antibodies
Communication

EMab-300 Detects Mouse Epidermal Growth Factor
Receptor-Expressing Cancer Cell Lines in Flow Cytometry
Nohara Goto 1 , Hiroyuki Suzuki 1,2, * , Tomohiro Tanaka 1 , Kenichiro Ishikawa 1 , Tsunenori Ouchida 2 ,Mika K. Kaneko 1,2 and Yukinari Kato 1,2, *1

2*

Citation: Goto, N.; Suzuki, H.;
Tanaka, T.; Ishikawa, K.; Ouchida, T.;
Kaneko, M.K.; Kato, Y. EMab-300
Detects Mouse Epidermal Growth

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Miyagi, Japan; s1930550@s.tsukuba.ac.jp (N.G.);
tomohiro.tanaka.b5@tohoku.ac.jp (T.T.); ken.ishikawa.r3@dc.tohoku.ac.jp (K.I.);
k.mika@med.tohoku.ac.jp (M.K.K.)
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan; tsunenori.ouchida.d5@tohoku.ac.jp
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: Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates thesustaining proliferative signaling, which is an important hallmark of cancer. Human EGFR-targeting
monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patientswith colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mousemodel is essential to further develop the mAb therapy against EGFR. Therefore, sensitive mAbs
against mouse EGFR (mEGFR) should be established. In this study, we developed a specific andsensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The
established anti-mEGFR mAb, EMab-300 (rat IgG1 , kappa), reacted with mEGFR-overexpressed
Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including
NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells, using flow cytometry. The kinetic analysis using flow cytometry indicated that the KD of EMab-300 for CHO/mEGFR
and NMuMG was 4.3 × 10−8 M and 1.9 × 10−8 M, respectively. These results indicated that EMab300 applies to the detection of mEGFR using flow cytometry and may be useful to obtain the proof ofconcept in preclinical studies.
Keywords: mouse EGFR; monoclonal antibody; Cell-Based Immunization and Screening (CBIS)

Factor Receptor-Expressing Cancer
Cell Lines in Flow Cytometry.
Antibodies 2023, 12, 42. https://doi.org/10.3390/antib12030042
Academic Editor: Itai Benhar
Received: 6 May 2023
Revised: 13 June 2023
Accepted: 19 June 2023
Published: 21 June 2023

Copyright: © 2023 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/).

1. Introduction
The Epidermal Growth Factor Receptor (EGFR) belongs to the ERBB family of receptor
tyrosine kinases. EGFR is a type I transmembrane glycoprotein, which is composed of an
extracellular ligand-binding domain and an intracellular tyrosine kinase domain [1]. Upon
ligand binding to the extracellular domain of EGFR, the downstream signaling pathways,
such as the mitogen-activated protein kinases, the phosphoinositide 3-kinase/Akt, theJanus kinase/signal transducer and activator of transcription, and the phospholipase Cγ/protein kinase C pathways are activated [2]. These pathways lead to the transcriptional
activation of target genes, which are important for cell proliferation, survival, migration,
invasion, and cancer-initiating properties [2]. In many carcinomas, EGFR overexpression
and its mutation are involved in sustaining proliferative signaling, which is an important
hallmark of cancer [3,4]. Therefore, EGFR has been considered as an important target forcancer therapy.
The EGFR-targeted therapies have been used in the clinic, including monoclonal
antibodies (mAbs) [5] and the tyrosine kinase inhibitors [6]. Cetuximab is a mouse/humanchimeric mAb that binds to the extracellular domain (domain III) of EGFR, which isimportant for neutralization activity [7]. Cetuximab has been approved by the Food andAntibodies 2023, 12, 42. https://doi.org/10.3390/antib12030042

