INTERNATIONAL JOURNAL OF MOlecular medicine 51: 18, 2023

Antitumor activities of a defucosylated anti‑EpCAM monoclonal
antibody in colorectal carcinoma xenograft models
GUANJIE LI1*, HIROYUKI SUZUKI1*, TOMOKAZU OHISHI2*, TEIZO ASANO3, TOMOHIRO TANAKA3,MIYUKI YANAKA3, TAKURO NAKAMURA3, TAKEO YOSHIKAWA4, MANABU KAWADA2,MIKA K. KANEKO3 and YUKINARI KATO1,3,4
1

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai,
Miyagi 980‑8575; 2Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Research Foundation,
Numazu, Shizuoka 410‑0301; Departments of 3Antibody Drug Development and 4Pharmacology,
Tohoku University Graduate School of Medicine, Sendai, Miyagi 980‑8575, Japan
Received August 14, 2022; Accepted December 7, 2022
DOI: 10.3892/ijmm.2023.5221
Abstract. Epithelial cell adhesion molecule (EpCAM) is a
type I transmembrane glycoprotein, which is highly expressedon tumor cells. As EpCAM plays a crucial role in cell adhe‑sion, survival, proliferation, stemness, and tumorigenesis, it
has been considered as a promising target for tumor diagnosis
and therapy. Anti‑EpCAM monoclonal antibodies (mAbs)
have been developed and have previously demonstrated
promising outcomes in several clinical trials. An anti‑EpCAM
mAb, EpMab‑37 (mouse IgG1, kappa) was previously devel‑
oped by the authors, using the cell‑based immunization andscreening method. In the present study, a defucosylated versionof anti‑EpCAM mAb (EpMab‑37‑mG2a‑f) was generated
to evaluate the antitumor activity against EpCAM‑positive
cells. EpMab‑37‑mG2a‑f recognized EpCAM‑overexpressing
CHO‑K1 (CHO/EpCAM) cells with a moderate binding‑affinity[dissociation constant (KD)=2.2x10‑8 M] using flow cytometry.
EpMab‑37‑mG2a‑f exhibited potent antibody‑dependent cellular

Correspondence to: Dr Hiroyuki Suzuki or Dr Yukinari Kato,

Department of Molecular Pharmacology, Tohoku University
Graduate School of Medicine, 2‑1 Seiryo‑machi, Aoba‑ku, Sendai,
Miyagi 980‑8575, JapanE‑mail: hiroyuki.suzuki.b4@tohoku.ac.jp
E‑mail: yukinari.kato.e6@tohoku.ac.jp*

Contributed equally

Abbreviations: EpCAM, epithelial cell adhesion molecule;mAb, monoclonal antibody; ADCC, antibody‑dependent cellularcytotoxicity; CDC, complement‑dependent cytotoxicity; FDA, Foodand Drug Administration; FcγR, Fcγ receptor; NK, natural killer;
RPMI, Roswell Park Memorial Institute; PBS, phosphate‑bufferedsaline; KD, dissociation constant

Key words: EpCAM monoclonal antibody, ADCC, CDC, colorectal
cancercytotoxicity (ADCC) and complement‑dependent cytotoxicity(CDC) for CHO/EpCAM cells by murine splenocytes andcomplements, respectively. Furthermore, the administrationof EpMab‑37‑mG2a‑f significantly suppressed CHO/EpCAM
xenograft tumor development compared with the control
mouse IgG. EpMab‑37‑mG2a‑f also exhibited a moderatebinding‑affinity (KD =1.5x10 ‑8 M) and high ADCC and CDC
activities for a colorectal cancer cell line (Caco‑2 cells). The
administration of EpMab‑37‑mG2a‑f to Caco‑2 tumor‑bearing
mice significantly suppressed tumor development comparedwith the control. By contrast, EpMab‑37‑mG2a ‑f neversuppressed the xenograft tumor growth of Caco‑2 cells inwhich EpCAM was knocked out. On the whole, these results
indicate that EpMab‑37‑mG2a‑f may exert antitumor activities
against EpCAM‑positive cancers and may thus be a promisingtherapeutic regimen for colorectal cancer.
Introduction
Epithelial cell adhesion molecule (EpCAM) is expressed on
the basolateral membrane of epithelial cells (1). EpCAM is aunique type I transmembrane glycoprotein which possesses a
different structure and functions compared to other classicaladhesion molecules, including cadherins, selectins and integ‑
rins. EpCAM‑mediated homophilic and intercellular adhesion
are essential for the maintenance of the epithelial integrity (2).
EpCAM also plays critical roles in intercellular signaling.
Following the cleavage of the EpCAM intracellular domain,
it functions as a transcriptional co‑factor with β‑catenin andregulates the transcriptional targets involved in cell prolifera‑tion, survival and stemness (3). Therefore, the overexpressionof EpCAM plays a key role in tumor development.
EpCAM is a critical marker for the isolation circulatingtumor cells (CTCs). CTCs provide critical prognostic informa‑tion as an indicator of micro‑metastasis, and determine theresponse to some cancer therapeutics (4). The US Food andDrug Administration (FDA) confirmed the clinical importanceof CTCs, and approved of CellSearch®, a platform that can beused for the isolation of EpCAM‑positive, CD45‑negative cells

