MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 1, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0031
Open camera or QR reader andscan code to access this article
and other resources online.

Epitope Mapping of an Anti-EpCAM Monoclonal Antibody(EpMab-37) Using the Alanine Scanning Method

Downloaded by Yukinari Kato from www.liebertpub.com at 03/09/23. For personal use only.

Guanjie Li,1,* Hiroyuki Suzuki,1,* Tomohiro Tanaka,2 Teizo Asano,2 Takeo Yoshikawa,3Mika K. Kaneko,2 and Yukinari Kato1–3

The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, and plays critical roles
in cell adhesion, proliferation, and tumorigenesis. EpCAM has been considered as a promising target for tumor
diagnosis and therapy. Anti-EpCAM monoclonal antibodies (mAbs) have been developed for EpCAMoverexpressed tumors, and several clinical trials have demonstrated promising outcomes. We previously
established an anti-EpCAM mAb, EpMab-37 (mouse IgG1, kappa), using the Cell-Based Immunization andScreening method. EpMab-37 was revealed to recognize the conformational epitope of EpCAM. In this study,
we determined the critical epitope of EpMab-37 by flow cytometry using the 1 · alanine scanning (1 · Ala-scan)and the 2 · alanine scanning (2 · Ala-scan) method. We first performed flow cytometry by 1 · Ala-scan using
one alanine (or glycine)-substituted EpCAM mutants, which were expressed on Chinese hamster ovary-K1
cells, and found that the EpMab-37 did not recognize the R163A mutant of EpCAM. We next performed flow
cytometry by 2 · Ala-scan using two alanine (or glycine) residues-substituted EpCAM mutants, and confirmedthat EpMab-37 did not recognize R163A-including mutants of EpCAM. The results indicated that the criticalbinding epitope of EpMab-37 includes Arg163 of EpCAM.
Keywords: EpCAM, epitope mapping, monoclonal antibody, flow cytometry, alanine scanning

EpCAM is an important marker to isolate circulatingtumor cells (CTCs), which play critical roles in tumor progression and metastasis.12 CTCs provide important prognostic information as an indicator of micrometastasis, anddetermine the response to some cancer therapies.13 The U.S.
Food and Drug Administration confirmed the clinical importance of CTCs, and approved of CellSearch, which uses ananti-EpCAM mAb to collect EpCAM-positive CTCs fromblood samples.14
The EpCAM N-terminal domain (24–63 amino acids [aa];ND) contains EGF-like domain and is known to possess
the high accessibility and antigenicity.15 Majority of antiEpCAM mAbs including HEA125 (used in CellSearch),
17-1A (edrecolomab), C215 (used in catumaxomab), andMOC31 (used in oportuzumab) recognize the EpCAM
ND.16,17 In contrast, the anti-EpCAM mAbs, which recognize the thyroglobulin-like domain (64–138 aa; TY) or
the extracellular C-terminal domain (139–265 aa; CD), areIntroductionEpithelial cell adhesion molecule (EpCAM) is aunique type I transmembrane glycoprotein and isexpressed on the basolateral membrane of epithelial cells.1
EpCAM-mediated intercellular adhesion is essential for the
maintenance of the epithelial integrity.2 Furthermore, overexpression of EpCAM is observed in breast, prostate, gastrointestinal, and colorectal cancers.3,4 EpCAM was the first
identified as human tumor antigen.3 After cleavage of the
EpCAM intracellular domain, it serves as a transcriptional
cofactor with b-catenin and regulates the transcriptionaltarget genes, which is involved in cell proliferation, survival,
and tumorigenesis.5
Therefore, EpCAM has been considered as a target of themonoclonal antibody (mAb) therapies. Several mAbs including adecatumumab,6 edrecolomab,7,8 oportuzumab monatox,9and catumaxomab10,11 have been clinically evaluated.

Departments of 1Molecular Pharmacology, 2Antibody Drug Development, and 3Pharmacology, Tohoku University Graduate School of
Medicine, Sendai, Japan.
*These authors contributed equally to this study.

41

42

rare.16 Among clinically tested mAbs, MT201 (adecatumumab) recognizes the EpCAM CD.18 Therefore, the detailed
determination of mAbs epitope could contribute the understanding of the property of mAbs.
We previously established an anti-EpCAM mAb, EpMab37 (mouse IgG1, kappa), by the Cell-Based Immunizationand Screening (CBIS) method.19 To clarify further characteristics of EpMab-37, we performed epitope mapping by
flow cytometry using the 1 · alanine scanning (1 · Ala-scan)method and the 2 · alanine scanning (2 · Ala-scan) method.
Materials and Methods

Downloaded by Yukinari Kato from www.liebertpub.com at 03/09/23. For personal use only.

