antibodies
ArticleA Defucosylated Anti-EpCAM Monoclonal Antibody(EpMab-37-mG2a-f) Exerts Antitumor Activity in
Xenograft Model
Teizo Asano 1,† , Tomohiro Tanaka 1,† , Hiroyuki Suzuki 2, * , Guanjie Li 2 , Tomokazu Ohishi 3,4 ,Manabu Kawada 4 , Takeo Yoshikawa 5 , Mika K. Kaneko 1 and Yukinari Kato 1,2,5, *
1

2

3

4

5

*
†

Citation: Asano, T.; Tanaka, T.;
Suzuki, H.; Li, G.; Ohishi, T.; Kawada,
M.; Yoshikawa, T.; Kaneko, M.K.;
Kato, Y. A Defucosylated
Anti-EpCAM Monoclonal Antibody(EpMab-37-mG2a -f) Exerts Antitumor
Activity in Xenograft Model.
Antibodies 2022, 11, 74. https://doi.org/10.3390/antib11040074
Academic Editors: Amita
Datta-Mannan and Brooke Rock
Received: 14 October 2022
Accepted: 22 November 2022
Published: 24 November 2022

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
18-24 Miyamoto, Numazu-shi 410-0301, Japan
Laboratory of Oncology, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Research
Foundation, 3-14-23 Kamiosaki, Shinagawa-ku 141-0021, Tokyo, Japan
Department of Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Japan
Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-22-717-8207 (H.S. & Y.K.)
These authors contributed equally to this work.

Abstract: The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, whichmediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low
expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, whichcorrelates with poor prognosis. Therefore, EpCAM has been considered as a promising target for
tumor diagnosis and therapy. Using the Cell-Based Immunization and Screening (CBIS) method,
we previously established an anti-EpCAM monoclonal antibody (EpMab-37; mouse IgG1 , kappa).
In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC), complementdependent cytotoxicity (CDC), and an antitumor activity by a defucosylated mouse IgG2a -type of
EpMab-37 (EpMab-37-mG2a -f) against a breast cancer cell line (BT-474) and a pancreatic cancer cellline (Capan-2), both of which express EpCAM. EpMab-37-mG2a -f recognized BT-474 and Capan2 cells with a moderate binding-affinity [apparent dissociation constant (KD ): 2.9 × 10−8 M and1.8 × 10−8 M, respectively] by flow cytometry. EpMab-37-mG2a -f exhibited ADCC and CDC for
both cells by murine splenocytes and complements, respectively. Furthermore, administration of
EpMab-37-mG2a -f significantly suppressed the xenograft tumor development compared with thecontrol mouse IgG. These results indicated that EpMab-37-mG2a -f exerts antitumor activities andcould provide valuable therapeutic regimen for breast and pancreatic cancers.

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with regard to jurisdictional claims inpublished maps and institutional affil-

Keywords: EpCAM; breast cancer; pancreatic cancer; antitumor activities; antibody-dependent
cellular cytotoxicity

iations.

1. Introduction
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

EpCAM is a unique type I transmembrane glycoprotein which is expressed on thebasolateral membrane of epithelial cells [1]. EpCAM mediates homophilic and intercellularadhesion through the extracellular domain, which is essential for the epithelial integrity [2].
EpCAM was the first identified human tumor antigen [3], and the expression is correlatedwith poor prognosis in various tumors [4–7]. EpCAM also functions as a signaling molecule.
The formation in intercellular EpCAM oligomers triggers the transmembrane proteolytic
cleavage by a membrane protease complex. The EpCAM intracellular domain serves as a

