antibodies
ArticleDevelopment of a Novel Anti-EpCAM Monoclonal Antibody
for Various Applications
Guanjie Li 1 , Hiroyuki Suzuki 1, * , Teizo Asano 2 , Tomohiro Tanaka 2 , Hiroyoshi Suzuki 3 , Mika K. Kaneko 2
and Yukinari Kato 1,2, *
1

2

3

*

Citation: Li, G.; Suzuki, H.; Asano, T.;
Tanaka, T.; Suzuki, H.; Kaneko, M.K.;
Kato, Y. Development of a Novel
Anti-EpCAM Monoclonal Antibody
for Various Applications. Antibodies
2022, 11, 41. https://doi.org/
10.3390/antib11020041
Academic Editor: Dimiter
S. Dimitrov

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Japan; kanketsu@med.tohoku.ac.jp
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1
Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; teizo.asano.a7@tohoku.ac.jp (T.A.);
tomohiro.tanaka.b5@tohoku.ac.jp (T.T.); k.mika@med.tohoku.ac.jp (M.K.K.)
Department of Pathology and Laboratory Medicine, Sendai Medical Center, 2-11-12, Miyagino, Miyagino-ku,
Sendai 983-0045, Japan; hirosuzsnh@gmail.com
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.)

Abstract: The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is
widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation,
survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target forcancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies(mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized themusing flow cytometry, Western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG1 , kappa), reacted with EpCAM-overexpressed
Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In
contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The KD of recEpMab-37
for CHO/EpCAM and Caco-2 was 2.0 × 10−8 M and 3.2 × 10−8 M, respectively. We observed that
EpCAM amino acids between 144 to 164 are involved in recEpMab-37 binding. In Western blot analysis, recEpMab-37 detected the EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37
could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry.
Taken together, recEpMab-37, established by the CBIS method, is useful for detecting EpCAM invarious applications.
Keywords: EpCAM; monoclonal antibody; recombinant antibody; colorectal carcinoma

Received: 28 March 2022
Accepted: 6 June 2022
Published: 8 June 2022

1. Introduction

Publisher’s Note: MDPI stays neutral

The epithelial cell adhesion molecule (EpCAM) is an approximately 40 kilodalton(kDa), type I transmembrane glycoprotein, which is expressed on the basolateral membraneof most epithelial cells [1]. EpCAM is a unique cell adhesion molecule that does notresemble other classical adhesion molecules (e.g., cadherins, selectins, and integrins) ineither structure or function. EpCAM forms homophilic and intercellular adhesion, whichplays critical roles in the maintenance of the epithelial integrity [2]. EpCAM also serves
as a signaling molecule. Upon cleavage of EpCAM at the cell membrane, the intracellulardomain of EpCAM serves as a transcriptional cofactor with β-catenin and regulates thetranscription of target genes involved with cell proliferation, survival, and the maintenanceof stem cell properties [3].
In addition to its role in normal epithelial tissues, EpCAM plays an important rolein tumor development, and was the first identified human tumor-associated antigen [4].
Studies have shown that EpCAM is overexpressed in many epithelial cancers including
pancreas, colorectal, and prostate cancers [5]. Due to high expression and antigenicity,with regard to jurisdictional claims inpublished maps and institutional affiliations.

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/).