Plasmids

EpCAM cDNAs plus an N-terminal PA tag that is recognized by the anti-PA tag mAb (NZ-1)20–23 were subcloned
into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical
Corporation, Osaka, Japan). An N-terminal PA-tagged
EpCAM19 and 1 · and 2 · alanine substituted mutants were
produced using a HotStar HiFidelity Polymerase Kit (Qiagen,Inc., Hilden, Germany), and subcloned into the pCAG-Ble
vector using the In-Fusion HD Cloning Kit (Takara Bio, Inc.,
Shiga, Japan).
Cell lines

Chinese hamster ovary-K1 (CHO-K1) cells were obtained
from the American Type Culture Collection (ATCC, Manassas, VA). Mutant EpCAM-overexpressed CHO-K1 cells
were established by transfecting pCAG/EpCAM mutant
vectors into CHO-K1 cells using the Neon Transfection
System (Thermo Fisher Scientific, Inc., Waltham, MA) as
described previously.24,25 All cells were cultured in a

LI ET AL.

Roswell Park Memorial Institute (RPMI) 1640 medium(Nacalai Tesque, Inc., Kyoto, Japan). The media were supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin,
100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B(Nacalai Tesque, Inc.). Cells were incubated at 37C in ahumidified atmosphere containing 5% CO2.
Flow cytometry

Cells were harvested after brief exposure to 0.25% trypsin
in 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.). After washing with 0.1% bovine serum albumin in
phosphate-buffered saline, cells were treated with 1 lg/mL ofrecombinant EpMab-3719 and 1 lg/mL of NZ-1 for 30 min
at 4C, and then with Alexa Fluor 488-conjugated anti-mouseor anti-rat IgG (1:2000; Cell Signaling Technology, Inc.,
Danvers, MA). Fluorescence data were collected using
EC800 Cell Analyzer (Sony Corp., Tokyo, Japan).
Results
Epitope mapping of EpMab-37 with alanine(glycine)-substituted EpCAM mutants

We previously established EpMab-37 by the CBIS method. We further constructed the deletion mutants of EpCAM,
and found that the EpMab-37 epitope exists in the EpCAM
CD (from 144 to 164 aa).19 To reveal the detailed binding
epitope of EpMab-37, we first established several cell lines,
which express one-alanine (or glycine)-substituted mutantsof EpCAM between 144 and 173 aa. The epitope mapping
using these one-alanine (or glycine)-substituted mutants iscalled as 1 · Ala-scan method (Fig. 1).

FIG. 1. Schematic illustration of EpCAM structure and the 1 · alanine (glycine)-substituted EpCAM mutants. The
N-terminal PA-tagged 1 · alanine-substituted EpCAM mutants were transfected with CHO-K1 cells, and the stable transfectants were generated. The EpMab-37 epitope involves Arg163 of EpCAM. EpCAM, epithelial cell adhesion molecule;
CD, C-terminal domain; CHO-K1, Chinese hamster ovary-K1; ND, N-terminal domain; TM, transmembrane domain;
TY, thyroglobulin-like domain.

Downloaded by Yukinari Kato from www.liebertpub.com at 03/09/23. For personal use only.

FIG. 2. Determination of the EpMab-37 epitope of EpCAM by flow cytometry using 1 · Ala-scan method. (A, B) The
wild type and 1 · alanine-substituted EpCAM-overexpressed CHO-K1 were incubated with (A) NZ-1 (1 mg/mL) and(B) EpMab-37 (1 mg/mL), followed by treatment using Alexa Fluor 488-conjugated anti-rat and anti-mouse IgG, respectively; black line, negative control. 1 · Ala-scan, 1 · alanine scanning.
43

Downloaded by Yukinari Kato from www.liebertpub.com at 03/09/23. For personal use only.