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LI et al: DEFUCOSYLATED ANTI-EpCAM mAb

from whole blood samples (5). The EpCAM‑based CellSearch®
CTC test has been studied in several clinical trials in lung (6),breast (7) and prostate (8) cancers.
Therapeutic monoclonal antibodies (mAbs) are the most
crucial biological therapeutics for the treatment of various
tumors (9) and inflammatory diseases, such as rheuma‑
toid arthritis (10). EpCAM was the first identified humantumor‑associated antigen, and plays a critical role in tumor
development (11). Therefore, EpCAM has been considered
as a target of mAb therapies, including Adecatumumab (12)and Edrecolomab (13,14) in EpCAM‑overexpressing breast,
prostate, gastrointestinal and colorectal cancers. A human‑
ized single chain Fv against EpCAM, fused to Pseudomonas
exotoxin A, Oportuzumab monatox, has been evaluated inbladder cancer, as have been previously reviewed (15). A
bispecific EpCAM/CD3‑antibody, Catumaxomab, functions
as the trifunctional antibody. When Catumaxomab recog‑
nizes EpCAM‑high tumors, it also recruits T‑cells to tumors.
This event promotes the formation of cytotoxic synapse andT‑cell‑mediated cytotoxicity. Furthermore, Catumaxomab
recruits natural killer (NK) cells and macrophages throughthe Fc domain and enhances antibody‑dependent cellularcytotoxicity (ADCC) (16,17). Catumaxomab has demonstrated
promising outcomes in several clinical trials (18‑20), and has
been approved by the European Union for the treatment ofpatients with malignant ascites (21).
ADCC is mediated by NK cells upon the binding of theFcγRIIIa to the Fc region of mAbs. The FcγRIIIa engagement
can stimulate NK cells, which attack and lyse target cells (9).
However, this function is affected by the N‑linked glycosylationin the Fc region (22). In particular, a core fucose deficiency on
the Fc N‑glycan has been revealed to enhance the Fc binding
to the FcγRIIIa on the effector cells (23). In a previous study
on recombinant mAb production using Chinese hamster
ovary (CHO) cells, the Fc N‑glycans were determined as anheterogeneous biantennary complex (22). Fucosyltransferase 8(FUT8) is the only α1,6‑fucosyltransferase transferring fucose
via an α1,6 linkage to the innermost N‑acetylglucosamine on
N‑glycans for core fucosylation. CHO cells subjected to FUT8
knockout have been revealed to produce completely defucosyl‑
ated recombinant antibodies. Furthermore, mAb producedby CHO cells subjected to FUT8 knockout strongly binds to
FcγRIIIa and potently enhances ADCC activity compared
to mAb produced by wild‑type CHO. Therefore, CHO cells
subjected to FUT8 knockout may be an ideal host cell for the
production of completely defucosylated high‑ADCC mAb fortherapeutic use (24).
An anti‑EpCAM mAb, EpMab‑37 (mouse IgG1, kappa) has
been previously established by the authors, using the cell‑based
immunization and screening method (25). In the present study,
a defucosylated anti‑EpCAM mAb (EpMab‑37‑mG2a‑f) was
produced by using FUT8‑deficient CHO cells to potentiate anti‑
tumor activity and investigated the ability of EpMab‑37‑mG2a‑f
to induce ADCC, complement‑dependent cytotoxicity (CDC)
and antitumor activity in EpCAM‑expressing cells.

HCT116 (ATCC CCL‑247), HT‑29 (ATCC HTB‑38), LS174T(ATCC CL‑188), COLO201 (ATCC CCL‑224), HCT‑8(ATCC CCL‑244) and SW1116 (ATCC CCL‑233), werepurchased from the ATCC. HCT‑15 (TKG 0504), COLO205(TKG 0457) and DLD‑1 (TKG 0379) were purchased fromthe Cell Resource Center for Biomedical Research Instituteof Development, Aging and Cancer at Tohoku University.
EpCAM cDNA plus a C‑terminal PA tag (EpCAM‑PA) was
subcloned into a pCAG‑Ble vector (FUJIFILM Wako Pure
Chemical Corporation). CHO/EpCAM was established bytransfecting the pCAG/EpCAM‑PA vector into CHO‑K1
cells using the Neon Transfection System (Thermo Fisher
Scientific, Inc.). CHO‑K1 cells (1.5x106) were transfected withthe pCAG/EpCAM‑PA vector. Positive cells for anti‑EpCAM
mAb (clone 9C4; cat. no. 324202; BioLegend, Inc.) were
sorted using an SH800 cell sorter (Sony Corporation) andselected as previously described (26,27). CHO‑K1 andCHO/EpCAM cells were cultured in Roswell Park Memorial
Institute (RPMI)‑1640 medium (Nacalai Tesque, Inc.),
supplemented with 10% heat‑inactivated fetal bovine serum(FBS; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin,
100 µg/ml streptomycin and 0.25 µg/ml amphotericin B(Nacalai Tesque, Inc.). Caco‑2 cells and Caco‑2 cells in which
EpCAM was knocked out (BINDS‑16), which were previously
established (26), were cultured in DMEM (Nacalai Tesque,Inc.), supplemented with 10% FBS, 100 units/ml penicillinand 100 µg/ml streptomycin. The COLO201, COLO205,
SW1116 and DLD‑1 cells were cultured in RPMI‑1640
medium (Nacalai Tesque, Inc.), supplemented with 10% FBS,
100 units/ml penicillin and 100 µg/ml streptomycin (Nacalai
Tesque, Inc.). The HCT116, HT‑29, LS174T and HCT‑8 were
cultured in DMEM (Nacalai Tesque, Inc.), supplemented
with 10% FBS, 100 U/ml penicillin and 100 µg/ml strepto‑
mycin. All cell lines were maintained at 37˚C in a humidified
atmosphere under 5% CO2.

Materials and methods

Antibodies. Anti‑EpCAM mAb, EpMab‑37 was previously
established (25). To generate recombinant EpMab‑37, V H
cDNA of EpMab‑37 and C H of mouse IgG2a was cloned
into the pCAG‑Ble vector. V L cDNA of EpMab‑37 and C L

Cell lines and cell culture. CHO‑K1 (ATCC CCL‑61) and thehuman colorectal cancer cell lines, Caco‑2 (ATCC HTB‑37),

Animal experiments. All animal experiments were performed
following regulations and guidelines to minimize animal
distress and suffering in the laboratory by the Institutional
Committee for Experiments of the Institute of Microbial
Chemistry (Numazu, Japan). The animal study protocol
was approved (approval no. 2022‑024) by the Institutional
Committee for Experiments of the Institute of Microbial
Chemistry (Numazu, Japan). BALB/c nude mice (female,a total of 70 mice) were maintained on an 11‑h light/13‑hdark cycle in a specific pathogen‑free environment, acrossthe experimental period. Food and water were supplied
ad libitum. The weight of the mice was monitored twice per
week and their health was monitored three times per week.
The loss of original body weight was determined to a point>25% (28) and/or a maximum tumor size >3,000 mm3 and/or
significant changes in the appearance of tumors as humane
endpoints for euthanasia. Cervical dislocation was used foreuthanasia. Mouse death was confirmed by respiratory arrest
and rigor mortis.