Antibodies 2022, 11, 74. https://doi.org/10.3390/antib11040074

https://www.mdpi.com/journal/antibodies

Antibodies 2022, 11, 74

2 of 14transcriptional cofactor by interacting with β-catenin, and regulates the transcriptional targets involved in cell proliferation, survival, and stemness [8]. Therefore, the overexpressionof EpCAM plays a critical role in the malignant progression of tumors.
Circulating tumor cells (CTCs) are an important indicator of micro-metastasis and provide prognostic and therapeutic information [9]. CellSearch® (Menarini Silicon Biosystems,
Inc., Huntington, PA, USA) is a platform that captures EpCAM-positive circulating tumor
cells (CTCs) from whole blood samples [10]. The U.S. Food and Drug Administration (FDA)
approved CellSearch® in 2009. The EpCAM-based CellSearch® CTC test has been studiedin several clinical trials in lung [11], pancreatic [12], and breast [13] cancers.
The clinicopathological classiffications of breast cancer are based on the expression of
estrogen receptor (ER), progesteron receptor (PR), human epidermal growth factor receptor
2 (HER2), and Ki-67. The intrinsic subtypes are classified into luminal A (ER+ and/or PR+,
HER2−, and low Ki-67), luminal B HER2-negative (ER+ and/or PR+, HER2−, and highKi-67), luminal B HER2-positive (ER+ and/or PR+, HER2+), HER2-positive non-luminal(ER− and PR−, HER2+), and triple negative (ER−, PR−, HER2−) [14]. EpCAM expressionin breast cancer was previously analyzed and showed varied clinical outcomes in the
intrinsic subtypes. EpCAM expression was especially associated with an unfavorable
prognosis in the luminal B HER2-positive and triple negative subtypes [15].
Pancreatic cancer is one of the most aggressive tumors that is difficult to detect inearly stages and has poor treatment options. Approximately 80% of pancreatic cancer
patients are in late stage when diagnosed and are not amenable to surgical resection. The
5-year survival is poor, estimated at 2% to 9%. Therapeutic options are largely limited tocytotoxic chemotherapy [16]. Serum carbohydrate antigen 19-9 is the only FDA-approved
tumor marker to manage pancreatic cancer status [17]. Therefore, it is necessary to develop
groundbreaking early diagnostic markers and new drugs with less toxicity. The antitumor
effects on pancreatic tumors of several mAbs have been evaluated in pre-clinical studies
and clinical trials [18].
EpCAM was the first target of monoclonal antibody (mAb) therapy in humans [19].
Adecatumumab is a human recombinant mAb [20], and exhibited antitumor activity in colorectal and prostate cancers through antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC) [21]. Catumaxomab is a bispecific and trifunctional anti-EpCAM/CD3-antibody. Catumaxomab recognizes EpCAM on tumors, recruits
T cells through the anti-CD3 arm, and recruits natural killer (NK) cells and macrophages
through the Fc domain. These events enhanced ADCC activity [22]. Catumaxomab exhibited promising outcomes in several clinical trials [23–25], and was approved by theEuropean Union for the treatment of patients with malignant ascites in 2009 [23].
The composition of the N-linked glycans on IgG plays a critical role in effector functions [26]. In other antibody classes, the influence of glycans, particularly sialic acid, havebeen suggested [27,28]. The binding of the FcγRIIIa on NK cells to the Fc region of mAbs
mediates ADCC [29]. However, the N-linked glycosylation in the Fc region is reported
to impair the binding to the FcγRIIIa on NK cells [30,31]. Fucosyltransferase 8 (FUT8)
plays a critical role in the N-linked glycosylation (core fucosylation) of the Fc region [31].
Therefore, FUT8-knockout Chinese hamster ovary (CHO) cells have been shown to produce
completely defucosylated recombinant antibodies in the Fc region [32]. The defucosylated
mAb potently binds to FcγRIIIa and exerts increased ADCC activity compared with aconventional mAb [30].
We previously established an anti-EpCAM mAb, EpMab-37 (mouse IgG1 , kappa)
using the Cell-Based Immunization and Screening (CBIS) method [33]. Because mouse
IgG2a can bind to FcγRIV, the murine orthologue of human FcγRIIIa, we produced asubclass-switched and a defucosylated EpMab-37 (EpMab-37-mG2a -f) using FUT8-deficient
CHO cells. In this study, we investigated the ability of EpMab-37-mG2a -f to induce ADCC,
CDC, and antitumor activities against a breast cancer cell line (BT-474) derived from theluminal B HER2-positive subtype [34], and a pancreatic cancer cell line (Capan-2), both ofwhich express EpCAM.

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2. Materials and Methods
2.1. Cell Lines
A human breast cancer cell line (BT-474) and a pancreatic cancer cell line (Capan-2) werepurchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). BT-474
was cultured in Dulbecco’s Modified Eagle Medium (Nacalai Tesque, Inc. (Nacalai), Kyoto,
Japan), supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.
(Thermo), Waltham, MA, USA), 100 units/mL of penicillin, 100 µg/mL streptomycin, and0.25 µg/mL amphotericin B (Nacalai). Capan-2 was cultured in McCOY’s medium (Cytiva,
Tokyo, Japan), supplemented with 10% FBS, 100 µg/mL streptomycin, 100 units/mL of
penicillin, and 0.25 µg/mL amphotericin B. The cell lines were maintained in a humidified
atmosphere under 5% CO2 at 37 ◦ C.
2.2. Animals
All animal experiments were performed following regulations and guidelines tominimize animal distress and suffering in the laboratory by the Institutional Committee
for Experiments of the Institute of Microbial Chemistry (Numazu, Japan) (approval no.
2022-024). Mice were maintained on an 11 h light/13 h dark cycle in a specific pathogen-freeenvironment across the experimental period. Food and water were supplied ad libitum.
Mice weight was monitored twice per week and health was monitored three times per week.
The loss of original body weight was determined to a point >25% and/or a maximum
tumor size >2000 mm3 and/or significant changes in the appearance of tumors as humane
endpoints for euthanasia. Cervical dislocation was used for euthanasia. Mice death was
confirmed by respiratory arrest and rigor mortis.
2.3. Antibodies
An anti-EpCAM mAb, EpMab-37, was established as previously described [27]. To
switch the subclass of EpMab-37 from mouse IgG1 to mouse IgG2a (EpMab-37-mG2a ),we cloned VH cDNA of EpMab-37 and CH of mouse IgG2a into the pCAG-Ble vector(FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka, Japan). VL cDNA of EpMab37 and CL cDNA of mouse kappa light chain were also cloned into the pCAG-Neo vector(Wako). To generate the defucosylated EpMab-37-mG2a , the vector for the EpMab-37-mG2a
was transfected into FUT8 knockout ExpiCHO-S (BINDS-09) cells using the ExpiCHO
Expression System (Thermo) [35–49]. Defucosytaled EpMab-37-mG2a (EpMab-37-mG2a -f)
was purified using Ab-Capcher (ProteNova Co., Ltd., Kanagawa, Japan). Mouse IgG (cat.
no. 140-09511) and IgG2a (cat. no. M7769) were purchased from Wako and Sigma-Aldrich(St. Louis, MO, USA), respectively. A 281-mG2a -f (a defucosylated anti-hamster podoplanin[PDPN] mAb, control mouse IgG2a for ADCC reporter assay) was previously described [50].
Trastuzumab was purchased from the R&D systems (Minneapolis, MN, USA).
2.4. Flow Cytometry
Cells were obtained by 0.25% trypsin/1 mM ethylenediamine tetraacetic acid (EDTA;
Nacalai). Cells were treated with 10 µg/mL of EpMab-37-mG2a -f or blocking buffer [0.1%bovine serum albumin (BSA; Nacalai) in phosphate-buffered saline (PBS)] (control) for
30 min at 4 ◦ C. Then, cells were incubated in Alexa Fluor 488-conjugated anti-mouse IgG(1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 4 ◦ C. Fluorescencedata were collected by the Cell Analyzer EC800, and analyzed by EC800 software ver. 1.3.6(Sony Corp., Tokyo, Japan).
2.5. Determination of Binding Affinity
Serially diluted EpMab-37-mG2a -f (0.006–100 µg/mL) was suspended with BT-474 andCapan-2 cells. Then, the cells were further treated with Alexa Fluor 488-conjugated antimouse IgG (1:200). Fluorescence data were obtained by BD FACSLyric (BD Biosciences, San
Jose, CA, USA). To determine the dissociation constant (KD ), the fitting binding isotherms

