‑16 in SAS cells was
1.1x10‑8 M, and the KD for EpMab‑16 against HSC‑2 cells was
1.9x10‑8 M (Fig. 4), indicating a moderate binding affinity of
EpMab‑16 against oral cancer cells.
ADCC and CDC activities of EpMab‑16 in oral cancer
cell lines. We then examined whether EpMab‑16 (mouse
IgG 2a) induced ADCC and CDC antitumor action in
EpCAM‑expressing SAS and HSC‑2 oral cancer cell lines.
EpMab‑16 exhibited higher ADCC (48% cytotoxicity) in
SAS cells than that of control mouse IgG2a (11% cytotoxicity;
P<0.01) or control PBS (9.4% cytotoxicity; P<0.01) treatment (Fig. 5A). Similarly, EpMab‑16 exhibited higher ADCC
(27% cytotoxicity) in HSC‑2 cells than that of control mouse

Figure 5. ADCC activity of an anti‑EpCAM mAb in OSCC cells. ADCC
activity of EpMab‑16, control mouse IgG2a, and control PBS in (A) SAS and(B) HSC‑2 cells. Values represent the mean ± SEM. **P<0.01 (determined by
ANOVA and Tukey's multiple comparisons tests). ADCC, antibody‑dependent
cellular cytotoxicity; EpCAM, epithelial cell adhesion molecule; mAb,
monoclonal antibody; OSCC, oral squamous cell carcinoma; SEM,
standard error of the mean; n.s., not significant.

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KANEKO et al: ANTITUMOR ACTIVITIES OF AN anti-EpCAM mAB

These results indicated that EpMab‑16 reduced the growth of
SAS xenografts, but did not altogether eliminate them.
EpMab‑16 antitumor effect in mouse xenografts of HSC‑2
oral cancer cells. On days 1, 8, and 14 after HSC‑2 cell injections, EpMab‑16 (100 µg) or control mouse IgG (100 µg) were
injected i.p. into HSC‑2 xenograft model mice. Tumor formation was observed in mice in the treatment and control groups.
Tumor volume was measured on days 7, 10, 14, and 18 after
HSC‑2 cell injection. The EpMab‑16‑treated mice exhibited
significantly less tumor growth on day 18 (P<0.01) than the
IgG‑treated control mice (Fig. 8A). Tumor volume reduction by EpMab‑16 treatment was 33% as of day 18. Tumors
from EpMab‑16‑treated mice weighed significantly less thantumors from IgG‑treated control mice (21% reduction, P<0.05;
Fig. 8B). Resected tumors on day 18 are presented in Fig. 8C.
Total body weights did not significantly differ between thetreatment and control groups (Fig. S1). These results indicatedthat EpMab‑16 reduced the growth of HSC‑2 xenografts,
although it did not eliminate them.
Discussion
Figure 6. CDC activity of an anti‑EpCAM mAb in OSCC cells. CDC activityof EpMab‑16, control mouse IgG2a, and control PBS in (A) SAS and (B) HSC‑2
cells. Values represent the mean ± SEM. **P<0.01 (determined by ANOVA
and Tukey's multiple comparisons tests). CDC, complement‑dependent
cytotoxicity; EpCAM, epithelial cell adhesion molecule; mAb, monoclonal
antibody; OSCC, oral squamous cell carcinoma; PBS, phosphate‑bufferedsaline; SEM, standard error of the mean; n.s., not significant.

IgG2a (11% cytotoxicity; P<0.01) or control PBS treatment(12% cytotoxicity; P<0.01) (Fig. 5B).
EpMab‑16 was also associated with more robust CDC
activity (56% cytotoxicity) in SAS cells than control mouse IgG2a(26% cytotoxicity; P<0.01) or control PBS treatment (23% cytotoxicity; P<0.01) (Fig. 6A). Similarly, EpMab‑16‑treated cells
exhibited more CDC activity (46% cytotoxicity) in HSC‑2
cells than control mouse IgG2a (24% cytotoxicity; P<0.01) orcontrol PBS treatment (23% cytotoxicity; P<0.01) (Fig. 6B).
These favorable ADCC and CDC activity results indicatedthat EpMab‑16 may induce strong antitumor action against
oral cancer cells in vivo as well as in vitro.
EpMab‑16 antitumor effect in mouse xenografts of SAS oral
cancer cells. On days 1, 8, and 14 after SAS cell injections into
the mice, EpMab‑16 (100 µg) or control mouse IgG (100 µg)
were injected i.p. in SAS xenograft model mice. Tumor formation was observed in mice from treatment and control groups.
Tumor volume was measured on days 7, 10, 14, and 17 after
SAS cell injection. EpMab‑16‑treated mice exhibited significantly less tumor growth on day 10 (P<0.05), day 14 (P<0.05)
and day 17 (P<0.01) compared with IgG‑treated control mice
(Fig. 7A). Tumor volume reduction by EpMab‑16 treatment was
37% as of day 17. Tumors from EpMab‑16‑treated mice weighed
significantly less than tumors from IgG‑treated control mice
(20% reduction, P<0.05; Fig. 7B). Resected tumors on day 17
are presented in Fig. 7C. Total body weights did not significantly differ between the treatment and control groups (Fig. S1).