Antibodies 2022, 11, 41. https://doi.org/10.3390/antib11020041

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

Antibodies 2022, 11, 41

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EpCAM has been considered as a target of monoclonal antibody (mAb) therapies incolorectal cancer [6,7]. More recently, a bispecific EpCAM/CD3-antibody, Catumaxomab,
serves as the trifunctional antibody. Catumaxomab binds to EpCAM-overexpressed tumors
and recruits T cells to tumor cells, which promote cytotoxic synapse formation and tumor
cell lysis. Furthermore, through the Fc domain, Catumaxomab also recruits natural killer
cells and macrophages and enhances antibody-dependent cellular cytotoxicity (ADCC) [8,9].
Several clinical trials have demonstrated promising outcomes [10–12], and Catumaxomab
has been approved by the European Union for the treatment of patients with malignant
ascites in EpCAM-positive carcinomas [13].
Moreover, anti-EpCAM mAbs are most widely used to collect circulating tumor cells(CTCs), which are useful indicators of micro-metastasis and provide important prognostic
information to determine the response to therapeutic interventions [14]. The clinical importance of CTCs was confirmed by the U.S. Food and Drug Administration (FDA) approval of
CellSearch, a platform that could extract EpCAM-positive, CD45-negative cells from whole
blood samples [15]. Therefore, the establishment of anti-EpCAM mAbs is important toimprove the efficacy of the abovementioned applications and to develop novel modalities
for tumor therapy. In this study, we developed anti-EpCAM mAbs using the Cell-Based Immunization and Screening (CBIS) method [16–31], and evaluated it for various applications,
including flow cytometry, Western blotting, and immunohistochemical analyses.
2. Materials and Methods
2.1. Plasmids
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. EpCAM cDNA plus a C-terminal PA tag that is recognized by
the anti-PA tag mAb (NZ-1), was subcloned into a pCAG-Ble vector (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan). N-terminal PA-tagged EpCAM deletion mutants(dN44, dN64, dN84, dN104, dN124, dN144, dN164, dN184, dN204, dN224, and dN244)
were produced using a HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany),and subcloned into the pCAG-Ble vector.
2.2. Cell lines
P3X63Ag8U.1 (P3U1), CHO-K1, and Caco-2 (human colorectal carcinoma) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). CHO/EpCAM
and CHO/EpCAM deletion mutants were established by transfecting pCAG/EpCAM vectors into CHO-K1 cells using the Neon Transfection System (Thermo Fisher Scientific, Inc.,
Waltham, MA, USA) as described previously [32]. EpCAM-knocked out Caco-2 (BINDS-16)
was established as described previously [33], and the related cell lines are available fromTohoku University Cell Bank (Kato’s Lab; http://www.med-tohoku-antibody.com/topics/
001_paper_cell.htm (accessed on 5 June 2022)). P3U1, CHO-K1, and CHO/EpCAM were
cultured in RPMI 1640 media (Nacalai Tesque, Inc., Kyoto, Japan). Caco-2 was cultured in
Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Inc., Kyoto, Japan). The
media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo
Fisher Scientific Inc., Waltham, MA, USA), 100 U/mL of penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (Nacalai Tesque, Inc.). Cells were incubated at
37 ◦ C in a humidified atmosphere containing 5% CO2 .
2.3. Animals
A BALB/c mouse was purchased from CLEA Japan (Tokyo, Japan). The animal washoused under specific pathogen-free conditions. All animal experiments were conductedfollowing the relevant guidelines and regulations to minimize animal suffering and distressin the laboratory. Animal experiments were approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001). The mouse was monitored
daily for health during the full four weeks duration of the experiment. A reduction of moreAntibodies 2022, 11, 41

