Communication

Development of Highly Sensitive Anti-Mouse HER2 Monoclonal
Antibodies for Flow Cytometry
Tsunenori Ouchida 1 , Hiroyuki Suzuki 2, * , Tomohiro Tanaka 2 , Mika K. Kaneko 2
1

2*

Citation: Ouchida, T.; Suzuki, H.;

and Yukinari Kato 1,2, *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
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-Ku, Sendai 980-8575, Miyagi, Japan; tomohiro.tanaka.b5@tohoku.ac.jp (T.T.);
k.mika@med.tohoku.ac.jp (M.K.K.)
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: Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer
is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to thedevelopment of trastuzumab–deruxtecan, an antibody-drug conjugate, the targetable HER2-positive
breast cancer patients have been expanded. To evaluate the developing modalities using antiHER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs againstmouse HER2 (mHER2) should be established. This study developed anti-mHER2 mAbs using
the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs,
H2 Mab-300 (rat IgG2b , kappa) and H2 Mab-304 (rat IgG1 , kappa), reacted with mHER2-overexpressed
Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG(a mouse mammary gland epithelial cell) via flow cytometry. Furthermore, these mAbs neverrecognized mHER2-knockout NMuMG cells. The kinetic analysis using flow cytometry indicatedthat the dissociation constant (KD ) values of H2 Mab-300 and H2 Mab-304 for CHO/mHER2 were
1.2 × 10−9 M and 1.7 × 10−9 M, respectively. The KD values of H2 Mab-300 and H2 Mab-304 for
NMuMG were 4.9 × 10−10 M and 9.0 × 10−10 M, respectively. These results indicated that H2 Mab300 and H2 Mab-304 could apply to the detection of mHER2 using flow cytometry and may be useful
to obtain the proof of concept in preclinical studies.

Tanaka, T.; Kaneko, M.K.; Kato, Y.
Development of Highly Sensitive

Keywords: mouse HER2; monoclonal antibody; Cell-Based Immunization and Screening; CBIS

Anti-Mouse HER2 Monoclonal
Antibodies for Flow Cytometry. Int. J.
Transl. Med. 2023, 3, 310–320.
https://doi.org/10.3390/ijtm3030022
Academic Editor: Pier Paolo Claudio
Received: 11 July 2023
Revised: 25 July 2023
Accepted: 4 August 2023
Published: 10 August 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
HER2 (human epidermal growth factor receptor 2) also called ERBB2, is a member ofthe ERBB family tyrosine kinase receptors for the epidermal growth factor (EGF) family.
Unlike other HER families, HER2 does not possess ligands and cannot form ligand-induced
homodimers. To activate the downstream signaling of HER2, the heterodimer formation
with other HER members in the presence of their specific ligands or ligand-independent
homodimer formation under the overexpressed condition are required [1]. After the homoand heterodimer formation, HER2 activates the downstream signaling pathways suchas the mitogen-activated protein kinases, the phosphoinositide 3-kinase/Akt, and thephospholipase C-γ/protein kinase C pathways [2].
HER2 is overexpressed in ~20% of breast cancers and is associated with poor prognosis including a higher recurrence rate and shorter overall survival [3]. Trastuzumab, ananti-HER2 monoclonal antibody (mAb), exhibited a growth inhibitory effect in vitro and apotent antitumor effect in vivo [4]. The combination of trastuzumab with chemotherapy
improves the response rates, progression-free survival, and overall survival in HER2overexpressing breast cancer patients with metastasis [5]. Trastuzumab has been a standard
therapy for HER2-overexpressing breast cancers for more than 20 years [6]. In the clinic,

