ONCOLOGY LETTERS 20: 383, 2020

The anti‑epithelial cell adhesion molecule (EpCAM)
monoclonal antibody EpMab‑16 exerts antitumor activity
in a mouse model of colorectal adenocarcinoma
HIDEKI HOSONO1*, TOMOKAZU OHISHI2*, JUNKO TAKEI1*, TEIZO ASANO1,YUSUKE SAYAMA1, MANABU KAWADA2, MIKA K. KANEKO1 and YUKINARI KATO1,3
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Miyagi 980‑8575; 2Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
Numazu, Shizuoka 410‑0301; 3New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi 980‑8575, Japan
Received June 18, 2020; Accepted October 6, 2020
DOI: 10.3892/ol.2020.12246
Abstract. The epithelial cell adhesion molecule (EpCAM),
which is a calcium‑independent homophilic intercellularadhesion factor, contributes to cell signaling, differentiation,
proliferation and migration. EpCAM is essential for carcino‑
genesis in numerous types of human cancer. The purpose of
the present study was to establish an anti‑EpCAM monoclonal
antibody (mAb) for targeting colorectal adenocarcinomas.
Thus, an anti‑EpCAM mAb, EpMab‑16 (IgG2a, κ), was estab‑lished by immunizing mice with EpCAM‑overexpressing
CHO‑K1 cells, and validated using flow cytometry, western
blot, and immunohistochemical analyses. EpMab‑16 reactedwith endogenous EpCAM specifically in a colorectal adeno‑carcinoma cell line as determined by flow cytometry andwestern blot analyses. Immunohistochemical analysis demon‑
strated that EpMab‑16 stained a plasma membrane‑like pattern
in clinical colorectal adenocarcinoma tissues. The dissociation
constant (KD) for EpMab‑16 in a Caco‑2 colorectal adenocarci‑
noma cell line determined by flow cytometry was 1.8x10‑8 M,suggesting moderate binding affinity of EpMab‑16 for EpCAM.
Whether the EpMab‑16 induced antibody‑dependent cellularcytotoxicity (ADCC) and complement‑dependent cytotoxicity(CDC) against Caco‑2 or antitumor activity was 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;

CBIS, cell‑based immunization and screening; CDC, complementdependent cytotoxicity; EpCAM, epithelial cell adhesion molecule;mAb, monoclonal antibody

Key words: EpCAM, monoclonal antibody, ADCC, CDC,
antitumor activity, colorectal adenocarcinoma

in a murine xenograft model. In vitro experiments revealed
strong ADCC and CDC induction in Caco‑2 cells by EpMab‑16
treatment. In vivo experiments in a Caco‑2 xenograft model
demonstrated that EpMab‑16 treatment significantly reduced
tumor growth compared with that in mice treated with thecontrol mouse IgG. These results suggested that EpMab‑16
may be a promising treatment option for EpCAM‑expressing
colorectal adenocarcinomas.
Introduction
Cellular junctions comprise a range of cell adhesion molecules(CAM) and are essential for maintaining tissue architec‑
ture (1). The four major CAM families are integrins, cadherins,
selectins and the immunoglobulin CAM superfamily (2).
Integrins are composed of two or more noncovalently‑asso‑
ciated membrane‑spanning subunits α and β (3). The specificcombination of α and β subunits confers specificity forvarious extracellular ligands and their respective intracellularsignaling events, and each combination of α and β repre‑
sents a significant receptor family within the context ofinteraction with the extracellular matrix (3). Cadherins arecalcium‑dependent glycoproteins, which possess an extra‑
cellular CAM domain with three to five internal repeats,a single‑span transmembrane domain and an intracellulardomain (2). The extracellular domain of selectins consistsof a calcium‑dependent lectin domain and an epidermal
growth factor (EGF)‑like domain (2). Selectins also contain ahydrophobic transmembrane domain and a short cytoplasmic
tail (2). The Ig‑CAMs are calcium‑independent, with an
extracellular domain comprising a ligand‑binding region offour to six Ig‑like repeats, one to five fibronectin‑like repeats,a transmembrane domain and an intracellular component (1).
Although these families are predominant, a number of CAMs
do not share any structural similarities with them, such as theepithelial cell adhesion molecule (EpCAM) (4).
EpCAM is one of the first identified human tumor‑asso‑
ciated biomarkers (5) and is now considered to be a marker
of tumor‑initiating cells (6). EpCAM is a transmembrane,
calcium‑independent, homophilic, intercellular adhesion glyco‑