3 of 13than 25% of the total body weight was defined as a humane endpoint. The mouse was
euthanized by cervical dislocation, and death was verified by respiratory and cardiac arrest.
2.4. Hybridoma Production
We used the CBIS method to develop mAbs against EpCAM. Briefly, one BALB/cmouse was intraperitoneally (i.p.) immunized with CHO/EpCAM cells (1 × 108 ) together
with Imject Alum adjuvant (Thermo Fisher Scientific Inc.). The procedure included three
additional immunizations, followed by a final booster injection-administered i.p. twodays before spleen cell harvesting. Spleen cells were then fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN, USA). Hybridomas were grown in RPMI
1640, supplemented with 10% FBS, 100 U/mL of penicillin, 100 µg/mL streptomycin, and0.25 µg/mL amphotericin B. Hypoxanthine, aminopterin, and thymidine (HAT; Thermo
Fisher Scientific Inc.) were also added for hybridoma selection. Culture supernatants werescreened using flow cytometry.
2.5. Production of the Recombinant Antibody
Variable (VH ) and constant (CH ) regions of heavy chain cDNAs of EpMab-37 were
subcloned into the pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan) along with variable (VL ) and constant (CL ) regions of light chain cDNAs of EpMab37 into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation) to produce
recombinant EpMab-37 (recEpMab-37). EpMab-37 vectors were transfected into ExpiCHOS cells using the ExpiCHO Expression System (Thermo Fisher Scientific Inc.). recEpMab-37
was purified using Ab-Capcher (ProteNova, Kagawa, Japan).
2.6. Flow Cytometry
CHO/EpCAM, CHO-K1, Caco-2, and BINDS-16 cells were harvested after brief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque,Inc.). After washing with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline(PBS), cells were treated with recEpMab-37 for 30 min at 4 ◦ C, and then with Alexa
Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers,
MA, USA). Fluorescence data were collected using SA3800 Cell Analyzer (Sony Corp.,
Tokyo, Japan).
2.7. Determination of the Binding Affinity
CHO/EpCAM and Caco-2 cells were suspended in 100 µL of serially diluted recEpMab37 (6 ng/mL–100 µg/mL), followed by the addition of Alexa Fluor 488-conjugated antimouse IgG (1:200). Fluorescence data were collected using BD FACSLyric (BD Biosciences,Franklin Lakes, NJ). The dissociation constant (KD ) was calculated by fitting binding
isotherms to built-in, one-site binding models in GraphPad Prism 8 (GraphPad Software,Inc., La Jolla, CA, USA).
2.8. Western Blot Analysis
Cell lysates of CHO/EpCAM, CHO-K1, Caco-2, and BINDS-16 cells were boiled in
sodium dodecyl sulfate sample buffer (Nacalai Tesque, Inc.). The samples were thenelectrophoresed on 5–20% polyacrylamide gels (Nacalai Tesque, Inc.) and transferred onto
polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). After
blocking with 4% skim milk (Nacalai Tesque, Inc.) for 1 h, the membrane was incubatedwith recEpMab-37 (1 µg/mL) or anti-β-actin (1 µg/mL) for 1 h, followed by incubation with
HRP-conjugated anti-mouse immunoglobulins (Agilent Technologies, Inc., Santa Clara, CA,
USA) at a 1:2000 dilution for 1 h. The membrane was developed using the ImmunoStar LD
Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) and a SayacaImager (DRC Co., Ltd., Tokyo, Japan). All Western blot procedures were performed atroom temperature.

