International Journal of

Molecular Sciences
ArticleA Cancer-Specific Monoclonal Antibody against HER2 Exerts
Antitumor Activities in Human Breast Cancer Xenograft Models
Mika K. Kaneko 1,† , Hiroyuki Suzuki 1,† , Tomokazu Ohishi 2,3 , Takuro Nakamura 1 , Tomohiro Tanaka 1
and Yukinari Kato 1, *
1

2

3

*
†

Citation: Kaneko, M.K.; Suzuki, H.;
Ohishi, T.; Nakamura, T.; Tanaka, T.;
Kato, Y. A Cancer-Specific Monoclonal

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; mika.kaneko.d4@tohoku.ac.jp (M.K.K.);
hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); takuro.nakamura.a2@tohoku.ac.jp (T.N.);
tomohiro.tanaka.b5@tohoku.ac.jp (T.T.)
Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Research Foundation, 18-24 Miyamoto,
Numazu 410-0301, Japan; ohishit@bikaken.or.jp
Laboratory of Oncology, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Research
Foundation, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
Correspondence: yukinari.kato.e6@tohoku.ac.jp; Tel.: +81-22-717-8207
These authors contributed equally to this work.

Abstract: Monoclonal antibody (mAb)-based and/or cell-based immunotherapies provide innovative
approaches to cancer treatments. However, safety concerns over targeting normal cells expressing
reactive antigens still exist. Therefore, the development of cancer-specific mAbs (CasMabs) thatrecognize cancer-specific antigens with in vivo antitumor efficacy is required to minimize the adverse
effects. We previously screened anti-human epidermal growth factor receptor 2 (HER2) mAbs andsuccessfully established a cancer-specific anti-HER2 mAb, H2 Mab-250/H2 CasMab-2 (IgG1 , kappa).
In this study, we showed that H2 Mab-250 reacted with HER2-positive breast cancer cells but did
not show reactivity to normal epithelial cells in flow cytometry. In contrast, a clinically approved
anti-HER2 mAb, trastuzumab, recognized both breast cancer and normal epithelial cells. We further
compared the affinity, effector activation, and antitumor effect of H2 Mab-250 with trastuzumab. The
results showed that H2 Mab-250 exerted a comparable antitumor effect with trastuzumab in the mouse
xenograft models of BT-474 and SK-BR-3, although H2 Mab-250 possessed a lower affinity and effector
activation than trastuzumab in vitro. H2 Mab-250 could contribute to the development of chimericantigen receptor-T or antibody–drug conjugates without adverse effects for breast cancer therapy.

Antibody against HER2 Exerts
Antitumor Activities in Human Breast
Cancer Xenograft Models. Int. J. Mol.

Keywords: HER2; cancer-specific monoclonal antibody; epitope; xenograft; breast cancer

Sci. 2024, 25, 1941. https://doi.org/
10.3390/ijms25031941
Academic Editor: Nam Deuk Kim
Received: 15 January 2024
Revised: 2 February 2024
Accepted: 2 February 2024
Published: 5 February 2024

Copyright: © 2024 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 overexpression of human epidermal growth factor receptor 2 (HER2) is observed
in approximately 20% of breast cancers [1] and 20% of gastric cancers [2], which areassociated with higher rates of recurrence and shorter overall survival. HER2 forms
heterodimers with other HER members and the ligands or ligand-independent homodimers
when overexpressed [3]. The formation of hetero- or homodimers leads to the activationof downstream signaling such as RAS-ERK and PI3K-AKT pathways, which promote
cancer cell proliferation, survival, and invasiveness [3]. A clinically approved anti-HER2
monoclonal antibody (mAb), trastuzumab, showed an anti-proliferative effect in vitro anda potent antitumor efficacy in vivo [4,5]. In the treatment of breast cancer patients withmetastasis, trastuzumab is administered in patients with HER2-overexpressed tumors,which are defined by strong and complete membranous staining of more than 10% of cells
in immunohistochemistry (IHC 3+) and/or in situ hybridization (ISH)-amplified [6]. The
combination therapy of chemotherapeutic agents with trastuzumab improves objective
response rates, progression-free survival, and overall survival in HER2-positive breast