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HOSONO et al: ANTITUMOR ACTIVITY OF AN ANTI‑EpCAM mAb

protein (314 amino acids; 40 kDa) with three distinct domains:An extracellular domain (EpEX, 265 amino acids), a transmem‑
brane domain and an intracellular domain (EpICD, 26 aminoacids) (7). The cleaved EpICD enters the nucleus, leading to theactivation of the β‑catenin/c‑Myc signaling pathway to promote
cancer cell proliferation (8). EpCAM functions include cellsignaling, differentiation and migration in addition to adhesion
and proliferation (4). EpCAM has been implicated in carcino‑
genesis and is expressed robustly in various types of humanepithelial cancers, such as lung, breast, ovarian, cervical andcolorectal cancer (CRC), suggesting that it may be a promisingtarget for cancer diagnosis and therapy (9‑11).
According to GLOBOCAN 2018 data, CRC is the third
most commonly occurring cancer and second leading causeof cancer‑associated death in the world, with ~881,000 deaths
estimated for 2018 (12). Although surgical removal of cancer
followed by adjuvant therapy is one of the most effective
treatments, recurrences are inevitable (13‑15). Antibody‑based
treatments are also currently used in patients with advanced
CRC; however, the prognosis and clinical outcomes of patientswith CRC remain poor (16). Therefore, new strategies are
required to improve the effectiveness of CRC treatment.
The present study developed an anti‑EpCAM monoclonal
antibody (mAb) using cell‑based immunization and screening(CBIS) methods (17) aiming to determine whether theseanti‑EpCAM mAbs induced antibody‑dependent cellular cyto‑
toxicity (ADCC), complement‑dependent cytotoxicity (CDC)or antitumor activity against CRC in a murine xenograft model.
Materials and methods
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 (Cytiva).

the Neon Transfection System (Thermo Fisher Scientific, Inc.).
CHO‑K1 cells (1.5x106) were transfected with 10 µg of plasmid
DNA using 100 µl Neon tip, at room temperature. After
4 days, cells were incubated with 1 µg/ml anti‑EpCAM mAb(clone 9C4; cat. no. 324202; BioLegend, Inc.) for 30 min on ice
and subsequently with Alexa Fluor 488‑conjugated anti‑mouse
IgG (1:1,000; cat. no. 4408; Cell Signaling Technology, Inc.) for
30 min on ice. Positive cells for anti‑EpCAM mAb were sorted
using an SH800 cell sorter (Sony Corporation), and stabletransfectants were cultivated in RPMI‑1640 medium (Nacalai
Tesque, Inc.) containing 0.5 mg/ml zeocin (InvivoGen). Using
TruGuide gRNA tool, gRNA of EpCAM (NM_002354)
was selected from GeneArt predesigned gRNAs database(Thermo Fisher Scientific, Inc.). gRNA sequence used was
GATCCTGACTGCGATGAGAG(cgg), which targeted exon 3of EpCAM (Assay ID, CRISPR701274). Double strand gRNA
sequence was subcloned into GeneArt CRISPR Nuclease
Vector with OFP Reporter (Thermo Fisher Scientific, Inc.).
Caco‑2/EpCAM‑knockout (BINDS‑16) cells were generated
by transfecting 10 μg of CRISPR/Cas9 plasmids for EpCAM(Thermo Fisher Scientific, Inc.) into Caco‑2 cells (1.5x106) for
7 days using a Neon transfection system with 100 µl Neontip. Stable transfectants were established by cell sorting as
aforementioned. P3U1, CHO‑K1 and CHO/EpCAM cells
were cultured in RPMI‑1640 medium (Nacalai Tesque, Inc.).
Caco‑2 and BINDS‑16 were cultured in Dulbecco's modified
Eagle's medium (DMEM; Nacalai Tesque, Inc.). The medium
was supplemented with 10% heat‑inactivated fetal bovine
serum (FBS; Thermo Fisher Scientific Inc.), 100 U/ml peni‑
cillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B(Nacalai Tesque, Inc.), and the cells were incubated at 37˚C ina humidified atmosphere containing 5% CO2.