Antibodies 2022, 11, 41

4 of 132.9. Immunohistochemical Analysis
Histologic sections that were 4 µm thick of colorectal carcinoma tissue array (Catalog
number: CO243b; US Biomax Inc., Rockville, MD, USA) were autoclaved in citrate buffer(pH 6.0; Agilent Technologies Inc.) for 20 min. Sections were then incubated with 1 µg/mLof recEpMab-37 for 1 h at room temperature and treated using an Envision+ kit (Agilent
Technologies, Inc.) for 30 min. The color was developed using 3,30 -diaminobenzidine
tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2 min, and sections were thencounterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation).
3. Results
3.1. Flow Cytometric Analysis of recEpMab-37 to EpCAM-Expressing Cells
In this study, EpMab-37 was established by immunizing one mouse with CHO/EpCAM
cells (Figure 1). We confirmed the reactivity of EpMab-37 to CHO/EpCAM cells, cloned
the cDNAs of EpMab-37, and produced the recombinant mAb, recEpMab-37.
We first confirmed the reactivity of recEpMab-37 against CHO/EpCAM by flow
cytometry. As shown in Figure 2A, recEpMab-37 recognized CHO/EpCAM cells in adose-dependent manner, but not CHO-K1 cells (Figure 2B). We next examined whether
recEpMab-37 could recognize endogenous EpCAM in colorectal carcinoma Caco-2 cells.
recEpMab-37 reacted with Caco-2 cells in a dose-dependent manner (Figure 2C). However,recEpMab-37 showed no reaction with EpCAM-knocked out Caco-2 (BINDS-16) cells(Figure 2D).
Then, we determined the apparent binding affinity of recEpMab-37 with CHO/EpCAM
and Caco-2 using flow cytometry. The KD of recEpMab-37 for CHO/EpCAM and Caco-2
was 2.0 × 10−8 M and 3.2 × 10−8 M, respectively, indicating that recEpMab-37 possesses
the moderate affinity for EpCAM-expressing cells (Figure 3).
We next generated eleven N-terminal PA tagged EpCAM deletion mutants transfected
CHO-K1 cells, namely dN44 (corresponding to 44–314 amino acids [aa]), dN64 (corresponding to 64–314 aa), dN84 (corresponding to 84–314 aa), dN104 (corresponding to
104–314 aa), dN124 (corresponding to 124–314 aa), dN144 (corresponding to 144–314 aa),dN164, (corresponding to 164–314aa), dN184 (corresponding to 184–314 aa), dN204, (corresponding to 204–314 aa), dN224, (corresponding to 224–314 aa), and dN244, (corresponding
to 244–314 aa). All deletion mutants of EpCAM containing the N-terminal PA tag weredetected by NZ-1 (an anti-PA tag mAb), indicating the expression level of each construct tobe similar (Figure 4A). Although recEpMab-37 detected dN44, dN64, dN84, dN104, dN124,
and dN144, it did not react with dN164, dN184, dN204, dN224, and dN244 (Figure 4B),
suggesting that the recEpMab-37 epitope exists between 144 and 164 aa of EpCAM.
3.2. Western Blot Analysis
Western blotting was performed to further assess the sensitivity of recEpMab-37.
Lysates of CHO-K1 and CHO/EpCAM cells were probed. As shown in Figure 5, recEpMab37 detected EpCAM as 35~40 kDa double bands. However, recEpMab-37 did not detect any
bands from lysates of CHO-K1 cells. These results indicate that recEpMab-37 specificallydetects exogenous EpCAM. Furthermore, recEpMab-37 could detect endogenous EpCAM
as a 35-kDa band from lysates of Caco-2 cells. In contrast, recEpMab-37 could not detect
any bands from lysates of EpCAM-knocked out Caco-2 (BINDS-16) cells. These results
suggest that recEpMab-37 specifically recognizes endogenous EpCAM in the colorectal
carcinoma cell line.
3.3. Immunohistochemical Analysis Using recEpMab-37 against Colorectal Carcinoma Tissues
We investigated whether recEpMab-37 is applicable for immunohistochemical analyses
using formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma sections. recEpMab37 strongly stained colorectal carcinoma cells in a colorectal carcinoma section (Figure 6A,B),
but not stromal tissues (Figure 6A). A cytoplasmic staining pattern in carcinoma cells wasobserved (Figure 6B). Hematoxylin and eosin (HE) staining was performed using the serial

Antibodies 2022, 11, 41, x FOR PEER REVIEW

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5 of 14sections (Figure 6C,D). In contrast, recEpMab-37 showed faint staining in the surface of
normal colorectal epithelium (Figure 6E,F). HE staining was also performed using theserial sections (Figure 6G,H). These results indicate that recEpMab-37 is useful for the
immunohistochemical analysis of FFPE tumor sections.

Figure 1. A schematic procedure of anti-EpCAM mAbs production. A mouse was intraperitoneally

Figure 1. A schematic
procedureof CHO/EpCAM
anti-EpCAMcells.
mAbs
A mousewasbyintraperitoneallyimmunizedwith theTheproduction.
screening was thenconducted
flow cytometry using
immunized with the
CHO/EpCAM
cells.
The
screeningwasthenconductedbyflowregions
cytometryparental cells and EpCAM-overexpressed CHO-K1 cells. Variable and constantcDNAsuswere
ing parental cells and
EpCAM-overexpressed
cells. Variable
constant regions cDNAs
clonedfrom an established clone,CHO-K1
and the recombinant
Ab wasandproduced.
were cloned from an established clone, and the recombinant Ab was produced.