Int. J. Mol. Sci. 2024, 25, 1941. https://doi.org/10.3390/ijms25031941

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

Int. J. Mol. Sci. 2024, 25, 1941

2 of 16cancer patients with metastasis [7]. Therefore, trastuzumab has become the most effective
therapy for HER2-positive breast cancers [8] and HER2-positive gastric cancers [9].
The trastuzumab-based antibody–drug conjugates (ADCs) such as trastuzumabderuxtecan (T-DXd) have been evaluated in various clinical trials [10]. Based on thestudies, T-DXd has been approved in not only HER2-positive breast cancer [11,12] but
also HER2-mutant lung cancer [13] and HER2-low (IHC 1+ or IHC 2+/ISH-non-amplified)advanced breast cancer [14]. Since approximately half of all breast cancers are classifiable
as HER2-low, a significant number of patients can benefit from T-DXd therapy [15].
Anti-HER2 therapeutic mAbs and the ADCs have common adverse effects such as
cardiotoxicity [16]. Patients must receive routine cardiac monitoring [17]. Moreover, ErbB2(ortholog of HER2)-knockout mice showed embryonic lethal phenotype because of thelack of cardiac trabeculae [18]. The ErbB2-conditional knockout mice in the ventriculararea displayed the features of dilated cardiomyopathy [19]. These results indicate that
HER2 is essential for normal heart development and homeostasis. Therefore, more selective
anti-HER2 mAbs against cancers are necessary to reduce heart failures.
We previously developed 278 clones of anti-HER2 mAbs using recombinant HER2
ectodomain (HER2ec) derived from glioblastoma LN229 [20] or HER2-overexpressed
LN229 [21] as antigens. We further screened the reactivity to HER2-positive breast cancers (BT-474 and SK-BR-3) and normal epithelial cells using flow cytometry. Finally, we
successfully developed a cancer-specific anti-HER2 mAb, H2 Mab-250/H2 CasMab-2 (IgG1 ,kappa) [22]. Importantly, H2 Mab-250 did not react with non-transformed normal epithelial
cells (HaCaT and MCF 10A) and immortalized normal epithelial cells derived from the
mammary gland, gingiva, lung bronchus, corneal, thymus, kidney proximal tubule, andcolon [22]. In contrast, most anti-HER2 mAbs including trastuzumab reacted with bothcancer and normal epithelial cells [22]. Furthermore, the results of IHC revealed that
H2 Mab-250 possesses high reactivity to the HER2-positive breast cancer tissues and did notreact with normal tissues, including the heart [22]. The epitope mapping demonstrated that
the Trp614 in HER2 domain IV mainly contributes to the recognition by H2 Mab-250 [22].
Trastuzumab is a humanized IgG1 mAb that binds to Fcγ receptors (FcγRs) on various immune cells [23]. The FcγR binding activates macrophages, dendritic cells, andneutrophils, which change adaptive immune responses by antigen presentation, cytokine
production, and chemotaxis [4]. Moreover, the FcγR engagement activates natural killer(NK) cells and macrophages, which can result in the target cell lysis, termed antibodydependent cellular cytotoxicity (ADCC) [4]. In mouse mAbs, IgG2a or IgG2b can bind to
FcγR with high affinity [24]. Furthermore, a core fucose deficiency on the Fc N-glycan
has been shown to enhance the binding of IgG to FcγR on effector cells [25] and exert
potent antitumor effects [26]. The defucosylated recombinant mAbs can be produced using
fucosyltransferase 8-knockout Chinese hamster ovary (CHO) cells [27].
In this study, we produced a mouse IgG2a -type mAb and a human IgG1 -type mAbfrom H2 Mab-250. We then compared the affinity, effector activation, and antitumor effectof H2 Mab-250 with trastuzumab.
2. Results
2.1. The Binding Affinity of H2 Mab-250 and Trastuzumab
H2 Mab-250 recognized HER2 expressed in breast cancers (BT-474 and SK-BR-3), but it
did not recognize HER2 in normal epithelial cells, although trastuzumab recognized bothtypes of HER2 [22]. Since H2 Mab-250 and trastuzumab are mouse IgG1 and human IgG1 ,respectively, we generated the same isotype of recombinant mAbs (mouse IgG2a or human
IgG1 ), which possess ADCC, to compare the antitumor activities between H2 Mab-250 andtrastuzumab. H2 Mab-250-mG2a and tras-mG2a are mouse IgG2a -type mAbs derived from
H2 Mab-250 and trastuzumab, respectively. H2 Mab-250-hG1 and trastuzumab are human
IgG1 -type mAbs.
We confirmed the reactivity of the mAbs against breast cancer cells and normal epithelial cells. As shown in Figure 1A, H2 Mab-250-mG2a reacted with HER2-overexpressed CHO-