Animals. All animal experiments were performed inaccordance with institutional guidelines and regulations tominimize animal suffering and distress in the laboratory.
The Institutional Committee for Experiments of the Instituteof Microbial Chemistry (Numazu, Japan) approved theanimal studies for ADCC and antitumor activity (approval
no. 2019‑066). Mice were monitored for health and weight
every 1‑5 days. Experiments on mice were conducted in≤3 weeks. Weight loss >25% or tumor size >3,000 mm3 wereidentified as humane endpoints for euthanasia. At humane andexperimental endpoints, mice were euthanized by cervicaldislocation, and death was verified by validating respiratory
and cardiac arrest.

Hybridoma production. The CBIS method (17) was used in
the present study to develop mAbs against EpCAM. Briefly,
one BALB/c mouse was intraperitoneally (i.p.) immunizedwith CHO/EpCAM cells (1x108 cells/500 µl) with Imject
Alum adjuvant (Thermo Fisher Scientific Inc.). The procedureincluded three additional immunizations, followed by a final
booster injection administered i.p. 2 days before spleen cell
harvesting. Spleen cells (1x108 cells) were then fused withmouse plasma cell myeloma P3U1 cells (1x107 cells) using
PEG1500 (Roche Diagnostics). The hybridomas were cultured
in RPMI‑1640 medium supplemented with hypoxanthine,aminopterin and thymidine for selection (50X solution;
Thermo Fisher Scientific Inc.). Cell culture supernatants ofhybridomas in each well of 96‑well plates were mixed with
1x105 CHO/EpCAM cells and were directly screened using
flow cytometry.

Cell lines. P3X63Ag8U.1 (P3U1), CHO‑K1 and Caco‑2 cells
were obtained from the American Type Culture Collection.
The Genome Network Project clone IRAK021G03 (EpCAM)
was provided by the RIKEN BioResource Research Center
through the National BioResource Project of the MEXT
and AMED agencies of Japan (18‑21). EpCAM DNA plus a
C‑terminal PA tag recognized by the anti‑PA tag mAb (NZ‑1)
was subcloned into a pCAG‑Ble vector (FUJIFILM Wako
Pure Chemical Corporation). CHO/EpCAM was establishedby transfecting pCAG/EpCAM‑PA into CHO‑K1 cells using

Flow cytometry. Caco‑2 cells (2.5x10 5 cells/ml) were
harvested after brief exposure to 0.25% trypsin in 1 mM
ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque,Inc.). Following washing with 0.1% bovine serum albumin(BSA; Nacalai Tesque, Inc.) in phosphate‑buffered saline(PBS), Caco‑2 cells were treated with 1 µg/ml of anti‑EpCAM
mAbs for 30 min at 4˚C, followed by Alexa Fluor 488‑conju‑
gated anti‑mouse IgG (1:1,000; cat. no. 4408; Cell Signaling
Technology, Inc.). Fluorescence data were obtained using
an EC800 Cell Analyzer (Sony Corporation), and analyzed