INTERNATIONAL JOURNAL OF MOlecular medicine 51: 18, 2023

cDNA of mouse kappa light chain were also subcloned into
the pCAG‑Neo vector (FUJIFILM Wako Pure Chemical
Corporation). The vector for the recombinant EpMab‑37 wastransduced into BINDS‑09 (FUT8‑knockout ExpiCHO‑S)
cells using the ExpiCHO Expression System (Thermo
Fisher Scientific, Inc.), as previously described (29‑36).
EpMab‑37‑mG2a‑f was purified using Ab‑Capcher (ProteNova
Co., Ltd.). Mouse IgG (cat. no. 140‑09511) and IgG2a (cat.
no. M7769) were purchased from FUJIFILM Wako Pure
Chemical Corporation and MilliporeSigma, respectively.
281‑mG2a‑f [defucosylated anti‑hamster podoplanin (PDPN)
mAb, control mouse IgG2a for ADCC reporter bioassay] waspreviously described (37).
Flow cytometry. CHO‑K1, CHO/EpCAM and Caco‑2cells were isolated using 0.25% trypsin and 1 mM ethyl‑enediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.)
treatment. The cells were treated with EpMab‑37‑mG2a‑f, or
blocking buffer [0.1% bovine serum albumin (BSA; Nacalai
Tesque, Inc.) in phosphate‑buffered saline (PBS)] (control)for 30 min at 4˚C. Subsequently, the cells were incubatedin Alexa Fluor 488‑conjugated anti‑mouse IgG (1:2,000;
cat. no. 4408; Cell Signaling Technology, Inc.) for 30 min
at 4˚C. Fluorescence data were collected using the SA3800
Cell Analyzer and analyzed using SA3800 software ver. 2.05
(Sony Corporation).
Determination of binding af f init y. Serially diluted
EpMab‑37‑mG2a‑f (0.006‑100 µg/ml) was suspended with
CHO/EpCAM and Caco‑2 cells. The cells were further treatedwith Alexa Fluor 488‑conjugated anti‑mouse IgG (1:200).
Fluorescence data were collected using BD FACSLyric
and analyzed using BD FACSuite software version 1.3 (BDBiosciences). To determine the dissociation constant (K D),
GraphPad Prism 8 (the fitting binding isotherms to built‑in
one‑site binding models; GraphPad Software, Inc.) was used.
A D CC of EpM a b ‑37‑ m G 2 a ‑f. A DCC i nduction by
EpMab‑37‑mG2a‑f was assayed as follows: Six female BALB/cnude mice (5 weeks old) were purchased from Charles River
Laboratories, Inc. The spleens were aseptically removed andsingle‑cell suspensions were obtained through a sterile cellstrainer (cat. no. 352360; BD Falcon). Erythrocytes were
removed with the treatment of ice‑cold distilled water. The
splenocytes were resuspended in DMEM with 10% FBS;
this preparation was designated as effector cells. Target cells(CHO‑K1, CHO/EpCAM and Caco‑2) were treated with
10 µg/ml Calcein AM (Thermo Fisher Scientific, Inc.) (38).
The target cells (2x10 4 cells) were plated in 96‑well plates
and mixed with effector cells (effector‑to‑target ratio, 100:1),
100 µg/ml of EpMab‑37‑mG2a ‑f or control mouse IgG2a.
Following incubation for 4.5 h at 37˚C, the Calcein release
into the medium was measured with an excitation wave‑
length (485 nm) and an emission wavelength (538 nm) using
a microplate reader (Power Scan HT; BioTek Instruments,Inc.).
The total percentage of cell lysis was determined asfollows: % lysis=(E‑S)/(M‑S) x100, where ‘E’ corresponds tothe fluorescence in the presence of both effector and target
cells, ‘S’ to the spontaneous fluorescence in the presence of3