250 and trastuzumab. H2Mab-250-mG2a and tras-mG2a are mouse IgG2a-type mAbs derived from H2Mab-250 and trastuzumab, respectively. H2Mab-250-hG1 and trastuzumab
are human IgG1-type mAbs.
Int. J. Mol. Sci. 2024, 25, 1941 We confirmed the reactivity of the mAbs against breast cancer cells and normal epi3 of 16
thelial cells. As shown in Figure 1A, H2Mab-250-mG2a reacted with HER2-overexpressed
CHO-K1 (CHO/HER2), HER2-positive BT-474, and SK-BR-3 cells, but not with triple-negative MDA-MB-468
cells. Importantly,
H2BT-474,
Mab-250-mG
2a did notreact
withwithspontaneously
K1 (CHO/HER2),HER2-positive
and SK-BR-3
cells,
but not
triple-negative
immortalized
normal epithelial
cells (HaCaT(keratinocyte)
10A spontaneously(mammary
MDA-MB-468
cells. Importantly,
H2 Mab-250-mG
not MCF
react with
2a didand
immortalized
normalepithelial
cells (HaCaT(keratinocyte)
andbronchus),
MCF 10A (mammary
gland)) andimmortalized
normalepithelial
cells (HBEC3-KT(lung
hTERTgland))
and
immortalized
normalepithelial
cells(HBEC3-KT(lungbronchus),
hTERTHME1 (mammary gland), hTCEpi (corneal), hTEC/SVTERT24-B (thymus), RPTEC/TERT1
HME1(mammary
gland),
hTCEpi(corneal),
hTEC/SVTERT24-B(thymus),
RPTEC/TERT1(kidney proximal tubule), and HCEC-1CT (colon)). In contrast, tras-mG2a showed the re(kidney proximal tubule), and HCEC-1CT (colon)). In contrast, tras-mG2a showed the reacactivity toboth breast cancer and normal epithelial cells. Similar reactivities were obtivity to both breast cancer and normal epithelial cells. Similar reactivities were observed
served in H2Mab-250-hG1 and trastuzumab (Figure 1B). These results indicated thatin H2 Mab-250-hG1 and trastuzumab (Figure 1B). These results indicated that H2 Mab-250H2Mab-250-mG
2a and H2Mab-250-hG1 retain the cancer specificity compared with trasmG2a andH2 Mab-250-hG1 retain the cancer specificity compared with tras-mG2a andtrastuzumab,
respectively.
mG2a andtrastuzumab,
respectively.

Figure 1. Flow cytometry using anti-HER2 mAbs against HER2-overexpressed cells, breast cancer
cell lines, and normal epithelial cell lines. (A) Flow cytometry using H2 Mab-250-mG2a (10 µg/mL;
red line) and tras-mG2a (10 µg/mL; red line) against CHO-K1, CHO/HER2, HER2-positive breast

Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW

4 of 17

Int. J. Mol. Sci. 2024, 25, 1941

4 of 16

Figure 1. Flow cytometry using anti-HER2 mAbs against HER2-overexpressed cells, breast cancer
cell lines, and normal epithelial cell lines. (A) Flow cytometry using H2Mab-250-mG2a (10 µg/mL;
red line) and tras-mG2a (10 µg/mL; red line) against CHO-K1, CHO/HER2, HER2-positive breast
cancers (BT-474 and SK-BR-3), a triple-negative breast cancer (MDA-MB-468), and spontaneously
cancers (BT-474 and SK-BR-3), a triple-negative breast cancer (MDA-MB-468), and spontaneously
immortalized(HaCaT
andandMCF
10A),
as wellas immortalized
normalepithelial
immortalizednormal
normalepithelial
epithelialcells
cells(HaCaT
MCF
10A),
as wellas immortalized
normalepicells
including
HBEC3-KT(lung bronchus),
hTERT-HME1(mammary
gland),
hTCEpi(corneal),
thelialcells including
HBEC3-KT(lung bronchus),
hTERT-HME1(mammary
gland),
hTCEpi(corhTEC/SVTERT24-B(thymus),
RPTEC/TERT1(kidney
proximaltubule),and and
HCEC-1CT(colon).
neal), hTEC/SVTERT24-B(thymus),
RPTEC/TERT1(kidney
proximaltubule),HCEC-1CT(co2
Mab-250-hG
1(10µg/mL;
red
line)
and
trastuzumab(10µg/mL;lon).
(B) cytometry
Flow cytometry
H
(B)
Flow
using Husing
Mab-250-hG(10µg/mL;
red
line)
and
trastuzumab(10µg/mL;
red
line)
2
1
red line)theagainstthe abovementioned
The cells
weretreatedwith
Alexa
Fluor 488-conjuagainst
abovementioned
cell lines. cell
Thelines.
cells weretreated
with
Alexa
Fluor
488-conjugatedantigatedanti-mouse
IgG
or
fluorescein(FITC)-conjugatedanti-human
IgG.
The
fluorescence
data
were
mouse IgG or fluorescein (FITC)-conjugated anti-human IgG. The fluorescence data were collectedcollectedusing an
SA3800Cell(Sony
Analyzer(Sony
Corp.,
Tokyo,
Japan),
data wereanalyzedusing
an SA3800
Cell
Analyzer
Corp.,
Tokyo,
Japan),and thedata andweretheanalyzed
using
FlowJousing FlowJo v10.8.1. The black line represents the negative control (blocking buﬀer).
v10.8.1. The black line represents the negative control (blocking buffer).

We next
next evaluatedevaluated thethebinding
bindingaffinity
aﬃnityofofHHMab-250-mG
2Mab-250-mG2a, H2Mab-250-hG1, trasWe
22a , H2 Mab-250-hG1 , trasmG
2a, and trastuzumab to HER2ec by enzyme-linked immunosorbent assay (ELISA). The
mG2a , and trastuzumab to HER2ec by enzyme-linked immunosorbent assay (ELISA).
KD valuesof H2Mab-250-mG
2a and tras-mG2a to HER2ec were determined to be 1.1 × 10−9The
KD valuesof H2 Mab-250-mG
2a and tras-mG2a to HER2ec were determined to be
−10
−
9
10 M
M (Figureand 1.92A)
× 10andM1.9(Figurerespectively.
KD values of
H2K
Mab-250-hG11.1
× 10 2A)
M (Figure× 10−2B),(Figure 2B),The
respectively.
The
D values of−9 M (Figure 2C) and−2.0
9andtrastuzumab
toHER2ecwere
also
determined
to
be
1.5
×
10
×
H2 Mab-250-hG1 and trastuzumab to HER2ec were also determined to be 1.5 × 10 M
10−10 M 2C)(FigureAlthoughthe binding Althoughaﬃnity of the
H2Mab-250-mG
2a and(Figureand 2D),
2.0 ×respectively.
10−10 M (Figure2D), respectively.
binding affinityofH2
Mab-250-hG
1
was
less
than
that
of
tras-mG2aandtrastuzumab,
respectively,
H
2
MabH2 Mab-250-mG2a and H2 Mab-250-hG1 was less than that of tras-mG2a and trastuzumab,
250-mG2a andHH
2Mab-250-hG1 exhibited high binding aﬃnity to HER2ec. Furthermore,
respectively,
2 Mab-250-mG2a and H2 Mab-250-hG1 exhibited high binding affinity to
the changes
in the mAbthe
formatdid notaﬀect
theformat
bindingdid
aﬃnitycomparedto the original
HER2ec.
Furthermore,changesin themAb
not affectthe binding
affinitymAbs [22].to the original mAbs [22].
compared