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

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Drug Administration for colorectal cancer [8] and head and neck squamous cell carcinoma(HNSCC) [9]. Although cetuximab has been used to treat patients with metastatic colorectal
cancers, the use is limited to tumors harboring wild-type RAS [10]. The cetuximab treatment
did not exhibit a benefit in patients with colorectal cancer harboring RAS mutations [10].
Moreover, the majority of HNSCC express EGFR; however, the benefit of therapy is limited
to only 15–20% of HNSCC patients [11].
Preclinical mouse models have been developed for the establishment of cancer therapy. The earliest models were built through the transplantation of murine tumors into
immunocompetent host mice [12]. Furthermore, tumors harvested from genetically modified mice can be transplanted and expanded into fully immunocompetent syngeneic
hosts [13]. These syngeneic models were used in preclinical studies to evaluate not only
small-molecule chemotherapeutic drugs but also immunotherapies including immune
checkpoint inhibitors [13]. Although several studies have shown that cetuximab can stimulate the innate or adaptive immune responses [14,15], the mechanisms have not been
fully understood, due to the lack of a preclinical model using anti-mouse EGFR (mEGFR)
mAb. The model is also thought to be important for the evaluation of novel antibody–drugconjugates and prediction of the side-effects.
The Cell-Based Immunization and Screening (CBIS) method includes immunization
with antigen-overexpressing cells and high-throughput hybridoma screening using flow cytometry. Using the CBIS method, we have developed mAbs against human antigens, including human epidermal growth factor receptor 2 (HER2) [16], human epidermal growth factor
receptor 3 (HER3) [17], epithelial cell adhesion molecule (EpCAM) [18,19], trophoblast cellsurface antigen 2 (TROP2) [20,21], programmed cell death ligand 1 (PD-L1) [22], podoplanin(PDPN) [23–34], the cluster of differentiation 19 (CD19) [35], CD20 [36,37], CD44 [38–41],
CD133 [42], killer cell lectin-like receptor G1 (KLRG1) [43], C-C motif chemokine receptor
9 (CCR9) [44], and T cell immunoreceptor with Ig and ITIM domains (TIGIT) [45] by the
immunization of antigen-overexpressing cells in mice. We also successfully developed
mAbs against mouse antigens, including mouse CCR3 [46] and mouse CCR8 [47], by theirimmunization in rats. In this study, we developed novel anti-mEGFR mAbs using the CBIS
method and evaluated its applications including flow cytometry.
2. Materials and Methods
2.1. Preparation of Plasmids
The expression plasmid of mEGFR (pCMV6-neo-mEGFR-Myc-DDK) is commerciallyavailable from OriGene Technologies, Inc. (Rockville, MD, USA). The cDNA encoding
mEGFR (NM_207655) was subcloned into pCAG-zeo_ssnPA and pCAG-zeo_MAP vectors,
which were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan),with the N-terminal PA tag [48–50] and MAP tag [51,52], respectively. The amino acidsequences of the tag system were as follows: PA tag, 12 amino acids (GVAMPGAEDDVV)
and MAP tag, 12 amino acids (GDGMVPPGIEDK). The PA tag can be recognized by NZ-1
(an anti-human PDPN mAb) [48–50,53–65].
2.2. Antibodies
Alexa-Fluor-488-conjugated anti-rat IgG was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
2.3. Cell Lines
P3X63Ag8U.1 (P3U1), Chinese hamster ovary (CHO)-K1, LN229, and NMuMG(a mouse mammary gland epithelial cell) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Lewis lung carcinoma was obtained from theJapanese Collection of Research Bioresources (JCRB; Osaka, Japan).
The pCAG-zeo_ssnPA-mEGFR and pCAG-zeo_MAP-mEGFR plasmids were transfected into LN229 and CHO-K1 cells, respectively. The stable transfectants were generated
as described previously [40,41].