only target cells and ‘M’ to the maximum fluorescence by
the treatment with a lysis buffer [10 mM Tris‑HCl (pH 7.4),
10 mM of EDTA and 0.5% Triton X‑100].
CDC of EpMab‑37‑mG 2a ‑f. Target cells (CHO‑K1,
CHO/EpCAM and Caco‑2) were treated with 10 µg/ml
Calcein AM (39). The target cells (2x104 cells) were mixed
with rabbit complement (final dilution 1:10; Low‑Tox‑M Rabbit
Complement; Cedarlane Laboratories) and 100 µg/ml control
mouse IgG2a or EpMab‑37‑mG2a‑f. Following incubation for
4.5 h at 37˚C, Calcein release into the medium was measured.
ADCC reporter bioassay. The ADCC reporter bioassay
was performed using an ADCC Reporter Bioassay kit (cat.
no. G7018; Promega Corporation), according to the manu‑
facturer's instructions (40). Target cells (12,500 cells perwell) were inoculated into a 96‑well white solid plate (cat.
no. 655083; Greiner Bio‑One). EpMab‑37‑mG2a‑f, EpMab‑37
and 281‑mG2a‑f were serially diluted and added to target
cells. Jurkat cells (a component of ADCC Reporter Bioassaykit) stably expressing the human FcγRIIIa receptor, and anuclear factor of activated T‑cells (NFAT) response element
driving Firefly luciferase, were used as effector cells. The
engineered Jurkat cells (75,000 cells in 25 µl) were then added
and co‑cultured with antibody‑treated target cells at 37˚C
for 6 h. Luminescence using the Bio‑Glo Luciferase Assay
System was measured with a GloMax luminometer (Promega
Corporation).
Antitumor activity of EpMab‑37‑mG 2a ‑f in xenografts of
CHO‑K1 and CHO/EpCAM. BALB/c nude mice (female,
32 mice) were purchased from Charles River Laboratories,
Inc. The CHO‑K1 and CHO/EpCAM cells (5x10 6 cells)suspended with BD Matrigel Matrix Growth Factor Reduced(BD Biosciences) were inoculated into the left flank of themice subcutaneously. On day 6 after the inoculation, 100 µg
EpMab‑37‑mG2a‑f (n=8) or control mouse IgG (n=8) in 100 µl
PBS were injected intraperitoneally. Additional antibody
injections were performed on day 14. The dose was based ona biodistribution study of mouse‑derived anti‑EpCAM mAb(MOC31) (41). The tumor volume was measured on days 6, 11,
14, 18 and 20, and determined as previously described (42). On
day 18, ulcerations began to appear in some tumors. As ulcer‑
ated tumors result in skin breakdown and a decrease in tumor
weight, it was decided to terminate the experiment on day 20prior to the diameter of the ulcers exceeding 4 mm. The healthand well‑being of the animals did not deteriorate during these
2 days. The xenograft tumors with or without ulceration were
carefully removed from the sacrificed mice, and then weighed
and photographed immediately.
Antitumor activity of EpMab‑37‑mG 2a ‑f in xenografts of
Caco‑2 and BINDS‑16 cells. The Caco‑2 and BINDS‑16 cells(5x106 cells) resuspended with BD Matrigel Matrix Growth
Factor Reduced (BD Biosciences) were inoculated into the left
flank of BALB/c nude mice (female, 32 mice) subcutaneously.
On day 6 after the inoculation, 100 µg EpMab‑37‑mG2a‑f (n=8)
or control mouse IgG (n=8) in 100 µl PBS were injected intra‑
peritoneally. Additional antibody injections were performedon days 14 and 20. The tumor volume was measured on

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LI et al: DEFUCOSYLATED ANTI-EpCAM mAb

Figure 1. Flow cytometry using EpMab‑37‑mG2a‑f. (A) Production of EpMab‑37‑mG2a‑f (core‑fucose‑deficient mouse IgG2a) from EpMab‑37 (mouse IgG1).
(B) CHO‑K1 and CHO/EpCAM cells were treated with EpMab‑37‑mG2a‑f (red) or buffer control (black), followed by Alexa Fluor 488‑conjugated anti‑mouse
IgG. Fluorescence data were analyzed using the SA3800 Cell Analyzer. (C) Determination of the binding affinity of EpMab‑37‑mG2a‑f using flow cytometry
for CHO/EpCAM cells. CHO/EpCAM cells were suspended in serially diluted EpMab‑37‑mG2a‑f. Alexa Fluor 488‑conjugated anti‑mouse IgG was thenadded. Fluorescence data were collected using the BD FACSLyric and the KD was calculated using GraphPad PRISM 8 software. CHO, Chinese hamster ovary;
EpCAM, epithelial cell adhesion molecule; KD. dissociation constant.

days 6, 11, 14, 18, 20, 25 and 27, and determined as previously
described (42). The xenograft tumors were removed, weighed
and photographed as described above.

Statistical analyses. All data are expressed as the mean ± stan‑
dard error of the mean (SEM). In ADCC, CDC and tumor
weight measurement, unpaired Welch's t‑test was conducted.

INTERNATIONAL JOURNAL OF MOlecular medicine 51: 18, 2023

5

Figure 2. Evaluation of ADCC and CDC elicited by EpMab‑37‑mG2a‑f. (A) ADCC elicited by EpMab‑37‑mG2a‑f and mIgG2a targeting CHO/EpCAM and
CHO‑K1 cells. (B) CDC elicited by EpMab‑37‑mG2a‑f and control mouse IgG2a targeting CHO/EpCAM and CHO‑K1 cells. Values are presented as themean ± SEM (*P<0.05 and **P<0.01; Welch's t‑test). (C) ADCC reporter bioassay by EpMab‑37‑mG2a‑f, EpMab‑37, and control (281‑mG2a‑f) in the presence of
CHO/EpCAM cells. ADCC, antibody‑dependent cellular cytotoxicity; CDC, complement‑dependent cytotoxicity; n.s., not significant; mIgG2a, mouse IgG2a;
CHO, Chinese hamster ovary; EpCAM, epithelial cell adhesion molecule.

ANOVA with Sidak's post hoc test were utilized for tumor
volume and mice weight. GraphPad Prism 8 (GraphPad
Software, Inc.) was used for all calculations. P<0.05 wasconsidered to indicate a statistically significant difference.
Results
Flow cytometric analysis against CHO/EpCAM cells using
EpMab‑37‑mG2a ‑f. In a previous study by the authors, ananti‑EpCAM mAb (EpMab‑37, mouse IgG1, kappa) was estab‑lished, using cancer‑specific mAb (CasMab) technology (25).
EpMab‑37 was revealed to be available for flow cytometry,western blotting and immunohistochemistry (25). In thepresent study, a defucosylated form of anti‑EpCAM mAb(EpMab‑37‑mG2a‑f) was produced by combining V H and V Lof EpMab‑37 with CH and CL of mouse IgG2a, respectively(Fig. 1A). EpMab‑37‑mG2a‑f detected CHO/EpCAM and notparental CHO‑K1 cells in a concentration‑dependent manner(Figs. 1B and S1A), indicating that EpMab‑37‑mG2a‑f reactedwith EpCAM.
A kinetic analysis of the interactions of EpMab‑37‑mG2a‑f
with CHO/EpCAM was performed using flow cytometry.
As demonstrated in Fig. 1C, the K D for the interaction of
EpMab‑37‑mG2a‑f with CHO/EpCAM cells was 2.2x10 ‑8 M,suggesting that EpMab‑37‑mG2a‑f may exhibit moderateaffinity for CHO/EpCAM cells.
E p M a b ‑37‑ m G 2 a ‑f‑ m e d i a te d A D C C a n d CD C i n
CHO/EpCAM cells. The present study then investigatedwhether EpMab‑37‑mG2a‑f was capable of mediating ADCC
against CHO/EpCAM cells. EpMab‑37‑mG2a‑f exhibited
ADCC (29.6% cytotoxicity) against CHO/EpCAM cells
more effectively than the control mouse IgG2a (5.0% cyto‑
toxicity; P<0.01). No notable difference was found between
EpMab‑37‑mG2a‑f and control mouse IgG2a in ADCC levels
against CHO‑K1 (Fig. 2A).