Figureand trastuzumab
2 Mab-250-mG
2aFigure 2.
2. Binding
Binding affinity
aﬃnityofofHH
2Mab-250-mG
2a,, H
H22Mab-250-hG
Mab-250-hG1,, tras-mG
tras-mG2a2a,, andtrastuzumab toto
HER2ec.
HER2ec. (A–D) HER2ec was immobilized on immunoplates and then incubatedincubated with thethe seriallyserially
Mab-250-mG2a2a (A), tras-mG2a2a (B),
diluted H
H22Mab-250-mG
diluted(B), H22Mab-250-hG
Mab-250-hG11 (C),(C), andand trastuzumab
trastuzumab (D), followed byperoxidase-conjugatedanti-mouseoranti-humanimmunoglobulins3). Enzymatic
reactionsperoxidase-conjugated anti-mouse or anti-human immunoglobulins (n =(n3). =Enzymatic
reactionswere
were
conducted,
andtheopticaldensity
at
655
nm
was
measured.
Valuesarepresented
as
the
mean
conducted, and the optical density at 655 nm was measured. Values are presented as the mean ±SD.
± SD. The binding isotherms were fitted into the built-in, one-site binding model in GraphPad
The binding isotherms were fitted into the built-in, one-site binding model in GraphPad PRISM 6 to
PRISM 6 to calculate the binding aﬃnity.
calculate the binding affinity.

Int. J. Mol. Sci. 2024, 25, 1941

Sci. 2024, 25, x FOR PEER REVIEW

5 of 162.2. The Ability of Effector Cell Activation by the Derivatives of H2 Mab-250 and Trastuzumab
The mAb-FcγRIIIa binding-mediated ADCC pathway activation in effector cells can bequantified by a bioluminescent reporter gene assay, called the ADCC reporter bioassay [28].
We previously showed that H2 Mab-250-mG2a selectively activates the effector cells against
breast cancer cells, but not against normal cells. In contrast, trastuzumab activated theeffector with a similar 50% effective concentration (EC50 ) against breast cancer and normal
cells [22]. We next examined whether the derivatives of H2 Mab-250 and trastuzumab
could activate the ADCC pathway in the presence of BT-474 and SK-BR-3 cells. To compare the ADCC pathway activation by the derivatives of H2 Mab-250 and trastuzumab,
we treated BT-474 and SK-BR-3 cells with serially diluted mAbs, and then we incubatedthem with effector Jurkat cells, which express the human FcγRIIIa, and a firefly luciferase
reporter gene driven by a nuclear factor of activated T cell (NFAT)-responsive element. As
shown in Figure 3A, H2 Mab-250-mG2a could activate the effector (EC50 : 1.6 × 10−5 g/mL),but it was less effective than tras-mG2a (EC50 : 3.7 × 10−8 g/mL) in the presence of BT474 cells. H2 Mab-250-mG2a and tras-mG2a also exhibited a similar relationship to EC50
(3.9 × 10−6 g/mL and 9.0 × 10−9 g/mL, respectively) in SK-BR-3 cells (Figure 3B). Furthermore, H2 Mab-250-hG1 could activate the effector (EC50 : 1.4 × 10−6 g/mL), but it was lesseffective than trastuzumab (EC50 : 1.5 × 10−8 g/mL) in BT-474 cells (Figure 3A). H2 Mab250-hG1 and trastuzumab also exhibited a similar relationship to EC50 (2.2 × 10−7 g/mL
and 1.5 × 10−9 g/mL, respectively) in SK-BR-3 cells (Figure 3B). These results indicated
6 of 17that H2 Mab-250-mG2a and H2 Mab-250-hG1 possess a lower ability of effector cell activation
than tras-mG2a and trastuzumab, respectively.

Figure 3. Cont.

Int. J. Mol. Sci. 2024, 25, 1941

6 of 16

Figure 3. The ADCC
assay reporterby H2Mab-250-mG, H2Mab-250-hG
1, tras-mG2a, andFigure reporter
3. The ADCC
assay by H2a2 Mab-250-mG
2a , H2 Mab-250-hG1 , tras-mG2a , andtrastuzumab in thepresence ofinBT-474
and SK-BR-3
cells.
HER2-positive
breast cancer cells
trastuzumab
the presenceof BT-474
andTarget
SK-BR-3
cells. Target HER2-positive
breast cancer cells
2Mab-250-mG2a,such as BT-474 (A)orSK-BR-3(B)
wereculturedina
96-well
white
solid
plate.
H
such as BT-474 (A) or SK-BR-3 (B) were cultured in a 96-well white solid plate. H2 Mab-250-mG2a ,H2Mab-250-hG1, tras-mG
2a, and trastuzumab were serially diluted and added to the target cells (n =H2 Mab-250-hG
1 , tras-mG2a , and trastuzumab were serially diluted and added to the target cells
3). The engineered Jurkat cells were then added and co-cultured with antibody-treated target cells.
(n = 3). The engineered Jurkat cells were then added and co-cultured with antibody-treated target
Luminescence using the Bio-Glo Luciferase Assay System was measured using a GloMax luminomcells. Luminescence using the Bio-Glo Luciferase Assay System was measured using a GloMax
eter (Promega Corporation, Madison, WI, USA). Values are presented as the mean ± SD. The EC50
luminometer (Promega Corporation, Madison, WI, USA). Values are presented as the mean ± SD.
was calculated using GraphPad PRISM 6.
The EC50 was calculated using GraphPad PRISM 6.