44

We first investigated the cell surface expression of the
EpCAM mutants on CHO-K1 cells using NZ-1 (an anti-PA
tag mAb), and confirmed the comparable expression levels(Fig. 2A). We next examined the reactivity of EpMab-37 to
those EpCAM mutants, which were expressed in CHO-K1
cells. As shown in Figure 2B, EpMab-37 exhibited reaction with I144A, I145A, I146A, E147A, L148A, K149A,
H150A, K151A, A152G, R153A, E154A, K155A, P156A,
Y157A, D158A, S159A, K160A, S161A, L162A, T164A,
A165G, L166A, Q167A, K168A, E169A, I170A, T171A,
T172A, R173A, and wild type. In contrast, EpMab-37 did notreact with EpCAM R163A.
Although the EpMab-37 reactivity completely disappearedin EpCAM R163A, several EpCAM mutants including
I144A, I146A, Y157A, L162A, and I170A exhibited the
reduced reactivity to EpMab-37. To confirm the involvementof these residues, we further established EpCAM mutants,
in which sequential two aa were substituted into two alanine(or glycine) residues; this method is named as 2 · Ala-scanmethod (Fig. 3).
We investigated the cell surface expression of the EpCAM
mutants on CHO-K1 cells using NZ-1, and confirmed thecomparable expression levels (Fig. 4A). We then examined the reactivity of EpMab-37 against those EpCAM
mutants, which were expressed in CHO-K1 cells. As shownin Figure 4B, EpMab-37 exhibited reaction with I144A–
I145A, I146A–E147A, L148A–K149A, H150A–K151A,
A152G–R153A, E154A–K155A, P156A–Y157A, D158A–
S159A, K160A–S161A, T164A–A165G, L166A–Q167A,
K168A–E169A, I170A–T171A, T172A–R173A, and wild
type. In contrast, EpMab-37 did not react with L162A–
R163A. These results also indicated that Arg163 is the critical epitope of EpMab-37.
Discussion

We previously performed the 1 · and 2 · Ala-scan epitope mapping methods for an anti-CXCR6 mAb, clone

LI ET AL.

Cx6Mab-1,26 using enzyme-linked immunosorbent assay(ELISA).27 Since Cx6Mab-1 can recognize linear peptide
epitope, ELISA could be applied for the epitope mapping.
In contrast, EpMab-37 never reacts with linear epitope,19suggesting that EpMab-37 recognizes the conformational
epitope.
Therefore, we first performed 1 · Ala-scan using one alanine (glycine)-substituted EpCAM mutants expressed on
CHO-K1 cells (Fig. 1). We found that the EpMab-37 reactivity disappeared completely in EpCAM R163A (Fig. 2),
suggesting that Arg163 is the most important residue of the
EpMab-37 epitope. We further performed the epitope mapping of EpMab-37 using 2 · Ala-scan, and confirmed that
Arg163 is the only critical epitope of EpMab-37 (Fig. 4B).
The Arg163 is located on the a-helix of EpCAM CD,
which is close to 167–QKEIT–171 sequence recognized by
MT201 (adecatumumab).18 Alanine substitution in the sequence did not lose the EpMab-37 recognition (Fig. 2B), suggesting that EpMab-37 possesses a unique epitope and adifferent mode of actions. Recently, CTC expansion methods including two-dimensional long-term expansion, 3D
organoids/spheroids culture, and xenografts have been developed to evaluate the character of CTCs.28 Because EpCAM
is an important antigen to collect CTCs, it would be worthwhile to investigate the effect of anti-EpCAM mAbs including EpMab-37 on the CTC expansion in vitro and metastasis
in vivo. Furthermore, the relationship between the epitopeand EpCAM-internalizing activity by anti-EpCAM mAbs
should be evaluated for the development of antibody–drugconjugates.
Anti-EpCAM mAb can be applied to a bispecific antibody
with anti-MET mAb.29 Because MET is one of the important oncogenes that promote tumorigenesis and metastasis,30
MET-targeting antibody therapeutics have been desired forclinic.31 However, the clinical development of MET-targeted
antibodies has been challenging because bivalent antibodies
exhibit agonistic properties, whereas monovalent antibodies
lack potency and the capacity to downregulate MET.32,33

FIG. 3. Schematic illustration of the 2 · alanine-substituted EpCAM mutants. The N-terminal PA-tagged 2 · alaninesubstituted EpCAM mutants were transfected with CHO-K1 cells, and the stable transfectants were generated.

Downloaded by Yukinari Kato from www.liebertpub.com at 03/09/23. For personal use only.

FIG. 4. Determination of the EpMab-37 epitope of EpCAM by flow cytometry using 2 · Ala-scan method. (A, B) The
wild type and 2 · alanine-substituted EpCAM-overexpressed CHO-K1 cells were incubated with (A) NZ-1 (1 mg/mL) and(B) EpMab-37 (1 mg/mL), followed by treatment using Alexa Fluor 488-conjugated anti-rat and anti-mouse IgG, respectively; black line, negative control. 2 · Ala-scan, 2 · alanine scanning.
45

46

LI ET AL.