ONCOLOGY LETTERS 20: 383, 2020

using the originally installed EC800 software v1.3.6 (SonyCorporation).
Western blot analysis. Cell pellets were lysed in PBS with
1% Triton X‑100 and 50 µg/ml aprotinin (cat. no. 03346‑84;
Nacalai Tesque, Inc.). Protein concentration was deter‑
mined using the BCA assay. Cell lysates of CHO‑K1,
CHO/EpCAM, Caco‑2 and BINDS‑16 cells were boiled
in sodium dodecyl sulfate sample buffer (Nacalai Tesque,Inc.). The samples (10 µg/lane) were then electrophoresedon 5‑20% polyacrylamide gels (Nacalai Tesque, Inc.) andtransferred to polyvinylidene difluoride membranes (Merck
KGaA). Following blocking with 4% milk (Nacalai Tesque,Inc.) for 1 h, the membrane was incubated with anti‑EpCAM(1 µg/ml) or anti‑β‑actin (1 µg/ml; clone AC‑15; cat no. A5441;
Sigma‑Aldrich; Merck KGaA) antibodies for 1 h, followed
by incubation with HRP‑conjugated anti‑mouse IgG (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 at room temperature. The membrane wasdeveloped using the ImmunoStar LD Chemiluminescence
Reagent (FUJIFILM Wako Pure Chemical Corporation) anda Sayaca‑Imager (DRC Co., Ltd.). All western blotting proce‑dures were performed at room temperature.
Immunohistochemical analyses. Histological sections (4 µm)of a colorectal adenocarcinoma tissue array (cat. no. CO243b;
US Biomax Inc.) were autoclaved directly in citrate buffer(pH 6.0; Nichirei Bioscience, Inc.) for 20 min. The sections
were then incubated with 1 μg/ml anti‑EpCAM mAb for
1 h at room temperature and treated using an Envision+ kit(Agilent Technologies, Inc.) for 30 min at room temperature.
The color was developed using 3,3'‑diaminobenzidine tetra‑hydrochloride (Agilent Technologies Inc.) for 2 min at room
temperature, and sections were then counterstained withhematoxylin (FUJIFILM Wako Pure Chemical Corporation)for 2 min at room temperature. Hematoxylin and eosin (H&E)
staining (FUJIFILM Wako Pure Chemical Corporation) was
performed using consecutive colorectal adenocarcinoma
tissue sections for 2 min at room temperature.
Determination of the binding affinity. Caco‑2 cells weresuspended in 100 µl serially diluted anti‑EpCAM mAb(0.006‑100 µg/ml), followed by the addition of Alexa Fluor
488‑conjugated anti‑mouse IgG (1:200; cat. no. 4408; Cell
Signaling Technology, Inc.). Fluorescence data were obtainedusing an EC800 Cell Analyzer, and analyzed using the origi‑
nally installed EC800 v1.3.6 software (Sony Corporation). 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 induction by EpCAM was assayed as follows:
A total of 6 female 5‑week‑old BALB/c nude mice werepurchased from Charles River Laboratories, Inc. Following
euthanasia by cervical dislocation, the spleens were removed
aseptically, and single‑cell suspensions were obtained byforcing spleen tissues through a sterile cell strainer (cat.
no. 352360; Corning, Inc.) with a syringe. Erythrocytes werelysed by 10‑sec exposure to ice‑cold distilled water. The