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to built-in one-site binding models in GraphPad Prism 9 (GraphPad Software, La Jolla, CA,
USA) was used.
2.6. ADCCADCC induction by EpMab-37-mG2a -f was assayed as follows. Six female BALB/cnude mice (five-week-old) were purchased from Charles River Laboratories, Inc. Spleens
were aseptically removed, and single-cell suspensions were obtained through a sterilecell strainer (352360, BD Falcon). Erythrocytes were removed with treatment of ice-cold
distilled water. The splenocytes were resuspended in the medium; this preparation was
designated as effector cells. Target cells (BT-474 and Capan-2) were treated with Calcein AM(10 µg/mL, Thermo Fisher Scientific, Inc.). The target cells (2 × 104 cells) were mixed witheffector cells (effector-to-target ratio, 100:1), 100 µg/mL of EpMab-37-mG2a -f or control
mouse IgG2a in 96-well plates. After incubation for 4.5 h at 37 ◦ C, the Calcein release
into the medium was measured with an excitation wavelength (485 nm) and an emission
wavelength (538 nm) using a microplate reader (Power Scan HT; BioTek Instruments, Inc.,
Winooski, VT, USA).
Cytolyticity (% lysis) was determined as follows: % lysis = (E − S)/(M − S) × 100.
“E” is the fluorescence in the presence of both effector and target cells. “S” is the spontaneous fluorescence in the presence of only target cells. “M” is the maximum fluorescenceby the treatment with a lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM of EDTA, and
0.5% Triton X-100).
2.7. CDC
BT-474 and Capan-2 cells were treated with Calcein-AM (10 µg/mL), resuspended
in medium and plated in 96-well plates, at 2 × 104 cells/well, with 10% rabbit complement (Low-Tox-M rabbit complement; Cedarlane Laboratories, Hornby, ON, Canada),
100 µg/mL of EpMab-37-mG2a -f, or control mouse IgG2a added to each well. Following
incubation for 4.5 h at 37 ◦ C, Calcein release into the medium was determined.
2.8. ADCC Reporter Bioassay
The ADCC reporter bioassay was performed using an ADCC Reporter Bioassay kit
from Promega (Madison, WI, USA), following the manufacturer’s instructions [51]. Target
cells (12,500 cells per well) were inoculated into a 96 well white solid plate. EpMab-37-mG2a f, EpMab-37, and 281-mG2a -f were serially diluted and added to the target cells. Jurkat
cells stably expressing the human FcγRIIIa receptor, and a Nuclear 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 (Promega) was measured with a GloMax luminometer (Promega).
2.9. Antitumor Activities in Xenografts of BT-474 and Capan-2
BALB/c nude mice (female, 4 weeks old) were purchased from Charles River Laboratories, Inc. The cells (5 × 106 cells) were resuspended with BD Matrigel Matrix Growth
Factor Reduced (BD Biosciences) and were subcutaneously injected into the left flank of
10 weeks old mice. On day 6 post-inoculation, 100 µg of EpMab-37-mG2a -f (n = 8) orcontrol mouse IgG (n = 8) in 100 µL PBS were injected intraperitoneally. On day 13 and 18,
additional antibodies were injected. The tumor volume was measured on days 6, 11, 13,
18, 22, and 24 after the injection of cells. Tumor volumes were determined as previously
described [35–49,52–55].
2.10. Statistical Analysis
All data are represented as mean ± standard error of the mean (SEM). Welch’s t test
was conducted for ADCC activity, CDC activity, and tumor weight. ANOVA with Sidak’s
post hoc test was conducted for tumor volume and mouse weight. GraphPad Prism 8

All data are represented as mean ± standard error of the mean (SEM). Welch’s t test
was conducted for ADCC activity, CDC activity, and tumor weight. ANOVA with Sidak’s
post hoc test was conducted for tumor volume and mouse weight. GraphPad Prism 8
Software, Inc.) was used for all calculations. p < 0.05 was considered to indicate
Antibodies 2022,(GraphPad
11, 745 of 14a statistically significant difference.
3. Results(GraphPad Software, Inc.) was used for all calculations. p < 0.05 was considered to indicate
a statistically significant difference.