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Figure 2. Flowcytometryof EpCAM
expressing
cells using recEpMab-37.
CHO/EpCAM
Figure 2. Flow cytometryof EpCAM
expressing
cellsusing recEpMab-37.
CHO/EpCAM(A), CHO- (A),CHO-K1(B),
Caco-2(C),
and
BINDS-16(EpCAM-knockedoutCaco-2)
(D)
cells
were
treatedK1 (B), Caco-2 (C), and BINDS-16 (EpCAM-knocked out Caco-2) (D) cells were treated with
0.01–with
0.01–10 µg/mL of recEpMab-37, followed by treatment with Alexa Fluor 488-conjugated anti-mouse
10 μg/mL of recEpMab-37,
followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG.
IgG. Black line represents the negative control.
Black line represents the negative control.

Antibodies 2022, 11, x FOR PEER REVIEW
Antibodies 2022, 11, 41

7
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Figure 3. The determination of the binding affinity of recEpMab-37. CHO/EpCAM and Caco-2

Figure3. The determination of the binding affinity of recEpMab-37. CHO/EpCAM and Caco-2
cells were suspended in 100 µL serially diluted recEpMab-37 (6 ng/mL to 100 µg/mL). Then, cells
weresuspended
100 Fluor
μL seriallydilutedanti-mouse
recEpMab-37(6 ng/mLdata
to 100Then, cells
weretreated withinAlexa
488-conjugated
IgG. Fluorescence
wereμg/mL).
subsequently
treated
with
Alexa
Fluor
488-conjugatedanti-mouse
IgG.
Fluorescence
data
were
subsequently
collected using a BD FACSLyric, following the calculation of the dissociation constant (KD ) bylectedusingPRISM
a BD8. FACSLyric, following the calculation of the dissociation constant (KDGraphPad
GraphPad PRISM 8.

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11, 11,41 x FOR PEER REVIEW
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Figureusingdeletiondeletionmutants
mutantsofof
EpCAM.
CHO-K1expressed
Figure4.4.Epitope
Epitopemapping
mapping ofof recEpMab-37
recEpMab-37 using
EpCAM.
CHO-K1expressed
EpCAMdeletiondeletionmutants
mutants were
were analyzed
analyzed using
mutants-transfected
EpCAM
using flow
flowcytometry.
cytometry.The
TheEpCAM
EpCAM
mutants-transfected
Antibodies 2022, 11, x FOR PEER REVIEW
9 of 14cells
wereincubatedwith
withNZ-1
NZ-1(anti-PA(anti-PA
tag;
red
line,
(A)),
recEpMab-37(anti-EpCAM(anti-EpCAMmAb;
mAb;redredline,cells
were
incubated
tag;
red
line,
(A)),
recEpMab-37
line, (B)), or buffer control (black line, (A,B)) for 30 min ◦at 4 °C, followed by secondary antibodies.
(B)), or buffer control (black line, (A,B)) for 30 min at 4 C, followed by secondary antibodies.

3.2. Western Blot Analysis
Western blotting was performed to further assess the sensitivity of recEpMab-37. Lysates of CHO-K1 and CHO/EpCAM cells were probed. As shown in Figure 5, recEpMab37 detected EpCAM as 35~40 kDa double bands. However, recEpMab-37 did not detect
any bands from lysates of CHO-K1 cells. These results indicate that recEpMab-37 specifically detects exogenous EpCAM. Furthermore, recEpMab-37 could detect endogenous
EpCAM as a 35-kDa band from lysates of Caco-2 cells. In contrast, recEpMab-37 could notdetect any bands from lysates of EpCAM-knocked out Caco-2 (BINDS-16) cells. These results suggest that recEpMab-37 specifically recognizes endogenous EpCAM in the colorectal carcinoma cell line.