3

splenocytes were washed with DMEM and resuspended in
DMEM with 10% FBS; this yield was designated as effector
cells. Caco‑2 cells were labeled with 10 µg/ml Calcein‑AM(Thermo Fisher Scientific, Inc.) and resuspended in DMEM
with 10% FBS. Caco‑2 cells were transferred to 96‑wellplates at 2x104 cells/well and mixed with the effector cells
at an effector‑to‑target ratio of 50:1, along with 100 µg/ml
anti‑EpCAM mAb or control mouse IgG2a. Following a 4‑h incu‑
bation, Calcein‑AM release into the supernatant was measured
in each well using a Power Scan HT microplate reader (BioTek
Instruments, Inc.) with an excitation wavelength of 485 nm
and an emission wavelength of 538 nm. Cytolytic activity wasdetermined as a percentage of lysis and calculated using thefollowing equation: Lysis (%) = (E ‑ S) / (M ‑ S) x 100, where
E is the fluorescence measured in the co‑cultures of target andeffector cells, S is the spontaneous fluorescence of the target
cells, and M is the maximum fluorescence measured after lysisof all cells with a buffer containing 0.5% Triton X‑100, 10 mM
Tris‑HCl (pH 7.4) and 10 mM EDTA.
CDC. CDC by EpCAM was assayed as follows: Caco‑2 cells
were labeled with 10 µg/ml Calcein‑AM and resuspended in
DMEM with 10% FBS. Caco‑2 cells were plated in 96‑wellplates at 2x104 cells/well, and 10% Low‑Tox‑M rabbit comple‑
ment (Cedarlane Laboratories) with 100 µg/ml anti‑EpCAM
mAb or control mouse IgG2a was added to each well. Following
a 4‑h incubation at 37˚C, Calcein‑AM release into the superna‑tant was determined in each well. Fluorescence intensity wascalculated as described in the ADCC section.
Antitumor activity of anti‑EpCAM mAb in colorectal adeno‑carcinoma xenografts. A total of 32 5‑week‑old female BALB/cnude mice were purchased from Charles River Laboratories, Inc.
After a 2‑week acclimation period, the mice were used in experi‑ments at 7 weeks of age. Caco‑2 cells (0.3 ml; 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 ofeach animal. On day 1 post‑inoculation, 100 µg anti‑EpCAM
mAb or control mouse IgG in 100 µl PBS was injected
i.p. Additional antibody inoculations were performed ondays 7 and 12. Tumor formation was measured in mice in thetreatment and control groups on days 7, 8, 12, 15 and 17 after
Caco‑2 cell injection. On day 17 after cell implantation, all mice
were euthanized by cervical dislocation, and tumor diametersand volumes were measured and recorded.
Statistical analyses. Data are presented as the 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.). P<0.05 was considered toindicate a statistically significant difference.
Results
Establishment and characterization of the anti‑EpCAM mAb.
The anti‑EpCAM mAb was established by immunizing one

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Figure 1. Recognition of EpCAM by EpMab‑16. (A) Flow cytometry analysis
using EpMab‑16. CHO/EpCAM, CHO‑K1, Caco‑2 and BINDS‑16 cells weretreated with EpMab‑16 (1 µg/ml) or buffer control, followed by secondary
antibodies. (B) Western blot analysis of EpCAM expression using EpMab‑16.
Red arrow denotes 35‑kDa EpCAM. EpCAM, epithelial cell adhesion
molecule; EpMab-16, anti-EpCAM monoclonal antibody; BINDS‑16,
Caco‑2/EpCAM‑knockout.

mouse with CHO/EpCAM cells and fusing its spleen cellswith P3U1 cells. Supernatants from hybridomas, which werepositive for CHO/EpCAM and negative for CHO‑K1, wereselected by flow cytometry. Further screening by immunohis‑
tochemistry and western blotting was performed for validation,
resulting in the establishment of EpMab‑16 (IgG2a, κ).
Flow cytometry was performed to assess the sensitivity of
EpMab‑16 in CHO/EpCAM cells and the Caco‑2 colorectal
adenocarcinoma cell line. As presented in Fig. 1A, EpMab‑16bound to CHO/EpCAM cells, but not CHO‑K1 cells. EpMab‑16
also bound to Caco‑2 but not BINDS‑16 cells, indicating that
EpMab‑16 was specific for EpCAM in the colorectal adeno‑carcinoma cell line.
Western blotting was performed to further assess thesensitivity of EpMab‑16. Lysates of CHO‑K1, CHO/EpCAM,
Caco‑2 and BINDS‑16 cells were probed. As demonstrated
in Fig. 1B, EpMab‑16 detected the 35‑kDa band of EpCAM
in lysates from CHO/EpCAM and Caco‑2 cells, whereas thisband was not present in lysates from CHO‑K1 and BINDS‑16
cells, indicating that EpMab‑16 specifically detected bothexogenous and endogenous EpCAM. The molecular weight of
EpCAM between CHO/EpCAM and Caco‑2 was different as
a PA tag (12 amino acids) was added to C‑terminus of EpCAM
in CHO/EpCAM cells.
EpMab‑16 detected membrane antigens in colorectal
adenocarcinoma tissues in the immunohistochemical analysis(Fig. 2A and B). Among six cases of colorectal adenocarci‑