Int. J. Transl. Med. 2023, 3, 310–320. https://doi.org/10.3390/ijtm3030022

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the HER2-overexpressing tumors are diagnosed through strong and complete immunohistochemistry (IHC) membranous staining of more than 10% of cells (IHC 3+) and/or in situ
hybridization (ISH)-amplified.
Trastuzumab–deruxtecan (T-DXd) is a trastuzumab-based antibody-drug conjugate(ADC). The cytotoxicity is achieved through the released topoisomerase I inhibitor, DXd
following the endocytosis and enzymatic cleavage of T-DXd [7]. T-DXd first showedbeneficial results in metastatic breast cancer patients who had received multiple anti-HER2targeting treatments [8]. Currently, various clinical trials have evaluated the efficacy of
T-DXd. Based on the studies, T-DXd has been approved in not only HER2-overexpressing
breast cancer [9,10] but also HER2-low (IHC 1+ or IHC 2+/ISH-non-amplified) advanced
breast cancer [11].
Since about half of all breast cancers are classifiable as HER2-low [12], a larger numberof patients will reap the benefits of T-DXd treatment. The future clinical diagnostics of
HER2-low breast cancer is thought to be important to maximize the patient’s benefit fromtreatment. Furthermore, novel modalities against HER2 will be developed and evaluatedin preclinical and clinical studies [13]. Therefore, preclinical mouse models are important
for the evaluation of novel modalities and the prediction of adverse effects.
Although the development of therapeutic modalities for tumors have been revolutionary, the number of newly approved cancer therapies is limited. Most failure is due to thelack of efficacy [14], suggesting that the current preclinical methods are not sufficient topredict successful outcomes. Preclinical mouse models are essential for the development ofcancer therapy. The first models were established through the transplantation of murine
tumors into immunocompetent host mice [15]. These models were successful preclinicalones for the evaluation of a chemotherapeutic agent before clinical trials [16]. Furthermore,tumors derived from genetically modified mice can be maintained in fully immunocompetent syngeneic hosts [14]. These syngeneic models were used in preclinical studies toevaluate not only molecular target therapies but also immunotherapies including immune
checkpoint inhibitors [14]. However, a preclinical model using anti-mouse HER2 (mHER2)
mAb has not been reported.
We have developed the Cell-Based Immunization and Screening (CBIS) method which
includes antigen-overexpressing cell immunization and high-throughput screening of
hybridoma supernatants using flow cytometry. Using the CBIS method, we have developed
mAbs against HER families, including human EGFR [17], HER2 [18], HER3 [19], and mouse
EGFR [20]. In this study, we developed novel anti-mHER2 mAbs using the CBIS method
and evaluated its application to flow cytometry.
2. Materials and Methods
2.1. Preparation of Cell Lines
LN229, Chinese hamster ovary (CHO)-K1, NMuMG (a mouse mammary gland epithelial cell), and P3X63Ag8U.1 (P3U1) were obtained from the American Type Culture
Collection (Manassas, VA, USA).
The cDNA encoding mHER2 (Accession No.: NM_001003817) with C-terminal mycDDK in a pCMV6 vector (cat# MR227307) was purchased from OriGene Technologies,
Inc. (Rockville, MD, USA). The mHER2 plasmid was transfected into CHO-K1 and LN229
cells, using a Neon transfection system (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Stable transfectants were established through cell sorting via a cell sorter, SH800 (SonyCorp., Tokyo, Japan) using an anti-mouse ErbB2/Her2 mAb (clone 666521, R&D systems,
Minneapolis, MN, USA), after which cultivation in a medium containing 0.5 mg/mL
of G418 (Nacalai Tesque, Inc., Kyoto, Japan) was conducted. The mHER2 knockout
NMuMG (BINDS-51) was generated by transfecting 10 µg of CRISPR/Cas9 plasmids
for mHER2 (Guide RNA: AGAGGTTGCGCATCGTGAGA, Thermo Fisher Scientific, Inc.)
into NMuMG cells using the Neon transfection system and established through cell sortingas described above.