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LI et al: DEFUCOSYLATED ANTI-EpCAM mAb

Figure 3. Antitumor activity of EpMab‑37‑mG2a‑f. (A and B) Measurement of tumor volume in (A) CHO/EpCAM and (B) CHO‑K1 xenograft models.
CHO/EpCAM and CHO‑K1 cells (5x106 cells) were injected into mice subcutaneously. On day 6, 100 µg EpMab‑37‑mG2a‑f or mIgG were injected into miceintraperitoneally. On day 14, additional antibodies were injected. On days 6, 11, 14, 18 and 20 following the inoculation, the tumor volume was measured.
Values are presented as the mean ± SEM. *P<0.05 and **P<0.01 (ANOVA and Sidak's multiple comparisons test). (C and D) The weight of the excised(C) CHO/EpCAM and (D) CHO‑K1 xenografts was measured on day 20. Values are presented as the mean ± SEM. **P<0.01 (Welch's t‑test). (E and F) The
resected tumors appearance of (E) CHO/EpCAM and (F) CHO‑K1 xenografts in the control mouse IgG and EpMab‑37‑mG2a‑f treated groups on day 20 (scalebar, 1 cm). n.s., not significant; CHO, Chinese hamster ovary; EpCAM, epithelial cell adhesion molecule; mIgG, mouse IgG.

Subsequently, it was examined whether EpMab‑37‑mG2a‑f
could exert CDC against CHO/EpCAM cells. As demonstrated
in Fig. 2B, EpMab‑37‑mG2a‑f induced a higher degree of CDC(33.2% cytotoxicity) in CHO/EpCAM cells compared with thatinduced by control mouse IgG2a (10.5% cytotoxicity; P<0.05).
There was no marked difference between EpMab‑37‑mG2a‑f
and control mouse IgG2a in the observed CDC levels against
CHO‑K1 (Fig. 2B).
The ADCC reporter bioassay is a bioluminescentreporter gene assay for the quantification of the biologicalactivity of the antibody via Fc γRIIIa‑mediated pathway

activation in an ADCC mechanism of action (40). In thepresent study, to compare the ADCC pathway activation by
EpMab‑37‑mG2a‑f and EpMab‑37, the CHO/EpCAM cells
were treated with serially diluted mAbs, and then incubatedwith effector Jurkat cells, which express the human FcγRIIIa
receptor and an NFAT response element driving Firefly
luciferase. Furthermore, defucosylated anti‑hamster PDPN
(mouse IgG2a Ab) (281‑mG2a‑f) was also used as a control
since defucosylated control mouse IgG2a was unavailable. It
was confirmed that 281‑mG2a‑f never recognized the target
cells. As demonstrated in Fig. 2C, EpMab‑37‑mG2a‑f activatedINTERNATIONAL JOURNAL OF MOlecular medicine 51: 18, 2023

7

Figure 4. Mouse body weights and appearance. (A and B) Body weights of (A) CHO/EpCAM and (B) CHO‑K1 xenografts‑implanted mice on days 6, 11, 14,18 and 20 (ANOVA and Sidak's multiple comparisons test). (C and D) Body appearance of (C) CHO/EpCAM and (D) CHO‑K1 xenografts‑implanted mice on
day 20 (scale bar, 1 cm). n.s., not significant; CHO, Chinese hamster ovary; EpCAM, epithelial cell adhesion molecule; mIgG, mouse IgG.

the effector (EC50; 31.7 ng/ml) in a concentration‑dependent
manner, whereas EpMab‑37 and 281‑mG2a‑f did not. These
results indicated that EpMab‑37‑mG2a‑f exhibits superior
ADCC activity against CHO/EpCAM cells compared with
EpMab‑37.
Antitumor effects of EpMab‑37‑mG2a‑f in the mouse xeno‑
grafts of CHO/EpCAM cells. In the CHO/EpCAM xenograft
tumors, EpMab‑37‑mG2a ‑f and control mouse IgG were
injected into mice intraperitoneally on days 6 and 14, following
CHO/EpCAM cell inoculation. On days 6, 11, 14, 18 and 20
following the inoculation, the tumor volume was measured.
The EpMab‑37‑mG2a‑f administration resulted in a significant
reduction in tumor volume on days 14 (P<0.05), 18 (P<0.01)
and 20 (P<0.01) compared with that of the control mouse IgG(Fig. 3A). The EpMab‑37‑mG2a‑f administration resulted ina 50% reduction in tumor volume compared with that of thecontrol mouse IgG on day 20.
The weight of CHO/EpCAM tumors treated with
EpMab‑37‑mG2a‑f was significantly lower than that of tumors
treated with control mouse IgG (57% reduction; P<0.01;
Fig. 3C). CHO/EpCAM tumors that were resected from miceon day 20 are depicted in Fig. 3E.
In the CHO‑K1 xenograft models, EpMab‑37‑mG2a‑f andcontrol mouse IgG were intraperitoneally injected into miceon days 6 and 14 following the inoculation of CHO‑K1 cells.
On days 6, 11, 14, 18 and 20 after the inoculation of cells, thetumor volume was measured. No marked differences wereobserved between EpMab‑37‑mG2a‑f and control mouse IgG as
regards CHO‑K1 tumor volume (Fig. 3B) and weight (Fig. 3D).
CHO‑K1 tumors that were resected from mice on day 20 are
demonstrated in Fig. 3F.