Figure 2. Immunohistochemical analysis of colorectal adenocarcinoma andnormal colon tissues using EpMab‑16. (A and B) FFPE tissue sections ofcolorectal adenocarcinoma stained with 1 µg/ml EpMab‑16. B is a magnified
version of A. (C and D) HE staining of consecutive colorectal adenocarci‑
noma tissue sections. D is a magnified version of C. (E and F) FFPE tissue
sections of normal colon stained with 1 µg/ml EpMab‑16. F is a magnified
version of E. (G and H) HE staining against consecutive normal colon tissue
sections. H is a magnified version of G. Scale bar, 100 µm. EpMab-16, antiepithelial cell adhesion molecule monoclonal antibody; FFPE, formalin‑fixedparaffin‑embedded; HE, hematoxylin and eosin.

noma, five (83%) were positively stained by EpMab‑16. No
staining was observed without the primary mAb (data notshown). H&E staining was performed to stain nucleus as blueand cytosol as pink, using consecutive colorectal adenocarci‑
noma tissue sections (Fig. 2C and D). Furthermore, EpMab‑16
weakly detected membrane antigens in normal colon tissues(Fig. 2E and F). Among six samples of normal colon tissues,
three tissues (50%) were stained by EpMab‑16. No staining
was observed without the primary mAb (data not shown).
H&E staining was performed to stain nucleus as blue andcytosol as pink, using consecutive normal colon tissue sections(Fig. 2G and H).
Kinetic analysis of the interactions of EpMab‑16 with
Caco‑2 cells was subsequently analyzed using flow cytometry.
The K D for EpMab‑16 in Caco‑2 cells was calculated to be
1.8x10‑8 M (Fig. 3A), indicating a moderate binding affinity of
EpMab‑16 to colorectal adenocarcinoma cells.
ADCC and CDC activities of EpMab‑16 in a colorectal
adenocarcinoma cell line. The present study further investi‑
gated whether EpMab‑16 induced ADCC and CDC antitumor
activity in an EpCAM‑expressing Caco‑2 colorectal adenocar‑
cinoma cell line. As presented in Fig. 3B, EpMab‑16 exhibited
higher ADCC (44% cytotoxicity) in Caco‑2 cells comparedONCOLOGY LETTERS 20: 383, 2020

Figure 3. Binding affinity, ADCC and CDC of EpMab‑16. (A) Determinationof binding affinity of EpMab-16 in Caco‑2 cells. (B) ADCC activity of
EpMab‑16, control mouse IgG2a and control PBS in Caco‑2 cells. (C) CDC
activity of EpMab‑16, control mouse IgG2a and control PBS in Caco‑2 cells.
**
P<0.01; n.s., not significant. EpMab-16, anti-epithelial cell adhesion mole‑cule monoclonal antibody; ADCC, antibody‑dependent cellular cytotoxicity;
CDC, complement‑dependent cytotoxicity; KD, dissociation constant.