Int. J. Transl. Med. 2023, 3

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CHO-K1, mHER2-overexpressed CHO-K1 (CHO/mHER2), and P3U1 cells were
cultured in a Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque,Inc.), with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc.),
100 units/mL of penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of amphotericin
B (Nacalai Tesque, Inc.). LN229 and mHER2-overexpressed LN229 (LN229/mHER2),
NMuMG, and BINDS-51 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)(Nacalai Tesque, Inc.), supplemented with 10% FBS, 10 µg/mL of insulin (Sigma-AldrichCorp., St. Louis, MO, USA), 100 U/mL of penicillin, 100 µg/mL streptomycin, and0.25 µg/mL amphotericin B.
All cells were grown in a humidified incubator at 37 ◦ C, in an atmosphere of 5% CO2
and 95% air.
2.2. Production 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 mHER2, we intraperitoneally immunized one rat with
LN229/mHER2 (1 × 109 cells) plus Imject Alum (Thermo Fisher Scientific Inc.). 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 [21]. The hybridoma supernatants were screenedusing flow cytometry with CHO/mHER2, CHO-K1, and NMuMG.
2.3. Purification of mAbs
The cultured supernatants of H2 Mab-300 and H2 Mab-304-producing hybridomas werecollected and filtered using Steritop (0.22 µm, Merck KGaA, Darmstadt, Germany). The
supernatants were applied to 1 mL of Ab-Capcher (ProteNova, Kagawa, Japan). After washing with phosphate-buffered saline (PBS), the antibodies were eluted with an IgG elution
buffer (Thermo Fisher Scientific Inc.). Finally, the eluates were concentrated, and the elution
buffer was replaced with PBS using Amicon Ultra (Merck KGaA, Darmstadt, Germany).
2.4. Flow Cytometric Analysis
CHO-K1, CHO/mHER2, and NMuMG cells were harvested after a brief exposure
to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA, Nacalai Tesque, Inc.).
The cells were subsequently washed with 0.1% bovine serum albumin (BSA) in PBS andtreated with 0.001, 0.01, 0.1, and 1 µg/mL of primary mAbs for 30 min at 4 ◦ C. The cells
were treated with Alexa Fluor 488-conjugated anti-rat IgG (1:2000). The fluorescence data
were collected using the SA3800 Cell Analyzer (Sony Corp.).
2.5. Determination of Dissociation Constant (KD ) through Flow Cytometry
CHO/mHER2 and NMuMG cells were suspended in 100 µL serially diluted H2 Mab300, H2 Mab-304, and clone 666521 for 30 min at 4 ◦ C. The cells were treated with 50 µL of
Alexa Fluor 488-conjugated anti-rat IgG (1:200). The GeoMean of each histogram including
primary mAb + secondary Ab (Alexa Fluor 488-conjugated anti-rat IgG) and only secondary
Ab (for background) was determined. We further withdrew the background from eachdata and determined the apparent KD by the fitting binding isotherms to built-in one-sitebinding models of GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA).
3. Results
3.1. Development of Anti-mHER2 mAbs using the CBIS Method
To develop anti-mHER2 mAbs, one rat was immunized with LN229/mHER2 cells.
(Figure 1A). The spleen was then excised from the rat, and splenocytes were fused withmyeloma P3U1 cells (Figure 1B). The developed hybridomas were subsequently seeded into

3. Results
3.1. Development of Anti-mHER2 mAbs using the CBIS Method
Int. J. Transl. Med. 2023, 3

To develop anti-mHER2 mAbs, one rat was immunized with LN229/mHER2 cells.
313(Figure 1A). The spleen was then excised from the rat, and splenocytes were fused withmyeloma P3U1 cells (Figure 1B). The developed hybridomas were subsequently seeded
into ten 96-well plates and cultivated for six days. The positive wells were screenedten 96-well plates and cultivated for six days. The positive wells were screened through thethrough the selection of mHER2-expressing cell-reactive and CHO-K1-non-reactive suselection of mHER2-expressing cell-reactive and CHO-K1-non-reactive supernatants using
pernatants using flow cytometry (Figure 1C). After the limiting dilution and several addiflow cytometry (Figure 1C). After the limiting dilution and several additional screenings,
tional screenings, anti-mHER2 mAbs, such as H2Mab-300 (rat IgG2b, kappa) and H2Mabanti-mHER2 mAbs, such as H2 Mab-300 (rat IgG2b , kappa) and H2 Mab-304 (rat IgG1 , kappa),
304 (rat IgG1, kappa), were finally established (Figure
1D).
were finally established (Figure 1D).

Figure 1. AAschematic
mAbs
production.
(A)(A)
mHER2
possesses
fourfourdoschematicillustration
illustrationofofanti-mHER2
anti-mHER2
mAbs
production.
mHER2
possesses
mains(I to(IIV)a tyrosine
kinasekinasedomain.
LN229/mHER2
cells were
immunized
into a Sprague–
domains
to andIV) anda tyrosine
domain.
LN229/mHER2
cells
were immunized
into aDawleyrat, using rat,
an intraperitoneal
injection. (B)The spleencellsspleen
were thenSprague–Dawleyusing an intraperitoneal
injection.
(B) Thecellsfused
were withthen P3U1
fused cells.
with(C) Subsequently, the culture supernatants were screened through flow cytometry to select antiP3U1 cells. (C) Subsequently, the culture supernatants were screened through flow cytometry tomHER2 mAb-producing hybridomas. (D) After limiting dilution and some additional screenings,
select anti-mHER2 mAb-producing hybridomas. (D) After limiting dilution and some additionalanti-mHER2 mAbs were finally established.
screenings, anti-mHER2 mAbs were finally established.