MM-131 is a bispecific antibody that is monovalent for
MET, but exhibits high avidity by concurrently binding
to EpCAM using single chain Fv of MOC31.34 MM-131
exhibits antagonistic antibody that blocks ligand-dependentand ligand-independent MET signaling by inhibiting HGF
binding to MET and inducing receptor downregulation.29 In
the future study, we would like to apply EpMab-37 for the
combination therapy with MET or bispecific mAbs.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

Downloaded by Yukinari Kato from www.liebertpub.com at 03/09/23. For personal use only.

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.).
References

1. Trzpis M, McLaughlin PM, de Leij LM, et al. Epithelial
cell adhesion molecule: More than a carcinoma marker andadhesion molecule. Am J Pathol 2007;171:386–395; doi:
10.2353/ajpath.2007.070152
2. Brown TC, Sankpal NV, Gillanders WE. Functional implications of the dynamic regulation of EpCAM during
epithelial-to-mesenchymal transition. Biomolecules 2021;
11:956; doi: 10.3390/biom11070956
3. Herlyn M, Steplewski Z, Herlyn D, et al. Colorectal
carcinoma-specific antigen: Detection by means of monoclonal antibodies. Proc Natl Acad Sci U S A 1979;76:1438–
1442; doi: 10.1073/pnas.76.3.1438
4. Momburg F, Moldenhauer G, Hämmerling GJ, et al.
Immunohistochemical study of the expression of a Mr
34,000 human epithelium-specific surface glycoprotein innormal and malignant tissues. Cancer Res 1987;47:2883–
2891.
5. Eyvazi S, Farajnia S, Dastmalchi S, et al. Antibody based
EpCAM targeted therapy of cancer, review and update.
Curr Cancer Drug Targets 2018;18:857–868; doi: 10.2174/
1568009618666180102102311
6. Baeuerle PA, Gires O. EpCAM (CD326) finding its role incancer. Br J Cancer 2007;96:417–423; doi: 10.1038/sj.bjc.6603494
7. Sears HF, Atkinson B, Mattis J, et al. Phase-I clinical trialof monoclonal antibody in treatment of gastrointestinal
tumours. Lancet 1982;1:762–765; doi: 10.1016/s01406736(82)91811-6
8. Riethmüller G, Holz E, Schlimok G, et al. Monoclonal
antibody therapy for resected Dukes’ C colorectal cancer:
Seven-year outcome of a multicenter randomized trial.
J Clin Oncol 1998;16:1788–1794; doi: 10.1200/jco.1998.16.5.1788
9. Kaplon H, Muralidharan M, Schneider Z, et al. Antibodies
to watch in 2020. MAbs 2020;12:1703531; doi: 10.1080/
19420862.2019.1703531
10. Zhang X, Yang Y, Fan D, et al. The development of bispecific antibodies and their applications in tumor immune
escape. Exp Hematol Oncol 2017;6:12; doi: 10.1186/
s40164-017-0072-7
11. Schönberger S, Kraft D, Nettersheim D, et al. Targeting
EpCAM by a bispecific trifunctional antibody exerts pro-found cytotoxic efficacy in germ cell tumor cell lines.
Cancers (Basel) 2020;12:1279; doi: 10.3390/cancers1205
1279
12. Baccelli I, Schneeweiss A, Riethdorf S, et al. Identificationof a population of blood circulating tumor cells frombreast cancer patients that initiates metastasis in a xenograft
assay. Nat Biotechnol 2013;31:539–544; doi: 10.1038/nbt.2576
13. Xiao J, Pohlmann PR, Isaacs C, et al. Circulating tumor
cells: Technologies and their clinical potential in cancer
metastasis. Biomedicines 2021;9:1111; doi: 10.3390/bio
medicines9091111
14. de Bono JS, Scher HI, Montgomery RB, et al. Circulatingtumor cells predict survival benefit from treatment inmetastatic castration-resistant prostate cancer. Clin Cancer
Res 2008;14:6302–6309; doi: 10.1158/1078-0432.Ccr-080872
15. Schön MP, Schön M, Mattes MJ, et al. Biochemical andimmunological characterization of the human carcinomaassociated antigen MH 99/KS 1/4. Int J Cancer 1993;55:
988–995; doi: 10.1002/ijc.2910550619
16. Pavšič M, Gunčar G, Djinović-Carugo K, et al. Crystal
structure and its bearing towards an understanding of key
biological functions of EpCAM. Nat Commun 2014;5:
4764; doi: 10.1038/ncomms5764
17. Gaber A, Lenarčič B, Pavšič M. Current view on EpCAM
structural biology. Cells 2020;9:1361; doi: 10.3390/cells
9061361
18. Münz M, Murr A, Kvesic M, et al. Side-by-side analysis offive clinically tested anti-EpCAM monoclonal antibodies.
Cancer Cell Int 2010;10:44; doi: 10.1186/1475-2867-10-44
19. Li G, Suzuki H, Asano T, et al. Development of a novelanti-EpCAM monoclonal antibody for various applications.
Antibodies (Basel) 2022;11:41; doi: 10.3390/antib1102
0041
20. Fujii Y, Kaneko M, Neyazaki M, et al. PA tag: A versatile
protein tagging system using a super high affinity antibody
against a dodecapeptide derived from human podoplanin.
Protein Expr Purif 2014;95:240–247; doi: 10.1016/j.pep.2014.01.009
21. Fujii Y, Matsunaga Y, Arimori T, et al. Tailored placementof a turn-forming PA tag into the structured domain of aprotein to probe its conformational state. J Cell Sci 2016;
129:1512–1522; doi: 10.1242/jcs.176685
22. Tamura R, Oi R, Akashi S, et al. Application of the NZ-1
Fab as a crystallization chaperone for PA tag-inserted target
proteins. Protein Sci 2019;28:823–836; doi: 10.1002/pro.3580
23. Tamura-Sakaguchi R, Aruga R, Hirose M, et al. Moving
toward generalizable NZ-1 labeling for 3D structure
determination with optimized epitope-tag insertion. Acta
Crystallogr D Struct Biol 2021;77:645–662; doi: 10.1107/
s2059798321002527
24. Kaneko MK, Ohishi T, Takei J, et al. Anti-EpCAM
monoclonal antibody exerts antitumor activity against oral
squamous cell carcinomas. Oncol Rep 2020;44:2517–2526;
doi: 10.3892/or.2020.7808
25. Hosono H, Ohishi T, Takei J, et al. The anti-epithelial
cell adhesion molecule (EpCAM) monoclonal antibody
EpMab-16 exerts antitumor activity in a mouse model ofcolorectal adenocarcinoma. Oncol Lett 2020;20:383; doi:
10.3892/ol.2020.12246
26. Kitamura K, Suzuki H, Kaneko MK, et al. Cx(6)Mab-1: A
novel anti-mouse CXCR6 monoclonal antibody establishedDownloaded by Yukinari Kato from www.liebertpub.com at 03/09/23. For personal use only.