EpCAM has been reported to be overexpressed in several
cancers (23‑27). EpCAM is overexpressed in 55‑75% of
ovarian cancers (24), and is overexpressed in over 90% of
ovarian cancer metastatic lesions (25). Overexpressionof EpCAM has been revealed to be correlated with poor
prognosis, therapeutic failure and early tumor recurrencein hepatocellular carcinoma (HCC) patients (26). EpCAM
has also been revealed to play important roles in proliferation, apoptosis, and metastasis during breast cancer
progression (27). Therefore, EpCAM should be a promisingnovel therapeutic target.
In the present study, it was investigated whetheranti‑EpCAM mAbs could be useful for treating oral cancers
via ADCC and CDC activity. We first developed a sensitiveand specific anti‑EpCAM mAb (EpMab‑16, mouse IgG2a),which exhibited favorable potential in flow cytometry, western
blot and immunohistochemical analyses. The present results
also suggested that EpMab‑16 had diagnostic efficacy in FFPE
tissues because pathological diagnosis utilizes FFPE tissues.
It was also demonstrated that EpMab‑16 was associated withstrong ADCC and CDC activity against SAS and HSC‑2
OSCC cell lines in vitro. The ADCC and CDC activity of
EpMab‑16 against OSCC was more pronounced in SAS cells
than HSC‑2 cells, presumably because the EpCAM expression
level and binding affinity are greater in SAS than in HSC‑2.
ADCC and CDC activities of EpMab‑16 against Caco‑2, acolon cancer cell line were also assessed, and values were
44 and 49%, respectively (data not shown), suggesting that
EpMab‑16 could induce observable ADCC and CDC in other
EpCAM‑expressing cancers in addition to the oral cancer celllines assessed in this study.
It was then investigated whether EpMab‑16 exerted antitumor function against OSCC xenografts in vivo. Although
EpMab‑16 significantly reduced not only the growth of SAS
and HSC‑2 xenografts, but also the tumor weight of SAS
and HSC‑2 xenografts, the tumor reduction was not sufficient to eliminate them. We previously investigated whether

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Figure 7. Antitumor activity of an anti‑EpCAM mAb in SAS xenografts. (A) Tumor volume of SAS xenografts. SAS cells (5x106 cells) were injected subcutaneously into the left flank, and 100 µg of EpMab‑16 or control mouse IgG in 100 µl PBS was injected i.p. into treatment and control mice, respectively.
Additional antibodies were injected on days 8 and 14. The tumor volume was measured on days 7, 10, 14, and 17. Values represent the mean ± SEM. *P<0.05
and **P<0.01 (determined by ANOVA and Sidak's multiple comparisons tests) (B) Tumor weights of SAS xenografts. Tumors of SAS xenografts were resectedfrom EpMab‑16 and control mouse IgG groups. The tumor weight on day 17 was measured from excised xenografts. Values represent the mean ± SEM. *P<0.05(determined by Welch's t‑test). (C) Resected tumors of SAS xenografts from EpMab‑16 and control mouse IgG groups on day 17. The tumor in the dotted region
was the largest tumor in this experiment. The white square grids under the tumors are 1x1 cm each. Scale bar, 1 cm. (D) A magnified image of the largest
tumor in the dotted region of C. The vertical and horizontal lengths for SAS cells were 2.4 and 1.3 cm, respectively (estimated tumor volume, 2028 mm3). All
mice possessed one tumor each. EpCAM, epithelial cell adhesion molecule; mAb, monoclonal antibody; PBS, phosphate‑buffered saline; SEM, standard errorof the mean; n.s., not significant.