with that of control mouse IgG2a (8.7% cytotoxicity; P<0.01) orcontrol PBS (9.0% cytotoxicity; P<0.01) treatment. EpMab‑16
was also associated with more robust CDC activity (49% cyto‑
toxicity) in Caco‑2 cells compared with that of control mouse
IgG2a (23% cytotoxicity; P<0.01) or control PBS (21% cytotox‑
icity; P<0.01) treatment (Fig. 3C). These results suggested that
EpMab‑16 induced strong ADCC and CDC antitumor activity
in vitro.
EpMab‑16 antitumor activity in mouse xenografts of Caco‑2colorectal adenocarcinoma cells. We further examined
whether EMab‑16 exerts antitumor activity in vivo. On
days 1, 7 and 12 after Caco‑2 cell injections into the mice,
the Caco‑2 xenograft mouse models were injected with
EpMab‑16 or control mouse IgG. During the animal experi‑ment, no apparent weight loss due to tumor burden or organ
failure was observed among the mice. EpMab‑16‑treatedmice exhibited significantly lower tumor growth on days 7, 8,
12, 15 and 17 (all P<0.01) compared with that in IgG‑treatedcontrol mice (Fig. 4A). Tumor volume reduction by EpMab‑16
treatment reached 66% relative to the control group on
day 17. Tumors from EpMab‑16‑treated mice weighed signif‑
icantly less compared with those from IgG‑treated control
mice (60% reduction; P<0.01; Fig. 4B). Resected tumors on
day 17 are depicted in Fig. 4C. Total body weights did not
significantly differ between the treatment and control groups(Fig. S1). These results indicated that EpMab‑16 reduced the5

Figure 4. Antitumor activity of EpMab‑16 in Caco‑2 xenografts. (A) Tumor
volumes of Caco‑2 xenografts. Caco‑2 cells were injected subcutaneously,
and 100 µg EpMab‑16 or control mouse IgG was injected into the treat‑ment and control mice, respectively; additional antibodies were injectedon days 7 and 12. Tumor volume was measured on days 7, 8, 12, 15 and 17.
(B) Tumor weights of Caco‑2 xenografts resected from the EpMab‑16 andcontrol mouse IgG groups on day 17. (C) Resected tumors of Caco‑2 xeno‑
grafts from the EpMab‑16 and control mouse IgG groups on day 17. Scalebar, 1 cm. **P<0.01. EpMab-16, anti-epithelial cell adhesion molecule mono‑clonal antibody.

growth of Caco‑2 xenografts, but did not altogether eliminateit.
Discussion
The present study aimed to determine whether a novelanti‑EpCAM mAb may be useful for treating colorectal adeno‑carcinoma. First, a sensitive and specific anti‑EpCAM mAb,
EpMab‑16, was developed, which exhibited high reactivity forcolorectal adenocarcinoma by flow cytometry, western blot‑
ting and immunohistochemical analyses. Notably, the results
also suggested that EpMab‑16 exhibited diagnostic efficacy in
FFPE tissues as pathological diagnosis utilizes FFPE tissues.
EpMab‑16 was demonstrated to possess strong ADCC and
CDC activity against the Caco‑2 colorectal adenocarcinoma
cell line in vitro, and significantly reduced not only the volume
but also the weight of Caco‑2 xenografts in vivo. Althoughtumor reduction was determined to be 66% by volume and60% by weight on day 17, the reduction was not sufficient toeliminate the tumor entirely.
Cancer stem cells (CSCs) exhibit specific characteristics
including decontrolled self‑renewal, tumor‑initiating andtumor‑promoting properties, and chemotherapy resistance.
Therefore, targeting CSCs is thought to be a promising
approach to treat cancer (22). Since CD133 is thought to be