3.1. Flow Cytometric Analysis

3. Results
In our previous
study, a sensitive and specific anti-EpCAM mAb, EpMab-37 (mouse
3.1. Flow Cytometric Analysis
IgG1, kappa), was established using the CBIS method [33]. We first performed flow cyIn our previous study, a sensitive and specific anti-EpCAM mAb, EpMab-37 (mouse
tometric analysis using
EpMab-37-mG2a-f against BT-474 and Capan-2 cells and found that
IgG1 , kappa), was established using the CBIS method [33]. We first performed flow cytoEpMab-37-mG2ametric-f recognizedbothcells (Figure-f1A).
A kinetic analysis of the interactions
analysis using
EpMab-37-mG
2a against BT-474 and Capan-2 cells and found thatof EpMab-37-mG
and Capan-2
cells
was1A).
conductedusing flow
2a-f with BT-474
EpMab-37-mG
both cells(FigureA kinetic analysisof thecytometry.
interactions
2a -f recognizedof
EpMab-37-mG-f
with
BT-474andCapan-2
cells
wasconducted
using
flow
cytometry.
The apparent dissociation constants(KD) of EpMab-37-mG2a-f to BT-474 and Capan-2
cells
2a
The
apparent
dissociation
constants(K)
of
EpMab-37-mG-fto
BT-474andCapan-2
cells
D(Figure 1B), indicating
2awere 2.9 × 10−8 M and 1.8 ×−810−8 M, respectively
moderatebinding−8
were 2.9 × 10 M and 1.8 × 10 M, respectively (Figure 1B), indicating moderate binding
2a-f against both cells.
affinity of EpMab-37-mG
affinity of EpMab-37-mG -f against both cells.
2aFigure 1. Flowcytometric(A) BT-474
and cells
Capan-2
cellstreated
were treated
10 µg/mL
Figure 1. Flow cytometricanalyses.
(A)analyses.
BT-474 andCapan-2
werewith with
10 μg/mLofof EpMab-37-mG2a -f (red line) or blocking buffer as a negative control (black line), followed by
EpMab-37-mG2a-f (red line) or blocking buffer as a negative control (black line), followed by addiaddition of Alexa Fluor 488-conjugated anti-mouse IgG. Data were collected using the EC800 Celltion of Alexa Fluor 488-conjugated anti-mouse IgG. Data were collected using the EC800 Cell AnaAnalyzer. (B) BT-474 and Capan-2 cells were suspended in 100 µL of serially diluted EpMab-37lyzer. (B) BT-474 and Capan-2 cells were suspended in 100 μL of serially diluted EpMab-37-mG2a-f
mG2a -f (0.006–100 µg/mL), followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG.
(0.006–100 μg/mL),followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG. Data
Data were collected using the BD FACS Lyric and analyzed by GraphPad PRISM 9 to determine thewere collected using
the dissociation
BD FACS constant
Lyric andby GraphPad PRISM 9 to determine the apapparent(KDanalyzed).
parent dissociation constant (KD).

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3.2.
-f in BT-474 and Capan-2
3.2. ADCCADCC andand CDC
CDC Activities
Activities ofof EpMab-37-mG
EpMab-37-mG2a2a -f in BT-474 and Capan-2
We-f-finduces
andCDC
activitiesin
We next
next examinedexamined whether
whetherEpMab-37-mG
EpMab-37-mG2a2a
inducesADCC
ADCC
and
CDC
activities
BT-474andCapan-2
cells.
EpMab-37-mG
2a-f exhibited much higher ADCC (64.0% cytoin BT-474 and Capan-2 cells. EpMab-37-mG2a -f exhibited much higher ADCC (64.0%
toxicity)in BT-474
cellscells
thanthanthat thatof control
mouse
IgG2aIgG(8.49%
cytotoxicity; p < 0.01) (Figcytotoxicity)
in BT-474
of controlmouse
2a (8.49% cytotoxicity; p < 0.01)
2a
ure
2A).
EpMab-37-mG-falso
showedhigherCDC
activity(64.2%
cytotoxicity)
in BT(Figure 2A). EpMab-37-mG2a -f also showed higher CDC activity (64.2%
cytotoxicity)in474
cellscells
thanthanthatthatof control
mouse
IgG2aIgG(11.1%
cytotoxicity;
p
<
0.01)
(Figure2B).
FurBT-474
of control
mouse(11.1%
cytotoxicity;
p
<
0.01)
(Figure2B).
2athermore,
EpMab-37-mGthat ADCC (24.8% cytotoxicity) against Capan-2 cells
2a-f showed
Furthermore,
EpMab-37-mG
2a -f showed that ADCC (24.8% cytotoxicity) against Capan-2
weremoremorepotentthan thancontrolmouse
IgG2aIgG(5.6%
cytotoxicity;p < 0.01)(Figure2C). 2C).
Epcells werepotentcontrolmouse
cytotoxicity;p < 0.01)(Figure2a (5.6%
Mab-37-mG-felicitedahigherdegreeofCDC(29.3%
cytotoxicity)inCapan-2cells
com2a
EpMab-37-mG2a -f elicited a higher degree of CDC (29.3% cytotoxicity) in Capan-2 cells
paredwithwith
thatthatelicited
by bycontrolmouse
IgG
cytotoxicity;
2a (6.8%
comparedelicited
controlmouse
IgG
cytotoxicity;p p<<0.05)
0.05)(Figure(Figure2D).
2D).
2a (6.8%
results demonstrated
demonstratedthat
thatEpMab-37-mG
EpMab-37-mG
exhibitedthe
theADCC
ADCC
and
CDC
activiThese results-f-fexhibited
andCDC
activities
2a2a
ties
againstBT-474
Capan-2
againstBT-474and and
Capan-2
cells.cells.