podocalyxin (PODXL) could be a therapeutic target in
OSCC using anti‑PODXL mAbs developed by converting ananti‑PODXL mAb of IgG1 subclass (PcMab‑47) into a mouse
IgG2a‑type mAb (47‑mG2a) to increase ADCC (28). This was
further developed into 47‑mG2a‑f, a core fucose‑deficientvariant of 47‑mG2a, also to increase its ADCC. In vivo

analyses demonstrated that 47‑mG2a ‑f, but not 47‑mG2a,exerted antitumor activity in SAS and HSC‑2 xenograftmodels at a dose of 100 µg/mouse/week administered threetimes, indicating that a core fucose‑deficient anti‑PODXL
mAb could be profitable for antibody‑based therapy against
PODXL‑expressing OSCCs (28). We thereby anticipate that

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KANEKO et al: ANTITUMOR ACTIVITIES OF AN anti-EpCAM mAB

Figure 8. Antitumor activity of an anti‑EpCAM mAb in HSC‑2 xenografts. (A) Tumor volume of HSC‑2 xenografts. HSC‑2 cells (5x106 cells) were injectedsubcutaneously into the left flank, and 100 µg of EpMab‑16 or control mouse IgG in 100 µl PBS was injected i.p. into treatment and control mice, respectively.
Additional antibodies were then injected on days 8 and 14. The tumor volume was measured on days 7, 10, 14, and 18. Values represent the mean ± SEM.
**
P<0.01 (determined by ANOVA and Sidak's multiple comparisons tests). (B) Tumor weights of HSC‑2 xenografts. Tumors of HSC‑2 xenografts were resectedfrom EpMab‑16 and control mouse IgG groups. The tumor weight on day 18 was measured from excised xenografts. Values represent the mean ± SEM. *P<0.05(determined by Welch's t‑test). (C) Resected tumors of HSC‑2 xenografts from EpMab‑16 and control mouse IgG groups on day 18. The tumor in the dotted
region was the largest tumor in this experiment. The white square grids under the tumors are 1x1 cm each. Scale bar, 1 cm. (D) A magnified image of the largest
tumor in the dotted region of C. The vertical and horizontal lengths for HSC‑2 cells were 1.8 and 1.2 cm, respectively (estimated tumor volume, 1296 mm3).
All mice possessed one tumor each. EpCAM, epithelial cell adhesion molecule; mAb, monoclonal antibody; PBS, phosphate‑buffered saline; SEM, standard
error of the mean; n.s., not significant.

developing a core fucose‑deficient variant of EpMab‑16 will
reveal a similar enhancement of ADCC activity in a future
study.

Results of this study and our previous work, which includes
the development of mAbs effective in SAS and HSC‑2 xenografts against epidermal growth factor receptor (EGFR)ONCOLOGY REPORTS 44: 2517-2526, 2020

(clone EMab‑17, mouse IgG2a) (29), human epidermal growth
factor receptor 2 (HER2) (clone H2Mab‑19, mouse IgG2b) (30),and cancer‑specific mAb (CasMab) against podoplanin(PDPN) (31), suggest that the targeting of several molecules,
such as PODXL, EGFR, HER2, PDPN, as well as EpCAM,
could be an effective therapy to cure OSCCs. In the future,
cancer‑specific anti‑EpCAM mAbs may also be developed thatcan reduce the adverse effects of traditional antibody therapy.
Acknowledgements
We would like to thank Ms. Saori Handa, Ms. Saki Okamoto,and Mr. Yu Komatsu (Department of Antibody Drug
Development, Tohoku University Graduate School of
Medicine) for technical assistance of in vitro experiments,and Ms. Akiko Harakawa (Institute of Microbial Chemistry(BIKAKEN), Numazu, Microbial Chemistry Research
Foundation) for technical assistance of animal experiments.
Funding
This research was supported in part by the Japan Agency
for Medical Research and Development (AMED) under
grant nos. JP20am0401013 (to YK), JP20am0101078 (to YK),
and JP20ae0101028 (to YK), and by the Japan Society for
the Promotion of Science (JSPS) Grants‑in‑Aid for Scientific
Research (KAKENHI) grant no. 17K07299 (to MKK),
grant nos. 19K07705 (to YK), and 20K16322 (to MS).
Availability of data and materials
The datasets used and/or analyzed during the study are available from the corresponding author on reasonable request.
Authors' contributions
HHo, JT, TO, TN, MS TA, YS, and MY performed the experiments. MKK analyzed the experimental data. MK, HHa, andYK designed the present study and wrote the manuscript. 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. 2019‑066).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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