2.3. Immunohistochemical Analysis by H2 Mab-250 and Trastuzumab in Breast Cancer Tissue
We previously showed that H2 Mab-250 could stain the HER2-positive breast cancer
tissue, but not normal tissues, including the heart, breast, stomach, lung, colon, kidney,
and esophagus in IHC [22]. We next compared the reactivity of H2 Mab-250 with tras-mG2ausing a formalin-fixed paraffin-embedded (FFPE) tissue of HER2-positive breast cancer. In
contrast to the binding affinity, H2 Mab-250 exhibited superior reactivity to the breast cancer
cells over tras-mG2a (Figure 4). H2 Mab-250 showed clear tumor staining even at 1/20
concentration of tras-mG2a (0.5 µg/mL) (Figure 4A). We also confirmed that H2 Mab-250
did not stain normal breast and heart tissues (Figure 4B).
2.4. Antitumor Activities by H2 Mab-250-mG2a and Tras-mG2a
We next investigated the in vivo antitumor effect using H2 Mab-250-mG2a and trasmG2a in the BT-474 and SK-BR-3 xenograft models. We injected H2 Mab-250-mG2a , trasmG2a , and a control mouse mAb (PMab-231) intraperitoneally on days 6, 13, and 20 after
BT-474 and SK-BR-3 inoculation. We measured the tumor volume on days 6, 13, 20, and27 following the inoculation. The H2 Mab-250-mG2a administration led to a significant
reduction in BT-474 and SK-BR-3 xenograft on days 20 (p < 0.01) and 27 (p < 0.01) comparedwith that of the control (Figure 5A,B). In contrast, the tras-mG2a administration also led
to a significant reduction in BT-474 and SK-BR-3 xenograft on days 20 (p < 0.01) and 27(p < 0.01) compared with that of the control (Figure 5A,B). The H2 Mab-250-mG2a andInt. J. Mol. Sci. 2024, 25, 1941

We previously showed that H2Mab-250 could stain the HER2-positive breast cancer
tissue, but not normal tissues, including the heart, breast, stomach, lung, colon, kidney,
and esophagus in IHC [22]. We next compared the reactivity of H2Mab-250 with tras-mG2a
7 of 16
using a formalin-fixed paraﬃn-embedded (FFPE) tissue of HER2-positive breast cancer.
In contrast to the binding aﬃnity, H2Mab-250 exhibited superior reactivity to the breast
cancer cells over tras-mG2a (Figure 4). H2Mab-250 showed clear tumor staining even at
tras-mG2a administration resulted in 58% and 56% reductions (BT-474) and 53% and 50%
1/20
concentrationof oftras-mG(0.5 µg/mL)(Figure4A).
We
confirmed
H2Mabreductions(SK-BR-3)
tumor2avolume
comparedwiththat
ofalsothe control
mAbthat(PMab-231)
250
did
not
stain
normalbreastandhearttissues(Figure4B).
on day 27, respectively.