Antibodies 2023, 12, 42

3 of 11

CHO-K1, mEGFR-overexpressed CHO-K1 (CHO/mEGFR), Lewis lung carcinoma,
and P3U1 were cultured in an RPMI-1640 medium (Nacalai Tesque Inc., Kyoto, Japan),with 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA, USA).
A cocktail of 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mLof amphotericin B (Nacalai Tesque Inc.) was added to the medium. LN229, mEGFRoverexpressed LN229 (LN229/mEGFR), and NMuMG were cultured in DMEM (Nacalai
Tesque Inc.), supplemented as indicated above. For NMuMG cells, 10 µg/mL of insulin(Sigma-Aldrich Corp., St. Louis, MO, USA) was further added. All cells were cultured
using a humidified incubator at 37 ◦ C, in an atmosphere of 5% CO2 and 95% air.
2.4. Development of Hybridomas
A five-week-old Sprague–Dawley rat was purchased from CLEA Japan (Tokyo, Japan).
The animal was housed under specific pathogen-free conditions. All animal experiments
were approved by the Animal Care and Use Committee of Tohoku University (Permit
number: 2022MdA-001).
To develop mAbs against mEGFR, we intraperitoneally immunized one rat with
LN229/mEGFR (1 × 109 cells) plus Imject Alum (Thermo Fisher Scientific Inc.). Inducing astrong immune response with immunogens can be a slow and inefficient process. Adding
an adjuvant, such as Imject Alum, to the antigen stimulates an improved immune responsecompared to the antigen alone. Adjuvants increase the immune response by localizing
antigens for an extended time and attracting the appropriate cells to interact with the
immunogen and each other. Adjuvants are mixed and injected along with antigens toprevent catabolism. Alum is frequently used as an alternative to Freund’s adjuvantsbecause alum is less hazardous. Although Freund’s complete and incomplete adjuvants
produce a stronger, longer-lasting immunogenic response compared to other adjuvants,
they are hazardous to the researcher and can produce lesions at the injection site.
After three additional injections every week (1 × 109 cells/rat), a final booster injection(1 × 109 cells/rat) was performed two days before harvesting spleen cells. The hybridomas
were produced, as described previously [46]. The hybridoma supernatants were screenedusing flow cytometry using CHO/mEGFR, CHO-K1, and NMuMG.
2.5. Purification of EMab-300
The cultured supernatant of EMab-300 hybridomas was applied to 1 mL of Ab-Capcher(ProteNova, Kagawa, Japan). Ab-capcher is a gel carrier in which the alkali-resistantantibody-binding protein Protein A-R28, developed by Protenova’s patented technology,
was immobilized at multiple points at high density. After washing with phosphate-bufferedsaline (PBS), the antibodies were eluted with an IgG elution buffer (Thermo Fisher ScientificInc.). Finally, the eluates were concentrated, and the elution buffer was replaced with PBS
using Amicon Ultra (Merck KGaA, Darmstadt, Germany).
2.6. Flow Cytometric Analysis
CHO/mEGFR, CHO-K1, NMuMG, and Lewis lung carcinoma were harvested aftera brief exposure to 1 mM ethylenediaminetetraacetic acid (EDTA, Nacalai Tesque Inc.).
The cells were treated with EMab-300 or blocking buffer (control) (0.1% BSA in PBS) for
30 min at 4 ◦ C, followed by treatment with Alexa-Fluor-488-conjugated anti-rat IgG. The
data were analyzed using the SA3800 Cell Analyzer and SA3800 software ver. 2.05 (SonyCorp., Tokyo, Japan).
2.7. Determination of KD using Flow Cytometry
We prepared 656 to 0.08 nM (diluted by 1/2) of EMab-300. The serially diluted EMab300 was suspended with CHO/mEGFR and NMuMG cells for 30 min at 4 ◦ C. The cells
were treated with 50 µL of Alexa Fluor 488-conjugated anti-rat IgG (1:200). The fluorescencedata were collected using the SA3800 Cell Analyzer. The KD was subsequently calculatedusing GraphPad PRISM 8 (GraphPad Software Inc., La Jolla, CA, USA).