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LI et al: DEFUCOSYLATED ANTI-EpCAM mAb

Figure 5. Flow cytometry of EpMab‑37‑mG2a‑f against the human colorectal cancer cell line, Caco‑2, and Caco‑2 cells in which EpCAM was knocked out(BINDS‑16). (A) Caco‑2 and BINDS‑16 cells were treated with EpMab‑37‑mG2a‑f (red) or buffer control (black), followed by Alexa Fluor 488‑conjugatedanti‑mouse IgG. Fluorescence data were analyzed using the SA3800 Cell Analyzer. (B) Determination of the binding affinity of EpMab‑37‑mG2a‑f for Caco‑2cells using flow cytometry. Caco‑2 cells were suspended in 100 µl serially diluted EpMab‑37‑mG2a‑f. Alexa Fluor 488‑conjugated anti‑mouse IgG was thenadded. Fluorescence data were collected using the BD FACSLyric and the K D was calculated by GraphPad PRISM 8. EpCAM, epithelial cell adhesion
molecule; KD, dissociation constant.

The loss of body weight was not observed in the
CHO/EpCAM (Fig. 4A) and CHO‑K1 (Fig. 4B) tumor‑implanted
mice. The mice on day 20 are demonstrated in Fig. 4C and D.
Flow cytometry of Caco‑2 cells using EpMab‑37‑mG2a‑f. As
demonstrated in Figs. 5A and S1B, EpMab‑37‑mG2a‑f detected
Caco‑2 cells in a concentration‑dependent manner. By contrast,
EpMab‑37 did not react with Caco‑2 cells in which EpCAM
was knocked out (BINDS‑16). The increased expression of

EpCAM could also be detected in other colorectal cancer
cell lines tested (Fig. S2). A kinetic analysis of the binding of
EpMab‑37‑mG2a‑f to Caco‑2 cells was performed using flow
cytometry. The K D for the interaction of EpMab‑37‑mG2a‑f
with Caco‑2 cells was 1.5x10 ‑8 M (Fig. 5B), suggesting that
EpMab‑37‑mG2a‑f exhibits moderate affinity for Caco‑2 cells.
EpMab‑37‑mG2a‑f‑mediated ADCC and CDC in Caco‑2 and
BINDS‑16 cells. The present study then investigated whether

INTERNATIONAL JOURNAL OF MOlecular medicine 51: 18, 2023

9

Figure 6. ADCC and CDC activity of EpMab‑37‑mG2a‑f against Caco‑2 and BINDS‑16 cells (Caco‑2 cells in which EpCAM was knocked out). (A) ADCC
elicited by EpMab‑37‑mG2a‑f and control mouse IgG2a (mIgG2a) targeting Caco‑2 and BINDS‑16 cells. (B) CDC elicited by EpMab‑37‑mG2a‑f and control
mouse IgG2a targeting Caco‑2 and BINDS‑16 cells. Values are presented as the mean ± SEM. *P<0.05 (Welch's t‑test). (C) ADCC reporter bioassay by
EpMab‑37‑mG2a‑f, EpMab‑37, and control (281‑mG2a‑f) in the presence of Caco‑2 cells. ADCC, antibody‑dependent cellular cytotoxicity; CDC, comple‑
ment‑dependent cytotoxicity; n.s., not significant; mIgG2a, mouse IgG2a; CHO, Chinese hamster ovary; EpCAM, epithelial cell adhesion molecule.

EpMab‑37‑mG2a‑f is capable of mediating ADCC against Caco‑2
and BINDS‑16 cells. As revealed in Fig. 6A, EpMab‑37‑mG2a‑fdemonstrated ADCC (45.7% cytotoxicity) against Caco‑2 cells,
more potently than the control mouse IgG2a (9.5% cytotoxicity;
P<0.05). It was then investigated whether EpMab‑37‑mG2a‑fexhibited CDC against Caco‑2 cells. EpMab‑37‑mG2a‑f induced
a higher degree of CDC (46.6% cytotoxicity) in Caco‑2 cells
compared with that induced by control mouse IgG2a (14.7%
cytotoxicity; P<0.05) (Fig. 6B). By contrast, ADCC and CDC
were not induced by EpMab‑37‑mG2a‑f in BINDS‑16 cells(Fig. 6A and B). The ADCC reporter bioassay for Caco‑2 cells
also revealed that the only EpMab‑37‑mG2a‑f exhibited the
ADCC effector activation (EC50; 15.9 ng/ml) (Fig. 6C). These
results demonstrated that EpMab‑37‑mG2a‑f exhibited potent
ADCC and CDC against Caco‑2 cells.
Antitumor effects of EpMab‑37‑mG 2a ‑f on Caco‑2 and
BINDS‑16 xenografts. In the Caco‑2 xenograft models,

EpMab‑37‑mG 2a ‑f and control mouse IgG were intra‑
peritoneally injected on days 6, 14 and 20, following
the inoculation of Caco‑2 cells. The tumor volume wasmeasured on days 6, 11, 14, 18, 20, 25 and 27 after the injec‑
tion. The administration of EpMab‑37‑mG2a‑f resulted in asignificant reduction in tumor growth on days 14 (P<0.05),
18 (P<0.01), 20 (P<0.01), 25 (P<0.01) and 27 (P<0.01) ascompared with the control mouse IgG (Fig. 7A). The admin‑
istration of EpMab‑37‑mG2a‑f resulted in a 42% reduction
in tumor volume compared with the control mouse IgG on
day 27. Tumors from the EpMab‑37‑mG2a ‑f‑treated miceweighed significantly less than those from the control
mouse IgG‑treated mice (28% reduction; P<0.01, Fig. 7C).
Tumors that were resected from mice on day 27 aredemonstrated in Fig. 7E. By contrast, the antitumor effects
of EpMab‑37‑mG 2a ‑f on BINDS‑16 were not observed(Fig. 7B and D). BINDS‑16 tumors that were resected frommice on day 27 are demonstrated in Fig. 7F.