3.2. Flow Cytometric Analyses
We conducted flow cytometry using three anti-mHER2 mAbs, H2 Mab-300, H2 Mab-304,
and a commercially available clone (666521) against CHO/mHER2. As shown in Figure 2A,
both H2 Mab-300 and H2 Mab-304 recognized CHO/mHER2 cells dose-dependently at 10,

Int. J. Transl. Med. 2023, 3, FOR PEER REVIEW

53.2. Flow Cytometric Analyses
Int. J. Transl. Med. 2023, 3

We conducted flow cytometry using three anti-mHER2 mAbs, H2Mab-300, H2Mab-304,
314
and a commercially available clone (666521) against CHO/mHER2. As shown in Figure 2A,
both H2Mab-300 and H2Mab-304 recognized CHO/mHER2 cells dose-dependently at 10,
1, 0.1,
0.1, andand 0.01
0.01 µg/mL.
µg/mL. In
666521 exhibited
exhibited low
low reactivity
reactivity against
against CHO/mHER2
CHO/mHER2
1,
In contrast,
contrast, 666521
comparedtoH
2Mab-300 and H2Mab-304. Parental CHO-K1 cells were not recognizedcompared to H2 Mab-300 and H2 Mab-304. Parental CHO-K1 cells were not recognized eveneven
10 µg/mL
all mAbs(Figureat 10 atµg/mLof allofmAbs(Figure2B). 2B).

Figure 2. Flow cytometry of mHER2-overexpressing
mHER2-overexpressing cells
cells using
using anti-mHER2
anti-mHER2mAbs.
mAbs. CHO/mHER2
CHO/mHER2(A) and CHO-K1 (B)(B) cells
cells were
were treatedtreatedwith
with0.01–10
0.01–10µg/mLµg/mL ofof H
H22Mab-300, H22Mab-304,
Mab-304, 666521,
666521, or
blocking
buﬀer (negative(negativecontrol,
control,black
blackline),line),
followed
treatment
Alexa
Fluor
488-conjublocking buffer
followed
by bytreatment
withwith
Alexa
Fluor
488-conjugated
gatedanti-rat
IgG.
The
red
line represents
each mAb-treatedanti-rat
IgG. Thered
line
represents
each mAb-treatedcell. cell.

We next investigated the reactivity of H2 Mab-300, H2 Mab-304, and 666521 against an
endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelialcell). The three mAbs reacted with NMuMG at more than 0.1 µg/mL (Figure 3A). H2 Mab300 and 666521 could not react with NMuMG at 0.01 µg/mL. In contrast, H2 Mab-304

Int. J. Transl. Med. 2023, 3, FOR PEER REVIEW

Int. J. Transl. Med. 2023, 3

6

We next investigated the reactivity of H2Mab-300, H2Mab-304, and 666521 against an
endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial
315cell). The three mAbs reacted with NMuMG at more than 0.1 µg/mL (Figure 3A). H2Mab300 and 666521 could not react with NMuMG at 0.01 µg/mL. In contrast, H2Mab-304 could
react with NMuMG at 0.01 µg/mL. Furthermore, we could not detect the reactivity against
could react with NMuMG at 0.01 µg/mL. Furthermore, we could not detect the reactivity
mHER2
knockout
NMuMG(BINDS-51,
Figure Figure3B) in 3B)
the inthree
TheseThese
resultsresultsindiagainst mHER2
knockout
NMuMG(BINDS-51,
the mAbs.
three mAbs.
cated
that
H
2Mab-300 and H2Mab-304 specifically recognize mHER2, and are also usefulindicated that H2 Mab-300 and H2 Mab-304 specifically recognize mHER2, and are also
fordetectingendogenous
mHER2
via flow
usefulfor detecting
endogenous
mHER2
viacytometry.
flow cytometry.

Figure 3. Flow
Flow cytometry
cytometry ofof endogenously
endogenously mHER2-expressing
mHER2-expressing cells
cells using
using anti-mHER2
anti-mHER2 mAbs.
mAbs.
NMuMG
(A)
and
mHER2
knockout
NMuMG(BINDS-51)
(B)
cells
were
treatedwith 0.01–10
0.01–10 µg/mLµg/mL
NMuMG (A) and mHER2 knockout NMuMG (BINDS-51) (B) cells were treated with
H2Mab-304, 666521, or blocking buﬀer (negative control, black line), followed byof
H2Mab-300,
of H
2 Mab-300, H2 Mab-304, 666521, or blocking buffer (negative control, black line), followed bytreatment with Alexa Fluor 488-conjugated anti-rat IgG. The red line represents each mAb-treatedtreatment with Alexa Fluor 488-conjugated anti-rat IgG. The red line represents each mAb-treated cell.
cell.