Antibodies 2023, 12, 42

We prepared 656 to 0.08 nM (diluted by 1/2) of EMab-300. The serially diluted EMab300 was suspended with CHO/mEGFR and NMuMG cells for 30 min at 4 °C. The cells
were treated with 50 μL of Alexa Fluor 488-conjugated anti-rat IgG (1:200). The fluorescence data were collected using the SA3800 Cell Analyzer. The KD was subsequently calculated using GraphPad PRISM 8 (GraphPad Software Inc., La Jolla, CA, USA).
4 of 11
3. Results
3.1.
Development of Anti-mEGFR mAbs Using the CBIS Method
3. Results
To develop anti-mEGFR
mAbs,
ratthewasimmunized
3.1. Developmentof Anti-mEGFR
mAbsoneUsing
CBIS
Method with LN229/mEGFR cells(Figure1A).
The
spleenwasthenexcised
from
the
rat,
and splenocytes
were fused with
To develop anti-mEGFR mAbs, one rat was immunizedwith LN229/mEGFR
cells
myeloma
P3U1
cells(Figure1B).
Thedeveloped
hybridomas
were
subsequently
seeded(Figure 1A). The spleen was then excised from the rat, and splenocytes were fused
with
into
96-wellplatesandcultivatedfor six
days. The
positive wells
screened seededby themyeloma
P3U1
cells(Figure1B). Thedeveloped
hybridomas
werewere
subsequently
selectionof mEGFR-expressing
cell-reactive
and TheCHO-K1-non-reactive
supernatants,
into 96-wellplates and cultivatedfor six days.
positive wells werescreened by ustheing
flow
cytometry(Figure
1C).
After
the
limiting
dilutionandseveraladditional
screenselection of mEGFR-expressing cell-reactive and CHO-K1-non-reactive supernatants, usingings,
anti-mEGFR
mAbs,
EMab-300(rat IgGdilution
1, kappa),
wasfinally
established(Figureflow ancytometry(Figure1C). Afterthe limiting
andseveraladditional
screenings,
1D).
an anti-mEGFR mAbs, EMab-300 (rat IgG1 , kappa), was finally established (Figure 1D).

Figure1.1.The
Theproduction
productionof
ofanti-mEGFR
anti-mEGFRmAb,
mAb,EMab-300.
EMab-300. (A)(A)LN229/mEGFR
LN229/mEGFRcells
cellswerewereimmunizedimmunized
Figureinto aa Sprague–Dawley
Sprague–Dawley rat.
rat. (B)(B)TheThespleenspleencells
cellswere
werefusedfusedwith
withP3U1P3U1cells.
cells.(C)(C)To
Toselectselectantiantiinto
mEGFR mAb-producing
mAb-producinghybridomas,
hybridomas,thethesupernatants
supernatantswere
werescreened
screened byby flow
flow cytometry
cytometry using
using
mEGFR
CHO-K1 and CHO/mEGFR cells. (D) After limiting dilution, an anti-mEGFR mAb, EMab-300
was finally established.

3.2. Flow Cytometric Analysis Using EMab-300
We conducted flow cytometry using EMab-300 against CHO/mEGFR, NMuMG,
and Lewis lung carcinoma cell lines. EMab-300 recognized CHO/mEGFR cells dosedependently at 10, 1, 0.1, and 0.01 µg/mL (Figure 2A). Parental CHO-K1 cells were notrecognized even at 10 µg/mL of EMab-300 (Figure 2B).

Antibodies 2023, 12, 42

Next, we investigated the reactivity of EMab-300 against endogenously mEGF
pressing cell lines, NMuMG and Lewis lung carcinoma. EMab-300 reacted with NM
and Lewis lung carcinoma in a dose-dependent manner (Figure 2C,D). These result
gested that EMab-300 specifically recognizes mEGFR, and it is also usefulfor det5 of 11endogenous mEGFR using flow cytometry.

Figure 2. Flow cytometry of mEGFR-expressing cells using EMab-300. CHO/mEGFR (A), CHO-K1 (B),Figure2. Flow cytometry of mEGFR-expressing cells using EMab-300. CHO/mEGFR (A), CH
NMuMG (C), and Lewis lung carcinoma (D) cells were treated with 0.01–10 µg/mL of EMab-300,(B), NMuMG (C), and Lewis lung carcinoma (D) cells were treated with 0.01–10 μg/mL offollowed by treatment with anti-rat IgG conjugated with Alexa Fluor 488. The black line represents
300, followed by treatment with anti-rat IgG conjugated with Alexa Fluor 488. The blac
the negative control.
represents the negative control.

Next, we investigated the reactivity of EMab-300 against endogenously mEGFRexpressing
lines, NMuMG
and Lewis
lungFlow
carcinoma.
EMab-300 reacted with NMuMG
3.3.
Kinetic cell
Analysisof EMab-300
Using
Cytometryand Lewis lung carcinoma in a dose-dependent manner (Figure 2C,D). These results
withmEGFR-expressing
cells,
we conduct
To determine
the specifically
KD of EMab-300
suggestedthat EMab-300
recognizes
mEGFR,and it is also useful for
detectingnetic
analysesby flowcytometryusing CHO/mEGFR and NMuMG cells. The KD v
endogenous
mEGFR
using
flow cytometry.