Figureof EpMab-37-mG
EpMab-37-mG2a
ADCC andand CDC
CDC activities
activitiesin
inBT-474
BT-474andandCapan-2
Capan2a-f
Figure 2.
2. Evaluation
Evaluation of-f mediatedmediated ADCC
2cells.
cells.(A,C)(A,C)ADCC
ADCCelicited
elicitedby
byEpMab-37-mG
EpMab-37-mG2a-f-f oror control
control mouse
mouse IgG
IgG2a against
against BT-474(A)and
BT-474 (A) and2a2aCapan-2 (C) cells. (B,D) CDC elicited by EpMab-37-mG2a-f or control mouse IgG2a against BT-474
Capan-2 (C) cells. (B,D) CDC elicited by EpMab-37-mG2a -f or control mouse IgG2a against BT(B) and Capan-2 (D) cells. Values are shown as mean ± SEM. Asterisks indicate statistical signifi474 (B) and Capan-2 (D) cells. Values are shown as mean ± SEM. Asterisks indicate statisticalcance (** p < 0.01, * p < 0.05; Welch’s t-test). ADCC, antibody-dependent cellular cytotoxicity; CDC,
significance (** p < 0.01, *cytotoxicity.
p < 0.05; Welch’s t-test). ADCC, antibody-dependent cellular cytotoxicity;
complement-dependent
CDC, complement-dependent cytotoxicity.

The ADCC reporter bioassay is a bioluminescent reporter gene assay to quantify the
The ADCC reporter bioassay is a bioluminescent reporter gene assay to quantify
biological activity of the antibody via FcγRIIIa-mediated pathway activation in an ADCC
the biological activity of the antibody via FcγRIIIa-mediated pathway activation in an
mechanism of action [51]. To compare the ADCC pathway activation by EpMab-37-mG2aADCC mechanism of action [51]. To compare the ADCC pathway activation by EpMabf, we treated BT-474 and Capan-2 cells with serially diluted mAbs, and then incubated
37-mG2a -f, we treated BT-474 and Capan-2 cells with serially diluted mAbs, and thenwith effector
Jurkat cells, which express the human FcγRIIIa receptor and an NFAT reincubated with effector Jurkat cells, which express the human FcγRIIIa receptor and an
sponse element driving firefly luciferase. Furthermore, we also used defucosylated antiNFAT response element driving firefly luciferase. Furthermore, we also used defucosylated
hamster PDPN mAb (mouse IgG2a) (281-mG2a-f) as a control since we could not obtain
anti-hamster PDPN mAb (mouse IgG2a ) (281-mG2a -f) as a control since we could not obtain
defucosylated control normal mouse IgG2a. We confirmed that 281-mG2a-f never recogdefucosylated control normal mouse IgG2a . We confirmed that 281-mG2a -f never recognized
nized
the target
cells.
As shown
in Figure
3A, EpMab-37-mG
2a-f dose-dependently actithe target
cells. As
shown
in Figure
3A, EpMab-37-mG
2a -f dose-dependently activated
vated
the
effector(EC
50; 45.8 ng/mL), but 281-mG2a-f did not. Similar results were obtained
the effector (EC50 ; 45.8 ng/mL), but 281-mG2a -f did not. Similar results were obtained ininpresence
Capan-2
cells
50; 10.9 ng/mL) (Figure 3B). We confirmed that origithethe
presenceof ofCapan-2
cells(EC(EC
50 ; 10.9 ng/mL) (Figure 3B). We confirmed that originalnal
EpMab-37
neveractivatedtheeffector(Supplementary
FigureFurthermore,
EpMab-37 never activated the effector cellscells(Supplementary
FigureS1).S1).
Furthermore,we
2a-f with an existing therawecomparedthe
ADCC
effector
activation
by
EpMab-37-mG
compared the ADCC effector activation by EpMab-37-mG2a -f with an existing therapeutic
peuticantibody,
Trastuzumab.
theBT-474
target,cells
BT-474cellsiswhich
known
HER2
antibody,
Trastuzumab.
We usedWetheusedtarget,which
knownisas
HER2aspositive
2a-f exhibited a superior EC50 compared topositive
breastcancer.
EpMab-37-mG
breast cancer. EpMab-37-mG2a -f exhibited a superior EC50 compared to trastuzumab
trastuzumab (Supplementary
FigureS2).indicated
These results
indicated that
EpMab-37-mG2a-f(Supplementary
Figure S2). These
results
that EpMab-37-mG
2a -f exhibits superior
exhibits
superior
ADCC
activation
activity
comparedwith
EpMab-37
andtrastuzumab.
ADCC activation activity compared with EpMab-37 and trastuzumab.