3.3. Kinetic Analyses against mHER2-Expressing Cells Using Flow Cytometry
To determine the KD of H2 Mab-300 and H2 Mab-304 with mHER2-expressing cells,
we conducted kinetic analysis via flow cytometry using CHO/mHER2 and NMuMG cells.
The geometric mean of the fluorescence intensity was plotted versus the concentrationof H2 Mab-300 and H2 Mab-304. The KD values of H2 Mab-300, H2 Mab-304, and 666521

3.3. Kinetic Analyses against mHER2-Expressing Cells Using Flow Cytometry
Int. J. Transl. Med. 2023, 3

To determine the KD of H2Mab-300 and H2Mab-304 with mHER2-expressing cells, we
conducted kinetic analysis via flow cytometry using CHO/mHER2 and NMuMG cells.
316
The geometric mean of the fluorescence intensity was plotted versus the concentration of
H2Mab-300 and H2Mab-304. The KD values of H2Mab-300, H2Mab-304, and 666521 for
CHO/mHER2 were determined as 1.2 × 10−9 M, 1.7
× 10−9 M, and 2.6 × 10−8 M, respectively
for CHO/mHER2 were determined as 1.2 × 10−9 M, 1.7 × 10−9 M, and 2.6 × 10−8 M,(Figure 4A–C). Furthermore, the KD values of H2Mab-300, H2Mab-304, and 666521 forrespectively (Figure 4A–C). Furthermore,
the K valuesof H2 Mab-300,−9H2 Mab-304, and
NMuMG were determined as 4.9 × 10−10 M, 9.0D ×−10
10−10 M and
5.0 × 10 M, respectively
666521 for NMuMG were determined as 4.9 × 10
M, 9.0 × 10−10 M and 5.0 × 10−9 M,(Figure 4D–F). These results indicated that H2Mab-300 and H2Mab-304 possess a high afrespectively (Figure 4D–F). These results indicated that H2 Mab-300 and H2 Mab-304 posfinity to CHO/mHER2 and NMuMG cells compared to 666521, which was established ussess a high affinity to CHO/mHER2 and NMuMG cells compared to 666521, which was
ing recombinant mHER2 as an antigen.
established using recombinant mHER2 as an antigen.

Figure 4.4.The
Thedetermination
determinationofofthe
thebinding
bindingaffinity
aﬃnityofofHH
2Mab-300 and H2Mab-304. CHO/mHER2
Figure2 Mab-300 and H2 Mab-304. CHO/mHER2
Mab-300, H
H2Mab-304,
Mab-304,
(A–C)
and
NMuMG
(D–F)
cells
were
suspendedin100µL
seriallydiluted
dilutedH
H2Mab-300,(A–C) and NMuMG (D–F) cells were suspended in 100 µL serially
22or666521attheindicatedconcentrations.
The
cells
were
treated
with
Alexa
Fluor
488-conjugatedor 666521 at the indicated concentrations. The cells were treated with Alexa Fluor 488-conjugatedanti-rat IgG. The fluorescence data were collected and the dissociation constant (KD) was calculated
anti-rat IgG. The fluorescence data were collected and the dissociation constant (KD ) was calculated
by GraphPad PRISM 8.
by GraphPad PRISM 8.

4. Discussion
The HER2 extracellular domain is composed of four domains (I–IV) (Figure 1A). The
domain II is essential for the formation of heterodimers with other HER members including
EGFR, HER3, and HER4 in the presence of their ligands, such as EGF [22] and neuregulin 1(NRG1, a HER3 ligand) [23]. The clinically approved anti-HER2 mAbs, trastuzumab andInt. J. Transl. Med. 2023, 3