of EMab-300 for CHO/mEGFR and NMuMG were determined as 4.3 × 10−8 M and3.3. Kinetic Analysis of EMab-300 Using Flow Cytometry
10−8 M, respectively (Figure 3A,B). These results indicate that EMab-300 posse
To determine the KD of EMab-300 with mEGFR-expressing cells, we conducted kinetic
moderateaffinity for both CHO/mEGFR and NMuMG cells.
analyses by flow cytometry using CHO/mEGFR and NMuMG cells. The K values
Dof EMab-300 for CHO/mEGFR and NMuMG were determined as 4.3 × 10−8 M and1.9 × 10−8 M, respectively (Figure 3A,B). These results indicate that EMab-300 possesses amoderate affinity for both CHO/mEGFR and NMuMG cells.

Antibodies
Antibodies 2023,
2023, 12,
12, x42FOR PEER REVIEW

6 6ofof11
11

Figure 3.
affinityof EMab-300.
CHO/mEGFR(A) and
NMuMG(B) cells weresuspended
Figure3. TheThebinding
bindingaffinityof EMab-300.
CHO/mEGFR(A)
and NMuMG(B) cells
weresuspendedin seriallydilutedas
EMab-300
inand“Materials
andThemethods”.
Thecells werein serially diluted
EMab-300
describedasindescribed“Materials
methods”.
cells weretreatedwithtreatedIgG conjugated488. The fluorescencewere subsequently
anti-ratwith
IgG anti-rat
conjugatedwith Alexa with
FluorAlexa
488. Fluor
The fluorescencedata weredata
subsequently
collected
GraphPad
collected
theCell
SA3800Cell followed
Analyzer,byfollowedby the calculationof theKD usingPRISM
using theusing
SA3800Analyzer,
the calculationof the KD using
GraphPad
8.
PRISM 8.

4. Discussion
4. Discussion
Revolutionary therapeutic strategies and modalities for cancer have been developed.

Revolutionary
therapeuticstrategies
and modalities
haveandbeenmost
developed.
However,
only about
five percentof new cancer
therapiesfor
arecancer
approved,
fail dueto the lackonlyof efficacy
These failuressignificant
amountsof money
reducetheHowever,
about [13].
five percentof new costcancertherapies
are approved,
andandmost
fail duepatient’sof life.[13].
TheThese
failuresalso indicatethat current
preclinicalmethods
are nottothe lackqualityof efficacy
failurescost significant
amountsof moneyand reduce
sufficient
to predict
Althoughis one ofthe important
targets
thepatient’s
quality successfulof life. Theoutcomes.
failures also
indicateEGFR
that current
preclinicalmethodsarefor cancer
therapy,anti-mEGFR
mAbs
for preclinicalnotofbeendeveloped.tarIn
notsufficient
to predict
successful
outcomes.
Althoughstudy
EGFRhaveis onethe important
this study,
we developed
a novel anti-mEGFR(EMab-300)
andgets
for cancer
therapy, anti-mEGFR
mAbs formAb
preclinicalstudyusing
have thenotCBISbeen method
developed.
showedthe application
to flowcytometry(Figures
2 and3). EMab-300
could
contributetoIn
this study,
we developed
a novelanti-mEGFR
mAb(EMab-300)
using
the CBIS
methodthepreclinicalstudy
toevaluatetheantitumor
effectsandpredict
theside-effectsoftheand showed the application to flow cytometry (Figures 2 and 3). EMab-300 could contribEGFR-targetingtherapy.
EMab-300
beand
usefulto obtain
the proof ofofute
to the preclinicalstudyFurthermore,
to evaluate theantitumorcould
effects
predictthe side-effects
concept
in EGFR-targeting
antibody–drugcombination
therapy
withtheimmunethe
EGFR-targetingtherapy.
Furthermore,conjugates
EMab-300orcould
be usefulto obtain
proof
checkpoint
in an immunocompetent
syngeneicormousemodel. therapy with imof
concept ininhibitors
EGFR-targeting
antibody–drug conjugatescombination
EMab-300
could
react
with
NMuMG
and
Lewis
lung
carcinomacells (Figure 2).
mune checkpoint inhibitors in an immunocompetent syngeneic mouse model.
NMuMG
has
been
used
in
the
study
of
the
epithelial-to-mesenchymaltransition(EMT),
EMab-300 could react with NMuMG and Lewis lung carcinoma cells (Figure
2).
which
is
inducedbyvariouscytokines,
such
as
TGF-β
[66]
and
transcriptional
factors[67].
NMuMG has been used in the study of the epithelial-to-mesenchymal transition (EMT),The activationofby
the
EMT program
the transcriptional
ability of migration,
which
is induced
variouscytokines,confers
such as tumor
TGF-β cells[66] andfactors inva[67].
sion,
extravasation,
andstemness
[68].
Once
the
EMT-induced
tumor
cells
reach
distant
The activation of the EMT program confers tumor cells the ability of migration, invasion,organs, these mesenchymal
to epithelial tumor
properties
thedistantmesenchymal–
extravasation,and stemnessproperties[68]. Oncerevert
the EMT-induced
cellsvia
reachorgans,epithelialtransition
(MET)
in
order
to
form
a
secondarytumor
indistant
organs[68]. The
these mesenchymal properties revert to epithelial properties via the mesenchymal–epiEMT-induced
NMuMG
can makespheresin the presenceof distant
EGF in vitro,andexhibitthelialtransition(MET) cells
in orderto forma secondary
tumor inorgans
[68].
The
the
tumorigenic
potential
in
vivo
[69].
Therefore,
we
canevaluatetheneutralization
activity
EMT-induced NMuMG cells can make spheres in the presence of EGF in vitro, and exhibitof EMab-300 in the sphere formation assay in a future study.
the tumorigenic potential in vivo [69]. Therefore, we can evaluate the neutralization activLewis lung carcinoma has been widely used as a syngenetic model [70]. The model
ity of EMab-300 in the sphere formation assay in a future study.
was a successful preclinical model for the evaluation of a chemotherapeutic agent, navel-