Figure 4.
4. Immunohistochemical
Immunohistochemical analysis
breastcancertissuesection2a2ainina a
FigureanalysisofofHH2 Mab-250
2Mab-250andandtras-mG
tras-mG
breastcancertissuesecandnormaltissues.
(A)
HER2-positive
breastcancertissuesections
weretreatedwithH
Mab-250
tion and normal tissues. (A) HER2-positive breast cancer tissue sections were treated with2 H2Mab250
1, 0.5andµg/mL)
0.5 µg/mL)or tras-mG(10 µg/mL).
(B) Sectionsof HER2-positive
breast cancer,(10, (10,
1, andor tras-mG(B) Sectionsof HER2-positive
breast cancer,
normal2a (102aµg/mL).
normalbreast,andwerehearttreated
were treated
H2Mab-250 (0.1 µg/mL). The sections were then treatedbreast, andheart
with Hwith
2 Mab-250 (0.1 µg/mL). The sections were then treated with
with
the Envision+kit (Agilent
Technologies,
Santa
Clara,
USA).
chromogenic
reaction
the Envision+kit (Agilent
Technologies,
Inc., Inc.,
Santa
Clara,CA, CA,USA).
The Thechromogenic
reactionwas
was
performed
using
DAB,
and
the
sections
were
counterstainedwithhematoxylin.
Scale
bar
100
performed using DAB, and the sections were counterstained with hematoxylin. Scale bar = 100= µm.
µm.

The BT-474 tumors from the H2 Mab-250-mG2a -treated and tras-mG2a -treated mice
2.4.
Antitumor
Activities lessby Hthan
2Mab-250-mG2a and Tras-mG2aweighedsignificantly
those from the control mouse mAb-treated mice (71%and We67%next
reduction,
respectively;
p < antitumor
0.01, Figure
5C,E).
TheHSK-BR-3
tumorsfromtrasthe
2Mab-250-mG
2a andinvestigated
the in vivo
eﬀect
using
H2 Mab-250-mG-treatedandtras-mG-treatedmice
weighedsignificantly
less
than
those
mG
2a in the BT-474
and
SK-BR-3
xenograftmodels.
We
injectedH2
Mab-250-mG
2a,
tras2a2afromcontrolmouse
mAb-treatedmice (53%intraperitoneallyand 46% reduction,
respectively;
0.01,
mG
2a, mouse
and a control
mAb (PMab-231)
on days6, 13, and p20<after
Figure 5D,F).
There wasno significant
difference
between
H2 Mab-250-mG
and
BT-474and SK-BR-3
inoculation.
We measured
thetumor volume
on days 2a6,-treated13, 20, andtras-mG
tumors. The H2Mab-250-mG2a administration led to a significant re27
following
the inoculation.
2a -treated
Theinbodyweightloss wasxenograftslightly observedon(pdays
6 andMab-250-mG
ductionBT-474and SK-BR-3
on days 20< 0.01)and1327in(pH<20.01)
compared
2a treated BT-474 xenograft-bearing mice (Figure 5G), but there was no significant differencein SK-BR-3 xenograft-bearing mice (Figure 5H).

Int. J. Mol. Sci. 2024, 25, 1941

mouse control mAb-treated mice (53% and 46% reduction, respectively; p < 0.01, Figure
5D,F). There was no significant diﬀerence between H2Mab-250-mG2a-treated and trasmG2a-treated tumors.
The body weight loss was slightly observed on days 6 and 13 in H2Mab-250-mG2a8 of 16
treated BT-474 xenograft-bearing mice (Figure 5G), but there was no significant diﬀerencein SK-BR-3 xenograft-bearing mice (Figure 5H).

Figure 5.
of of
H2Mab-250-mG
2a andtras-mG
2a against
BT-474and SK-BR-3
xeno5. Antitumor
Antitumoractivity
activity
H2 Mab-250-mG
tras-mG
BT-474and SK-BR-3
2a and2a against
grafts.
(A,B)
BT-474(A)
and
SK-BR-3
(B)
cells
were
injected
into
BALB/c
nude
mice
(day
0).
On
xenografts. (A,B) BT-474 (A) and SK-BR-3 (B) cells were injected into BALB/c nude mice (day 0).dayOn
day 6, 100 µg of tras-mG2a (n = 8), H2 Mab-250-mG2a (n = 8), or a control mAb (PMab-231) (n = 8)
were injected into mice. On days 13 and 20, additional antibodies were injected. On days 6, 13, 20, and27, the tumor volume was measured. Values are presented as the mean ± SEM. ** p < 0.01 (two-way
ANOVA and Tukey’s multiple comparisons test). (C,D) The tumor weight of BT-474 (C) and SK-BR-3(D) xenograft tumors on day 27. Values are presented as the mean ± SEM. ** p < 0.01 (ANOVA
and Tukey’s multiple comparisons test). (E,F) The BT-474 (E) and SK-BR-3 (F) xenograft tumors on
day 27 (scale bar, 1 cm). (G,H) The body weight of BT-474 (G) and SK-BR-3 (H) xenograft-bearing
mice treated with control mAb (PMab-231), tras-mG2a , and H2 Mab-250-mG2a . * p < 0.05 (two-way
ANOVA and Tukey’s multiple comparisons test).