317

pertuzumab, recognize domains IV and II, respectively [24,25]. Pertuzumab was shown to
inhibit the NRG1-induced heterodimerization with HER3 and intracellular signaling [24].
In this study, we developed novel anti-mHER2 mAbs (H2 Mab-300 and 304) using the CBIS
method and showed the application via flow cytometry (Figures 2–4). We should identify
these epitopes and determine the property of mAbs including the effect on the homodimerand heterodimer formation of mHER2. We established the RIEDL insertion for epitope
mapping (REMAP) method [26–29] to identify the conformational epitope because mostof the mAbs developed using the CBIS method recognized the conformational epitopes.
We determined the conformational epitopes of anti-human EGFR mAbs (EMab-51 andEMab-134) [27,29] using the REMAP method.
In a preclinical study, the antitumor activity of anti-HER2 mAbs have been evaluatedon the HER2-positive tumor-bearing immunodeficient mice [30] or HER2-overexpressedmurine breast cancer-transplanted syngeneic models [31]. Furthermore, mouse mammary
tumor virus (MMTV)-driven overexpression of HER2 was generated to investigate the rolesof HER2 in mammary adenocarcinoma development and therapeutic effects of anti-HER2
mAb such as 4D5 (murine version of trastuzumab) [32]. The incidence of mammary tumors
and tumor growth were significantly reduced in 4D5-treated mice [33].
Currently, targetable HER2-positive tumors are expanded from IHC3+ to HER2-low
in breast cancer, suggesting that other tumor types could apply to HER2-targeting therapy.
Pancreatic cancer is a difficult-to-treat disease with critical unmet needs. HER2 expressionis also observed in pancreatic cancers and is associated with poor prognosis [34]. Recently,
new drugs that target KRASG12D , a common mutation in PDAC, have been developed [35].
One of the drugs, MRTX1133, exhibited specific efficacy in tumor cells harboring KRASG12D
mutations [36,37]. However, MRTX1133 promoted the expression and activation of ERBB receptors including HER2, which is expected as a mechanism of resistance to MRTX1133 [38].
Similar ErbB receptors upregulation was also observed in Kras+/LSL-G12D ; Trp53+/LSL-R172H ;Pdx1-Cre (KPC) mice, a murine model of pancreatic cancer [39], treated with MEK and
AKT inhibitors [40]. Therefore, specific kinase inhibitors or mAbs against ErbB receptors
could potentiate the efficacy of the treatment. H2 Mab-300 and H2 Mab-304 may be applied
to obtain the proof of concept in the above mouse model system. For the preclinical study,
the conversion from H2 Mab-300 and H2 Mab-304 (rat IgG) to mouse IgG is essential toevaluate the efficacy in mice.
A major adverse effect of anti-HER2 therapeutic mAbs is cardiotoxicity, which ispotentiated by co-treatment with chemotherapeutic agents. The cardiotoxic surveillancethrough routine cardiac monitoring is essential in clinic [41]. Furthermore, ErbB2-deficient
mice were embryonic lethal due to cardiac dysfunctions [42]. Mice lacking ErbB2 inventricular displayed the characteristics of dilated cardiomyopathy [43]. These results
indicated that HER2 is essential in normal heart development and homeostasis. However,the detailed mechanism of cardiotoxicity has not been investigated. Therefore, H2 Mab-300
and H2 Mab-304 could be used to analyze the cardiotoxicity in various combinations withchemotherapeutic agents in mice. Furthermore, clinical trials combining immunotherapies
with HER2-targeted therapies have been conducted [13]. H2 Mab-300 and H2 Mab-304 arealso important to predict the side effect of the treatment.
5. Conclusions
H2 Mab-300 and 304 could be used in preclinical studies to obtain the proof of conceptof antitumor effects and predict the side effects of HER2-targeting mAb therapy.
We have established other mAb clones against mHER2 and updated them in our
original “Antibody Bank” [http://www.med-tohoku-antibody.com/topics/001_paper_
antibody_PDIS.htm#HER2 (accessed on 8 August 2023)].

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Author Contributions: T.O. and T.T. performed the experiments. M.K.K. and Y.K. designed theexperiments. T.O., 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 ofany part of the work is appropriately investigated and resolved. All authors have read and agreed tothe published version of the manuscript.
Funding: This research was supported in part by the Japan Agency for Medical Research andDevelopment (AMED) under grant numbers: JP23ama121008 (to Y.K.), JP23am0401013 (to Y.K.),
23bm1123027h0001 (to Y.K.), and JP23ck0106730 (to Y.K.); and by the Japan Society for the Promotionof Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant numbers 22K06995 (to H.S.),
21K07168 (to M.K.K.), and 22K07224 (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: 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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