EPITOPE MAPPING USING ALA-SCAN

by N-terminal peptide immunization. Monoclon AntibImmunodiagn Immunother 2022;41:133–141; doi: 10.1089/
mab.2022.0010
27. Isoda Y, Tanaka T, Suzuki H, et al. Epitope mapping ofan anti-mouse CXCR6 monoclonal antibody (Cx6Mab-1)
using the 2 · Alanine scanning method. Monoclon AntibImmunodiagn Immunother 2022;41(5):275–278; doi:
10.1089/mab.2022.0019
28. Rupp B, Ball H, Wuchu F, et al. Circulating tumor cells in
precision medicine: Challenges and opportunities. Trends
Pharmacol Sci 2022;43:378–391; doi: 10.1016/j.tips.2022.02.005
29. Casaletto JB, Geddie ML, Abu-Yousif AO, et al. MM-131,
a bispecific anti-Met/EpCAM mAb, inhibits HGFdependent and HGF-independent Met signaling throughconcurrent binding to EpCAM. Proc Natl Acad Sci U S A
2019;116:7533–7542; doi: 10.1073/pnas.1819085116
30. Trusolino L, Bertotti A, Comoglio PM. MET signalling:
Principles and functions in development, organ regeneration and cancer. Nat Rev Mol Cell Biol 2010;11:834–848;
doi: 10.1038/nrm3012
31. Gherardi E, Birchmeier W, Birchmeier C, et al. Targeting
MET in cancer: Rationale and progress. Nat Rev Cancer
2012;12:89–103; doi: 10.1038/nrc3205
32. Prat M, Crepaldi T, Pennacchietti S, et al. Agonistic
monoclonal antibodies against the Met receptor dissect the47

biological responses to HGF. J Cell Sci 1998;111 (Pt 2):
237–247; doi: 10.1242/jcs.111.2.237
33. Pacchiana G, Chiriaco C, Stella MC, et al. Monovalency
unleashes the full therapeutic potential of the DN-30 antiMet antibody. J Biol Chem 2010;285:36149–36157; doi:
10.1074/jbc.M110.134031
34. Willuda J, Honegger A, Waibel R, et al. High thermal
stability is essential for tumor targeting of antibody fragments: Engineering of a humanized anti-epithelial
glycoprotein-2 (epithelial cell adhesion molecule) singlechain Fv fragment. Cancer Res 1999;59:5758–5767.

Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: September 2, 2022
Accepted: January 17, 2023