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Figureassay
3. ADCC
assay by EpMab-37-mG-f in the presenceof BT-474
2a -f andFigure 3. ADCC reporterby reporter
EpMab-37-mG
in the 2apresenceof BT-474
and and
2a-f and 281-mG
2a-f 281-mG
Capan-2
cells.
(A,B)
Targetcells(BT-474(A)
andCapan-2(B))
weretreatedwithserially
dilutedCapan-2 cells. (A,B) Target cells (BT-474 (A) and Capan-2 (B)) were treated with serially diluted
EpMab-37-mG-f and 281-mG-f (mouseThen,
IgG2a control).
Then,stably
Jurkat expressing
cells stably expressing
EpMab-37-mG2a-f and
281-mG2a-f2a(mouse
IgG2a2acontrol).
Jurkat cells
the hu- thehuman FcγRIIIa receptor and an NFAT response element driving firefly luciferase were added andman FcγRIIIa receptor and an NFAT response element driving firefly luciferase were added and coco-cultured. Luminescence was measured using the Bio-Glo Luciferase Assay System. ADCC,
cultured. Luminescence was measured using the Bio-Glo Luciferase Assay System. ADCC, antiantibody-dependent cellular cytotoxicity.
body-dependent cellular cytotoxicity.

3.3. Antitumor Effects of EpMab-37-mG2a -f in Mouse Xenograft Models of BT-474 and Capan-2
cells were2ainoculated
into
the left flankof mice,followed
the intraperi3.3. Antitumor Effects ofBT-474
EpMab-37-mG-f in Mouse
Xenograft
Modelsof BT-474
andbyCapan-2
toneally injection of EpMab-37-mG -f or control mouse IgG on days 6, 13, and 18. The

2aBT-474 cells were
inoculated into the left flankof mice, followed by the intraperitotumor volume was measured after the inoculation. EpMab-37-mG2a -f-treated mice exhib2a-f tumor
neally injection of EpMab-37-mGor control
mouse
daysday
6, 13,
and18. Thetumor
ited significantly lessvolumeon dayIgG
11 (pon< 0.01),13 (p< 0.01),day 18(p < 0.01),
day 22after(p < 0.01),
and day 24 (p EpMab-37-mG< 0.01), compared2awith
control micemouseexhibited
IgG-treatedsigcontrol
volume was measured
the inoculation.
-f-treatedmice(Figure on
4A).day
The 11EpMab-37-mG-f treatment
resultedin a1851.3%
of thenificantly less tumor
volume(p < 0.01), 2aday
13 (p < 0.01),
day(p < reduction0.01), daytumor volume compared with that of the control mouse IgG on day 24 (Figure 4C). Tumors
22 (p < 0.01), and day 24 (p < 0.01), compared with control mouse IgG-treated control micefrom EpMab-37-mG2a -f-treated mice weighed significantly less than tumors from control(Figure 4A). The EpMab-37-mG
2a-f treatment resulted in a 51.3% reduction of the tumor
IgG-treated control
mice (52.4% reduction, p < 0.01; Figure 4E). Resected tumors on day 24
volume compared arewiththat ofinthe
control
mouse
IgG onday did
24 (Figure4C). Tumors
from thepresented
Figure4E. Thetotal bodyweights
not significantly
differ between
EpMab-37-mG-f-treatment
andthecontrolgroups(Figure
5A).
The
body
appearance
EpMab-37-mG2a-f-treated mice2aweighed significantly less than tumors from control IgG- ofmiceon dayreduction,
24 post inoculationis shown
Figure5C, and the
body weights’
treated control mice
(52.4%
p < 0.01;
Figurein4E).
Resectedtumorson dayloss
24 andare skindisorder were not observed.
presented in Figure 4E. The total body weights did not significantly differ between the
EpMab-37-mG2a-f-treatment and the control groups (Figure 5A). The body appearance ofmice on day 24 post inoculation is shown in Figure 5C, and the body weights’ loss andskin disorder were not observed.
In the Capan-2 xenograft, EpMab-37-mG2a-f and control mouse IgG were injected intraperitoneally into mice on days 6, 14, and 18 after the inoculation of Capan-2 cells. The
tumor volume was measured on days 6, 11, 14, 18, 20, 25, and 27. The treatment of EpMab-decreased than that of control IgG-treated mice (49.1% reduction; p < 0.01, Figure 4D). The
excised tumors of control and EpMab-37-mG2a-f -treated groups on day 27 are shown in
Figure 4F. Total body weights were almost similar in both groups (Figure 5B). Appearanceof mice on day 27 after inoculation of cells are shown in Figure 5D.
Antibodies 2022, 11, 74 These results indicate that administration of EpMab-37-mG2a-f effectively suppresses 8 of 14
the tumor growth of BT-474 and Capan-2 xenografts.

Figure 4. Antitumor activity of EpMab-37-mG2a -f. (A,B) Measurement of tumor volume in (A) BT-474
Figure 4. Antitumor
activity of EpMab-37-mG2a-f. (A,B) Measurement of tumor volume in (A) BTand (B) Capan-2 xenograft models. BT-474 and Capan-2 cells6 (5 × 106 cells) were inoculated into
474 and (B) Capan-2
xenograft models. BT-474 and Capan-2 cells (5 × 10 cells) were inoculated intomice subcutaneously. On day six, 100 µg of EpMab-37-mG2a -f or control mouse IgG were injected
mice subcutaneously.
On day six, 100 μg of EpMab-37-mG2a-f or control mouse IgG were injected
into mice intraperitoneally. On day 13 and 18 (BT-474) or 14 and 18 (Capan-2), additional antibodies
into mice intraperitoneally. On day 13 and 18 (BT-474) or 14 and 18 (Capan-2), additional antibodies
were injected. On the indicated days after the inoculation, the tumor volume was measured. Values
were injected. On the indicated days after the inoculation, the tumor volume was measured. Values
are presented as the mean ± SEM. ** p < 0.01, * p < 0.05 (ANOVA and Sidak’s multiple comparisonsare presented as the mean ± SEM. ** p < 0.01, * p < 0.05 (ANOVA and Sidak’s multiple comparisonstest). (C,D) The weight of excised (C) BT-474 and (D) Capan-2 xenografts was measured on day 24test). (C,D) The weight of excised (C) BT-474 and (D) Capan-2 xenografts was measured on day 24
and 27, respectively. Values are presented as the mean ± SEM. ** p < 0.01 (Welch’s t test). (E,F) The
and 27, respectively. Values are presented as the mean ± SEM. ** p < 0.01 (Welch’s t test). (E,F) The
resected tumors appearance of (E) BT-474 and (F) Capan-2 xenografts in the control mouse IgG andresected tumors appearance of (E) BT-474 and (F) Capan-2 xenografts in the control mouse IgG and
EpMab-37-mG2a -f treated groups on day 24 and 27, respectively (scale bar, 1 cm). n.s., not significant.
EpMab-37-mG2a-f treated groups
on day 24 and 27, respectively (scale bar, 1 cm). n.s., not significant.