Antibodies 2023, 12, 42

7 of 11bine, prior to its implementation in clinical trials [70]. Recently, syngenetic models havealso been important for the evaluation of combination therapy with immune checkpoint
inhibitors [71,72]. Therefore, the conversion of EMab-300 (rat IgG1 , kappa) to mouse IgG
is essential for evaluating antitumor effects and developing antibody–drug conjugates orchimeric antigen receptor T cell therapies. We previously produced recombinant antibodies,
which were converted into the mouse IgG2a subclass from mouse IgG1 . Furthermore, we
produced defucosylated IgG2a mAbs using fucosyltransferase 8-deficient CHO-K1 cells toenhance the antibody-dependent cellular cytotoxicity activity [73–80]. The defucosylated
mAbs showed an antitumor effect in xenograft models. Therefore, a class-switched anddefucosylated type of EMab-300 could contribute to the treatment of syngenetic mousetumors and spontaneous mice tumors in the future.
The identification of the epitope is also important to assess the property of mAbs. We
established anti-human EGFR mAbs using the CBIS method. Most of the mAbs recognized
the conformational epitopes. Therefore, we developed the RIEDL insertion for epitope mapping (REMAP) method [81–84] to identify the conformational epitope. We determined theconformational epitopes of anti-human EGFR mAbs (EMab-51 and EMab-134) [82,84] andanti-CD44 mAbs (C44 Mab-5 and C44 Mab-46) [81,83] using the REMAP method. Therefore,
further investigations are required to determine the epitope of EMab-300.
Author Contributions: N.G., H.S., T.T., K.I. and T.O. performed the experiments. M.K.K. and Y.K.
designed the experiments. H.S. and Y.K. analyzed the data. H.S. and Y.K. wrote the manuscript.
All authors agreed to be accountable for all aspects of the research in ensuring that the accuracy or
integrity of any part of the work is appropriately investigated and resolved. All authors have readand agreed to the published version of the manuscript.
Funding: This research was supported in part by the Japan Agency for Medical Research and Development (AMED) under grant nos. JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.), JP22bm1004001(to Y.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.), and by the Japan Society for the
Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI), grant nos. 21K20789(to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.). And the APC was fundedby AMED.
Institutional Review Board Statement: The animal study protocol was approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2022MdA-001) for studies
involving animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: All related data and methods are presented in this paper. Additional
inquiries should be addressed to the corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest.

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