Figure5.5.Western
Westernblotting
blotting
recEpMab-37.
lysatesof CHO-K1,
CHO/EpCAM,
Caco-2,
Figurebyby
recEpMab-37.
CellCelllysatesof CHO-K1,
CHO/EpCAM,
Caco-2,and and
BINDS-16
(10
μg)
were
electrophoresedandtransferredonto
polyvinylidene
fluoride
membranes.
BINDS-16 (10 µg) were electrophoresed and transferred onto polyvinylidene fluoride membranes.
The
incubatedwith
1 μg/mLof recEpMab-37
and and
1 μg/mLof anti-β-actin
and and
Themembranes
membraneswere
were
incubatedwith
1 µg/mLof recEpMab-37
1 µg/mLof anti-β-actin
subsequently with peroxidase-conjugated anti-mouse immunoglobulins. Black arrows indicate thesubsequently with peroxidase-conjugated anti-mouse immunoglobulins. Black arrows indicate thepredicted size of EpCAM (~35 kDa).
predicted size of EpCAM (~35 kDa).

3.3. Immunohistochemical Analysis Using recEpMab-37 against Colorectal Carcinoma Tissues
We investigated whether recEpMab-37 is applicable for immunohistochemical analyses using formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma sections. recEpMab-37 strongly stained colorectal carcinoma cells in a colorectal carcinoma section (Fig-Antibodies 2022, 11, x FOR PEER REVIEW
Antibodies 2022, 11, 41

10 o
9 of 13

Figure 6. Immunohistochemical analysis using recEpMab-37 against colorectal carcinoma tissues.
Figure6. Immunohistochemical analysis using recEpMab-37 against colorectal carcinoma tissu(A,B,E,F) After antigen retrieval, sections were incubated with 1 µg/mL of recEpMab-37 followed by(A,B,E,F) After antigen retrieval, sections were incubated with 1 μg/mL of recEpMab-37 follow
treatment with the Envision+ kit. Color was developed using DAB, and sections were counterstainedby treatment with the Envision+ kit. Color was developed using DAB, and sections were counwith hematoxylin. Cytoplasmic brown staining indicates the EpCAM positive cells. (C,D,G,H)
stained with hematoxylin. Cytoplasmic brown staining indicates the EpCAM positive ce
Hematoxylin and eosin (HE) staining. Scale bar = 100 µm.
(C,D,G,H) Hematoxylin and eosin (HE) staining. Scale bar = 100 μm.
4. Discussion

4. Discussion
In this study, we first developed EpMab-37 using the CBIS method and next produced

its recombinant
mAbwe(recEpMab-37)(Figure1). Then, weinvestigatedthemethod
usefulnessof next p
In this study,
first developed
EpMab-37
using
the CBIS
andrecEpMab-37 for flow cytometry (Figure 2), Western blotting (Figure 5), and immunohistoduced its recombinant mAb (recEpMab-37) (Figure 1). Then, we investigated the usef
chemistry (Figure 6). Since recEpMab-37 does not detect any bands in EpCAM-knocked out
nessof cells,
recEpMab-37
forexhibited
flow cytometry(Figure2), Western
blottingthe(FigureCaco-2
recEpMab-37
high specificity
to EpCAM.
To determine
epitope5), and imunohistochemistry(Figure6).
Since
recEpMab-37
does
notdetectany
bands
in EpCAof recEpMab-37, we first performed an enzyme-linked immunosorbent assay using linear
peptides in
theCaco-2
EpCAMcells,
extracellulardomain.exhibited
However, EpMab-37
never recognized
any To det
knockedout
recEpMab-37
high specificity
to EpCAM.

mine the epitope of recEpMab-37, we first performed an enzyme-linked immunosorb
assay using linear peptides in the EpCAM extracellular domain. However, EpMabnever recognized any peptides including 144 to 163 aa of EpCAM (Supplemental Figu
S1). Next, we confirmed that the 144 to 164 aa of EpCAM are involved in recEpMabAntibodies 2022, 11, 41