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one of CSC markers for CRC (23‑25), we previously developed
an anti‑CD133 mAb (CMab‑43, mouse IgG2a) (17) and inves‑tigated whether CD133 may represent a therapeutic target incolorectal adenocarcinoma using CMab‑43 (26). Importantly,
CMab‑43 exerted antitumor activity in Caco‑2 xenograftmodels at a dose of 100 µg/mouse/week administered threetimes, suggesting that CMab‑43 may be useful for antibody
therapy against CD133‑positive CRC (26). In the present study,
the effects of an anti‑EpCAM mAb, EpMab‑16, were exam‑
ined in vitro and in vivo, as EpCAM is also known to be a
CSC marker for CRC (25). In the present study, EpMab‑16 also
showed antitumor activity against CRC in a murine xenograftmodel. CSC marker‑expressing cells may be heterogenous;
therefore, the combination of mAbs or anticancer drugs tokill the CSC marker‑expressing cancer cells could be an ideal
strategy to treat CRC.
In another recent study, we established an anti‑HER2
mAb (H 2Mab‑19, mouse IgG2b) (27). Our previous study
demonstrated that H 2Mab‑19 significantly reduced tumor
development in HER2‑expressing breast cancer cells (BT‑474),
oral cancer cells (HSC‑2 and SAS) and CRC cells (Caco‑2)
xenograft models, suggesting that treatment with H2Mab‑19
may be a useful therapy for patients with HER2‑expressing
cancers (27,28). Although H 2 Mab‑19 showed significant
antitumor activity, immunohistochemical analysis revealed
that HER2 expression was diminished in the remaining
cancer cells after H 2Mab‑19 treatment (28). These results
suggest that H2Mab‑19 treatment may not be fully effective forpatients with HER2‑expressing cancers. In the current study,
an EpCAM‑targeting mAb, EpMab‑16, was developed. To
enhance the therapeutic effects in vivo, the combinational useof different mAbs, such as H2Mab‑19 and EpMab‑16 may berequired to treat HER2 and/or EpCAM‑expressing CRC cells.
The success of antibody‑based therapeutics of cancer
depends on the target antigen and the antibody to be used.
It has been reported that EpCAM is upregulated in 94% of
CRCs (29), whereas EpCAM expression is restricted to only
the basolateral membrane of epithelial cells in normal tissues,and therapeutic agents have limited access to it (30), under‑
scoring the importance of EpCAM as a therapeutic target.
Since EpMab‑16 appears to react with not only the basolateral
membrane, but also the apical membrane of epithelial cells
according to the results of the present study, the expres‑sion pattern of EpCAM should be further investigated bycomparing EpMab‑16 with other anti‑EpCAM mAbs.
A number of antibody‑based therapeutic approaches
targeting EpCAM have been developed (10). Previously,
Liao et al (31) generated an anti‑EpCAM mAb EpAb2‑6.
Using a colon cancer xenograft model, they demonstrated
that EpAb2‑6 induced CRC cell death by inhibiting EpCAM
signaling rather than by acting through the ADCC or CDC (31).
On the other hand, EpMab‑16 generated in the present study
possessed high ADCC and CDC activities against CRC cells
and exhibited antitumor activity in vivo. Another previous study
demonstrated that an anti‑EpCAM toxin‑conjugated antibody
Oportuzumab monatox (also termed VB4‑845) was effective
against squamous cell carcinomas of the head and neck andnon‑muscle invasive bladder cancer, and well‑tolerated inclinical trials (phase Ⅰ and Ⅱ) (32). Considering that EpCAM is
also expressed in tumor‑initiating cells, antibody‑based thera‑pies against EpCAM may kill not only proliferating cancer
cells, but also drug‑resistant dormant CRC cells (6). These
results suggest that the EpCAM‑targeted immunotherapy
may be a promising therapeutic strategy for CRC. To further
develop EpCAM‑targeted cancer therapy, further studies are
required to elucidate the precise role of EpCAM inhibition
among various cancer cells.
Acknowledgements
The authors would like to thank Mr. Takuro Nakamura,
Ms. Miyuki Yanaka, Ms. Saori Handa, Ms. Saki Okamoto and
Mr. Yu Komatsu (Department of Antibody Drug Development,
Tohoku University Graduate School of Medicine, Sendai,
Japan) for technical assistance with the in vitro experiments,and Ms. Akiko Harakawa [Institute of Microbial Chemistry(BIKAKEN), Microbial Chemistry Research Foundation,
Numazu, Japan] for technical assistance with the animal
experiments.
Funding
This research was supported in part by the Japan
Agency for Medical Research and Development (grantnos. JP20am0401013 to YK, JP20am0101078 to YK
and JP20ae0101028 to YK).
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
HH, TA, YS, and MKK performed the in vitro experiments. TO
performed in vivo experiments. JT analyzed the experimental
data. MK and YK designed the study and wrote the manu‑
script. All 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 Experimentsof the Institute of Microbial Chemistry (Numazu, Japan;
approval no. 2019‑066).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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