10

LI et al: DEFUCOSYLATED ANTI-EpCAM mAb

Figure 7. Antitumor activity of EpMab‑37‑mG2a‑f against Caco‑2 and BINDS‑16 xenografts. (A and B) Measurement of tumor volume in (A) Caco‑2 and(B) BINDS‑16 (Caco‑2 cells in which EpCAM was knocked out) xenograft‑implanted mice. Caco‑2 and BINDS‑16 cells (5x106 cells) were inoculated intomice subcutaneously. On day 6, 100 µg EpMab‑37‑mG2a‑f or mIgG were injected into mice intraperitoneally. On days 14 and 20, additional antibodies were
injected. On days 6, 11, 14, 18, 20, 25 and 27 following the inoculation, the tumor volume was measured. Values are presented as the mean ± SEM. *P<0.05
and **P<0.01 (ANOVA and Sidak's multiple comparisons test). (C and D) The weight of excised xenografts of (C) Caco‑2 and (D) BINDS‑16 was measured
on day 27. Values are presented as the mean ± SEM. **P<0.01 (Welch's t‑test). (E and F) The resected tumors appearance of (E) Caco‑2 and (F) BINDS‑16
xenografts in the control mouse IgG and EpMab‑37‑mG2a‑f treated groups on day 27 (scale bar, 1 cm). n.s., not significant; mIgG, mouse IgG; EpCAM,
epithelial cell adhesion molecule.

The loss of body weight was not observed in Caco‑2 and
BINDS‑16 tumor‑implanted mice (Fig. 8A and B). The miceon day 27 are demonstrated in Fig. 8C and D.
Discussion
In the present study, a mouse IgG1 subclass of EpMab‑37
was converted into a mouse IgG2a, and a defucosylated form

(EpMab‑37‑mG2a‑f) was produced in order to enhance ADCC
activity. In fact, EpMab‑37‑mG2a‑f exhibited a high ADCC
activity in vitro (Figs. 2 and 6), and exhibited potent antitumor
activity against CHO/EpCAM (Fig. 3A and C) and Caco‑2
xenografts (Fig. 7A and C). Notably, EpMab‑37‑mG2a‑f did
not affect the growth of tumors derived from Caco‑2 cells inwhich EpCAM was knocked out (BINDS‑16) (Fig. 7B and D),
indicating that EpMab‑37‑mG2a‑f can target EpCAM‑positiveINTERNATIONAL JOURNAL OF MOlecular medicine 51: 18, 2023

11

Figure 8. Mouse body weights and appearance. (A and B) Body weights of (A) Caco‑2 and (B) BINDS‑16 xenografts‑implanted mice on days 6, 11, 14, 18,
20, 25 and 27 (ANOVA and Sidak's multiple comparisons test). (C and D) Body appearance of (C) Caco‑2 and (D) BINDS‑16‑xenografts‑implanted mice on
day 27 (scale bar, 1 cm). n.s., not significant.

tumors selectively. Additionally, it was observed that ahigher EpCAM expression could be detected in HCT‑15,
COLO201 and COLO205 than Caco‑2 cells (Fig. S2). Further
studies are required to investigate the antitumor activity of
EpMab‑37‑mG2a‑f against these cell lines.
The EpCAM N‑terminal domain (residues 24‑63) contains
the EGF‑like domain, which is targeted by the vast majority ofanti‑EpCAM mAbs, including HEA125 (used in CellSearch®),17‑1A (Edrecolomab), C215 (used in Catumaxomab) and MOC31(used in Oportuzumab) probably due to its high accessibility
and antigenicity on plasma membrane (43,44). By contrast, theanti‑EpCAM mAbs targeting the thyroglobulin‑like domain(residues 64‑138) or the extracellular C‑terminal domain (resi‑
dues 139‑265; CD) are rare (43). Among the clinically tested
mAbs, MT201 (Adecatumumab) was reported to recognize
the 167‑QKEIT‑171 sequence in the EpCAM CD (45). The

epitope mapping of EpMab‑37 was previously performed by
the authors and revealed that EpCAM (residues 144‑164) are
involved in its recognition (25). In crystal structure analysis,
this domain forms β‑sheet and loop structures, and exposedon the molecular surface when EpCAM forms cis‑dimeror trans‑tetramer (43). Therefore, EpMab‑37 has a uniqueepitope, and is expected a different mode of action. However,
EpMab‑37 never recognized EpCAM peptides including
144 to 163 amino acids, suggesting that EpMab‑37 has theconformational epitopes (25). To determine the conforma‑tional epitopes, the epitope mapping strategy was developed,
including Arg, Ile, Glu, Asp and Leu (RIEDL)‑insertion for
epitope mapping (REMAP) (46‑49) and His‑tag insertion for
the epitope mapping (HisMAP) method (50,51). The detailed
determination of EpMab‑37 epitope could contribute theunderstanding of recognition by EpMab‑37.