Antibodies
2022,
11,11,
74x FOR PEER REVIEW
Antibodies
2022,

99 of 14
14

Figure5.5.Mice
Micebody
bodyweights
weightsand
andappearance.
appearance. (A,B)(A,B) Body
Body weights
weights inin (A)(A) BT-474
BT-474 andand (B)(B) Capan-2
Capan-2
Figurexenografts-implanted
mice
on
the
indicated
days(ANOVAand
Sidak’smultiplecomparisonstest).
xenografts-implanted mice on the indicated days (ANOVA and Sidak’s multiple comparisons test).
(C,D) Body appearance in (C) BT-474 and (D) Capan-2 xenografts-implanted mice on day 24 and(C,D) Body appearance in (C) BT-474 and (D) Capan-2 xenografts-implanted mice on day 24 and 27,
27, respectively (scale bar, 1 cm). n.s., not significant.
respectively (scale bar, 1 cm). n.s., not significant.

4. Discussion
In the Capan-2 xenograft, EpMab-37-mG2a -f and control mouse IgG were injected
The impact of
EpCAM
expression
on breast
prognosis
is dependent
on intrinintraperitoneallyintomice ondays 6, 14,
and 18cancer
after theinoculationof Capan-2
cells.
sic subtype.
In the luminal
B HER2-positive
negative
EpCAM
expresThetumor volume
was measured
on days 6,and
11,triple
14, 18,20, 25, subtypes,
and 27. The
treatmentofsion is associatedwithanunfavorable
prognosis.
In
contrast,
EpCAM
expressionisassoEpMab-37-mG-fresultedinasignificant
inhibitionintumor
development
on
days
11
2a
with
non-luminal
There(pciated< 0.05),14 a(pfavorable< 0.01), 18prognosis(p < 0.01),in20the(p HER2-positive< 0.01), 25 (p < 0.01),
and 27 subtype(p < 0.01)[15].
comparedfore,thatthe ofluminalB HER2-positive
and triple
subtypes areofpotential
groups2afor
withthe control
mouse IgG (Figure4B).negative
The administration
EpMab-37-mG-ftreatment
EpCAM-targetingtherapy.
In thisstudy, with
we investigated
the antitumor
resultedin awith
51.3%
reduction of tumor
volumecomparedthat of the controlmouseeffecta defucosylated
anti-EpCAM(EpMab-37-mG
breast cancercell2a-f) against
IgG
on of
day
27. Furthermore,
the tumormAb
weightof the EpMab-37-mG
mice was
2a -f a-treatedline, BT-474 decreased
derived fromluminalHER2-positive
subtype[34].
EpMab-37-mG
significantly
thanthat ofB control
IgG-treatedmice(49.1%
reduction;2ap-f<exhib0.01,
Figure4D). TheADCC
excisedtumorscontrol inandEpMab-37-mG
groups onactivity
day 27
ited superior
and
CDC ofactivities
vitro(Figures 2 and3), and antitumor
2a -f -treated
are
shown
in Figure
4F. Totalbody miceweights
were4).
almostsimilar in developed
both groups
5B).
againstBT-474
xenograftin nude(FigureWe previouslyan(Figureanti-HER2
Appearanceof miceandon day27 afterADCC,
inoculation
cellsantitumor
are shownactivities
in Figureagainst
5D.
mAb (H2Mab-19)
examined
CDC,ofandBT-474indicatethat administrationof2Mab-19
EpMab-37-mG
cellsThese[45]. results
Althoughthe binding
affinity of H
and EpMab-37-mG
to BT-474
2a-fsuppresses
2a -f effectively
the
tumor
of BT-474
and Capan-2
xenografts.
cells
weregrowthcomparable,
EpMab-37-mG
exerted more potent ADCC activity and anti2a-ftumor effect in vivo. These results are probably due to the defucosylation in EpMab-37mG2a-f, but not in H2Mab-19. Moreover, EpCAM forms a cis-dimer which further makes