10 of 13

peptides including 144 to 163 aa of EpCAM (Supplemental Figure S1). Next, we confirmedthat the 144 to 164 aa of EpCAM are involved in recEpMab-37 binding using the deletionmutant-expressed CHO-K1 cells (Figure 4). Because EpMab-37 was established by the immunization of EpCAM-overexpressed CHO-K1 cells, EpMab-37 could recognize the nativeand conformational epitope. However, we cannot rule out the possibility that the deletionof EpCAM affects the native conformation and collapse the recEpMab-37 epitope. We havedeveloped the Arg, Ile, Glu, Asp, and Leu (RIEDL)-insertion for epitope mapping (REMAP)
and His-tag insertion for the epitope mapping (HisMAP) method to determine the conformational epitopes. We successfully determined the epitopes of anti-EGFR mAbs (EMab-51
and EMab-134) [34,35] and anti-CD44 mAbs (C44 Mab-5 and C44 Mab-46) [36,37] by the
REMAP method, and anti-CD20 mAbs (C20 Mab-11 and C20 Mab-60) using the HisMAP
method [38,39]. The determination of EpMab-37 epitope may provide useful information
for future applications.
EpCAM plays critical roles in tumorigenesis to promote tumor cell proliferation,
survival, and tumor-initiating potential [2]. EpCAM has been reported to be overexpressedin several tumors [40–44], which correlates with poor prognosis, therapeutic failure, andearly tumor recurrence [43]. Therefore, EpCAM has been considered as a promisingtherapeutic target. A humanized scFv against EpCAM, fused to Pseudomonas exotoxin A,
Oportuzumab monatox (Vicinium, VB4-845), was developed [3]. After Vicinium targets
EpCAM on tumor cells, it is internalized, receives the cleavage of the linker, releases thepayload exotoxin, and exhibits cytotoxicity. Vicinium was granted the FDA fast track
designation for the treatment of bacillus Calmette-Guérin (BCG)-unresponsive non-muscle
invasive bladder cancer (NMIBC) [45]. Vicinium was evaluated in the single-arm Phase
3 VISTA study (NCT02449239) for the treatment of patients with high-grade NMIBC insitu and high-grade papillary disease previously treated with BCG. However, FDA did not
approve Vicinium for BCG-unresponsive NMIBC in August 2021 [46].
As mentioned above, clinical applications of anti-EpCAM mAbs are still limited
since anti-EpCAM mAbs might generate a degree of toxicity against normal tissues. We
previously established cancer-specific mAbs (CasMabs) targeting podoplanin [47–50] andpodocalyxin [51], which are expressed on many tumors [52–55]. It is worthwhile to develop
cancer-specific anti-EpCAM mAbs using the CasMab method. We previously converted
a mouse IgG1 subclass into a mouse IgG2a , and produced a defucosylated version which
exhibited high ADCC activities in vitro, and significantly reduced tumor growth [56].
Therefore, the production of a class-switched and defucosylated version of EpMab-37 iswarranted to evaluate in vivo antitumor activity.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antib11020041/s1, Figure S1: Determination of the recEpMab-37
epitope for EpCAM by ELISA using synthesized peptides of EpCAM extracellular domain.
Author Contributions: G.L., T.T. and T.A. performed the experiments; H.S. (Hiroyoshi Suzuki),
M.K.K. and Y.K. designed the experiments; G.L. analyzed the data; G.L., H.S. (Hiroyuki Suzuki)
and Y.K. wrote the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was supported in part by Japan Agency for Medical Research and Development (AMED) under Grant Numbers: JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.).
Institutional Review Board Statement: The animal study protocol was approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001) for studies
involving animals.
Data Availability Statement: The data presented in this study are available in the article andsupplementary material.
Conflicts of Interest: The authors declare no conflict of interest.

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