12LI et al: DEFUCOSYLATED ANTI-EpCAM mAb

It was observed that tumors derived from Caco‑2 cells inwhich EpCAM was knocked out exhibited a larger weight andvolume compared to parental ones (Fig. 7). Huang et al (52)reviewed the effect of EpCAM depletion in Caco‑2 cells
and Xenopus embryos. In EpCAM‑depleted Caco‑2 cells,
the elevated PKC activity resulted in the downregulationof E‑cadherin. A similar phenomenon was observed in
Xenopus embryos (53). Huang et al (52) mentioned that these
events were ‘two noteworthy exceptions’ in the regulation of
Cadherins by EpCAM. E‑cadherin downregulation sometimes
leads to epithelial‑to‑mesenchymal transition and an increasein tumorigenicity (54). Although the in vitro cell proliferationof parental and EpCAM knocked out Caco‑2 is similar in theexperiments of the present study, the aforementioned phenom‑
enon may influence the growth in vivo.
In the present study, mouse IgG2a and 281‑mG2a‑f wereused as controls in ADCC/CDC and ADCC reporter assays,
respectively, which are mAbs that could recognize unknown(mouse IgG2a) and known (281‑mG2a ‑f) targets. It was
confirmed that they were not specific for the target cells.
Therefore, they are suitable as negative controls in vitro
for ADCC/CDC and ADCC reporter assays. However,concerning the in vivo analysis, it could not be excluded thatthey recognize unidentified target and exhibit the side effects
in mice. For the aforementioned reasons, normal mouse IgG(a mixture of diverse antibodies) was used as the control
in the xenograft assays. In future xenograft studies, it willbe also necessary to test IgG, IgG2a and/or 281‑mG2a‑f and
EpMab‑37‑mG2a‑f.
EpCAM‑overexpressing CTCs exhibit cancer stem cellfeatures (55). CTCs, which express EpCAM, CD44, CD47 and
MET, identify as a subset with increased metastasis‑initiating
phenotype (56), suggesting that EpCAM plays a crucial rolein cancer stemness and metastasis. Recently, CTC expan‑
sion methods, including two‑dimensional (2D) long‑term
expansion, 3D organoids/spheroids culture, and xenografts,
have been developed to evaluate the character of CTCs (57).
Therefore, the biological characteristic of affecting cell prolif‑
eration and invasion need to be investigated as Adecatumumab,
which has an epitope close to that of EpMab‑37, exerts weakanti‑proliferative effects in the absence of complement andimmune effector cells (45). Moreover, it would be worthwhile
to investigate the effect of EpMab‑37‑mG2a‑f on the CTC
expansion in vitro and metastasis in vivo.
Antibody‑drug conjugate (ADC) exhibits cytotoxicity
through the contents released from endocytic receptor‑boundmAbs‑drug conjugate (9). Oportuzumab monatox (Vicinium,
VB4‑845) is an EpCAM‑targeting scFv, conjugated with
Pseudomonas exotoxin A, an inhibitor of translation throughADP‑ribosylation of eukaryotic elongation factor‑2 (58).
Vicinium has been developed for the treatment of bacillus
Calmette‑Guérin (BCG)‑unresponsive non‑muscle invasive
bladder cancer (NMIBC) (15). Vicinium was approved by the
FDA as fast track designation and evaluated in the single‑arm
Phase 3 VISTA study (NCT02449239) for the patients withhigh‑grade NMIBC previously treated with BCG. However,
FDA did not approve Vicinium for BCG‑unresponsive
NMIBC (59). An anti‑dog PDPN mAb conjugated anti‑
body with emtansine was developed as the contents(P38B‑DM1) (60‑62) P38B‑DM1 exhibited cytotoxicity to

dog PDPN expressed CHO‑K1 cells, and demonstrated more apotent anti‑tumor effect than P38B in the xenograft model (63).
Moreover, anti‑trophoblast cell‑surface antigen 2 (TROP2)
ADC (Sacituzumab govitecan‑hziy) has been approved by
the FDA (64). TROP2 is a paralogue of EpCAM (65). As
Sacituzumab possesses the potent internalization activityof TROP2 (66), the capacity of EpCAM internalization by
EpMab‑37‑mG2a‑f needs to be further investigated for thedevelopment of the ADC.
The applications of anti‑EpCAM mAbs in the clinic are still
limited, since anti‑EpCAM mAbs may generate side‑effects
by affecting normal tissues. CasMabs targeting PDPN (67‑70)
and podocalyxin (71) have been previously developed by
the authors, which are currently applied to chimeric antigen
receptor T‑cell therapy in mice models (72‑74). It will be ofgreat value to establish cancer‑specific anti‑EpCAM mAbs
using the CasMab method. In the future, anti-EpCAM
CasMab production may be applicable as a basis for designingand optimizing potent immunotherapy modalities, including
ADCs and chimeric antigen receptor T‑cell therapy.
Acknowledgements
The authors would like to thank Ms. Saori Okuno and Ms. Saori
Handa (Department of Antibody Drug Development, Tohoku
University Graduate School of Medicine) for providing tech‑nical assistance for the completion of the in vitro experiments,and Mr. Shun‑ichi Ohba and Ms. Akiko Harakawa [Instituteof Microbial Chemistry (BIKAKEN), Numazu, Microbial
Chemistry Research Foundation] for providing technicalassistance in performing the animal experiments.
Funding
The present study was supported in part by Japan Agency
for Medical Research and Development (AMED; grantnos. JP22ama121008, JP21am0401013, JP22bm1004001,
JP22ck0106730 and JP21am0101078).
Availability of data and materials
The datasets used and/or analyzed during the current study areavailable from the corresponding author on reasonable request.
Authors' contributions
GL, TO, TT, MY, TN, TA and TY performed the experiments.
MKK, MK and YK designed the experiments. GL, TO, HS
and YK analyzed the data. GL, HS, and YK wrote the manu‑
script. All authors have read and approved the final manuscript
and agree to be accountable for all aspects of the researchin ensuring that the accuracy or integrity of any part of thework are appropriately investigated and resolved. HS and YK
confirm the authenticity of all the raw data.
Ethics approval and consent to participate
The animal study protocol was approved (approval
no. 2022‑024) by the Institutional Committee for Experimentsof the Institute of Microbial Chemistry (Numazu, Japan).

INTERNATIONAL JOURNAL OF MOlecular medicine 51: 18, 2023

Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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