Antibodies 2022, 11, 74

10 of 14

4. Discussion
The impact of EpCAM expression on breast cancer prognosis is dependent on intrinsic
subtype. In the luminal B HER2-positive and triple negative subtypes, EpCAM expression
is associated with an unfavorable prognosis. In contrast, EpCAM expression is associatedwith a favorable prognosis in the HER2-positive non-luminal subtype [15]. Therefore, theluminal B HER2-positive and triple negative subtypes are potential groups for treatment
with EpCAM-targeting therapy. In this study, we investigated the antitumor effect ofa defucosylated anti-EpCAM mAb (EpMab-37-mG2a -f) against a breast cancer cell line,
BT-474 derived from luminal B HER2-positive subtype [34]. EpMab-37-mG2a -f exhibitedsuperior ADCC and CDC activities in vitro (Figures 2 and 3), and antitumor activity against
BT-474 xenograft in nude mice (Figure 4). We previously developed an anti-HER2 mAb(H2 Mab-19) and examined ADCC, CDC, and antitumor activities against BT-474 cells [45].
Although the binding affinity of H2 Mab-19 and EpMab-37-mG2a -f to BT-474 cells werecomparable, EpMab-37-mG2a -f exerted more potent ADCC activity and antitumor effectin vivo. These results are probably due to the defucosylation in EpMab-37-mG2a -f, but notin H2 Mab-19. Moreover, EpCAM forms a cis-dimer which further makes a biologically
relevant oligomeric state (e.g., cis-tetramer, trans-tetramer, and trans-octamer) according
to several experimental observations [56]. These oligomeric structures of EpCAM could
promote the clustering of anti-EpCAM mAbs, which might help the FcγRIIIa engagementon effector cells, and potentiate the ADCC activity.
EpCAM is an important cell surface molecule to collect CTCs [10]. In a prospective
study of pancreatic cancer patients, the CTC in the peripheral blood affect the outcome ofpatients independent from other risk factors, including adjuvant chemotherapy [12]. Furthermore, EpCAM-sorted pancreatic adenocarcinoma cells from surgically resected tumors
could be applied to the analysis of tumor-cell-intrinsic chromatin accessibility patterns. A
chromatin accessibility signature and associated transcriptional factors (ZKSCAN1 and
HNF1β) are significantly correlated with pancreatic cancer prognosis [57]. This information
could contribute to the selection of patients to be applied to anti-EpCAM mAb therapy.
Recently, CTC expansion techniques have been developed to evaluate the characteristics of CTCs. The techniques include the two-dimensional (2D) long-term expansion, 3D
organoids/spheroids culture, and in vivo xenografts/metastasis formation in immunodeficient mice [58]. It would be worthwhile to investigate the effect of EpMab-37-mG2a -f on
the 2D and 3D CTC expansion in vitro and anti-metastatic activity in vivo.
We have been investigating the critical epitope of EpMab-37 [33], and recently identified that Arg163 of EpCAM is the most important residue of the EpMab-37 epitope(submitted). Among clinically tested mAbs, no mAb recognized above region, suggestingthat EpMab-37 possesses a unique epitope and a different mode of actions. In this study, we
did not examine the EpCAM-internalizing activity by EpMab-37-mG2a -f. Furthermore, therelationship between the internalizing activity and the epitope has not been investigated. In
future study, we would like to evaluate it for the development of antibody-drug conjugates.
Anti-EpCAM mAb can be used for a bispecific Ab with anti-MET mAb [59]. MM-131
is a bispecific Ab that is monovalent against MET, but it exhibits high avidity to EpCAM
through binding to a single chain Fv of an anti-EpCAM mAb, MOC31 [60]. MM-131 exhibits
antagonistic activity that interferes both ligand-dependent and ligand-independent MET
signaling and induces the receptor down-regulation [59]. MCLA-128 is a bispecific Ab for
HER2 and HER3. MCLA-128 can inhibit heregulin (a HER3 ligand)-mediated signaling of
HER2/HER3 heterodimer and suppress tumor cell growth via the suppression of PI3K/Akt
signaling [61]. Clinical studies on MCLA-128 are ongoing in patients with breast cancer,
pancreatic cancer, and non-small cell lung cancers [62]. In the future study, we would like toapply EpMab-37 for the combination therapy with anti-HER2 mAbs or develop a bispecificmAb targeting EpCAM and HER2.

Antibodies 2022, 11, 74

11 of 14

Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antib11040074/s1, Figure S1: ADCC reporter bioassay by EpMab37 in the presence Capan-2 cells; Figure S2: ADCC reporter bioassay by EpMab-37-mG2a -f andtrastuzumab in the presence BT-474 cells.
Author Contributions: Methodology, M.K. and M.K.K.; Investigation, T.A., T.T., G.L., T.O. and T.Y.;
Writing—original draft, T.A., T.T. and H.S.; Writing—review & editing, H.S. and Y.K.; Supervision,
Y.K.; Project administration, Y.K.; Funding acquisition, Y.K. All authors have read and agreed to the
published version of the manuscript.
Funding: The present study was supported in part by the Japan Agency for Medical Research andDevelopment (AMED; grant nos: JP22ama121008, JP21am0401013, JP22bm1004001, JP22ck0106730
and JP21am0101078).
Institutional Review Board Statement: All animal experiments were approved (no. 2022-024) by theInstitutional Committee for Experiments of the Institute of Microbial Chemistry (Numazu, Japan).
Data Availability Statement: Data is contained within the article or Supplementary material.
Acknowledgments: The authors would like to thank Saori Okuno and Saori Handa (Departmentof Antibody Drug Development, Tohoku University Graduate School of Medicine) for technicalassistance of in vitro experiments, and Shun-ichi Ohba and Akiko Harakawa [Institute of Microbial Chemistry (BIKAKEN), Numazu, the Microbial Chemistry Research Foundation] for technicalassistance with animal experiments.
Conflicts of Interest: The authors declare no conflict of interest.

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