2.5. Antitumor Activities by H2 Mab-250-hG1 and Trastuzumab
We next evaluated the antitumor activity of H2 Mab-250-hG1 and trastuzumab in the
BT-474 and SK-BR-3 xenograft models. We injected H2 Mab-250-hG1 and trastuzumab,
as well as control human IgG, intraperitoneally on days 7, 14, and 21 after BT-474 andSK-BR-3 inoculation. Furthermore, human NK cells were injected around the tumors on
the same days of the mAb injection. We measured the tumor volume on days 7, 14, 21,
and 28 following the inoculation. The H2 Mab-250-hG1 administration led to a significant

Int. J. Mol. Sci. 2024, 25, 1941

9 of 16

reduction in BT-474 and SK-BR-3 xenograft on days 14 (p < 0.05 (BT-474), p < 0.01 (SK-BR-3)),
21 (p < 0.01), and 28 (p < 0.01) compared with that of the control (Figure 6A,B). In contrast,
the trastuzumab administration also led to a significant reduction in BT-474 and SK-BR-3
xenograft on days 14 (p < 0.01 (BT-474), p < 0.05 (SK-BR-3)), 21 (p < 0.01), and 28 (p < 0.01)
compared with that of the control (Figure 6A,B). The H2 Mab-250-hG1 and trastuzumab
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
of 17
administration resulted in 62% and 62% reductions (BT-474) and 53% and 53%10reductions(SK-BR-3) of tumor volume compared with that of the control on day 28, respectively.

Figureactivityof Hof2Mab-250-hG
1 and trastuzumab
against against
BT-474 andSK-BR-3
Figure6.6.Antitumor
Antitumor
activity
H2 Mab-250-hG
BT-474and xenSK-BR-3
1 and trastuzumab
ografts.
(A,B)(A,B)
BT-474(A) and
SK-BR-3(B) cells(B)wereinjected
BALB/cnude
mice (day0).miceOn dayxenografts.
BT-474(A)
and SK-BR-3
cells
wereinto
injected
into
BALB/cnude(day 0).
7, 100 µg of H2Mab-250-hG1 (n = 8), trastuzumab (n = 8), or a control human IgG (hIgG) (n = 8) were
On day 7, 100 µg of H2 Mab-250-hG1 (n = 8), trastuzumab (n = 8), or a control human IgG (hIgG)
injected into mice. On days 14 and 21, additional antibodies were injected. Human NK cells were(n = 8) were
injected
into mice.
days
14ofandadditional antibodies
Human
injectedaroundthe tumors
on theOnsame
days
Ab 21,
administration.
On days 7,were14, 21,injected.
and 28, theNK cells
werewasinjectedaroundthe tumors
on theassamedays ±ofSEM.
Ab administration.
On daystumor
volumemeasured.
Valuesare presented
the mean* p < 0.05, ** p < 0.01
(two- 7, 14,21, and28, thetumor
volumewascomparisonsmeasured. Values
are presented
the mean± SEM.
p < 0.05,
way
ANOVA
and
Tukey’smultipletest). (C,D)
The tumorasweightof BT-474(C)*and
SK-BR-3(D)
xenograftANOVA
tumors andon day28. Values
are comparisonspresented as themean± SEM.
** p <weight0.01 of**p < 0.01(two-way
Tukey’smultipletest).
(C,D)
The tumor(ANOVAand
Tukey’smultiplecomparisonstest).
BT-474 are(E) and
SK-BR-3
BT-474 (C)andSK-BR-3(D) xenograft
tumorson(E,F)
day The28. Values
presented
as (F)
thexenograft
mean ± SEM.
tumors on day 28 (scale bar, 1 cm). (G,H) The body weight of BT-474 (G) and SK-BR-3 (H) xenograft** p < 0.01 (ANOVA and Tukey’s multiple comparisons test). (E,F) The BT-474 (E) and SK-BR-3
bearing mice treated with trastuzumab, H2Mab-250-hG1, or a control hIgG.
(F) xenograft tumors on day 28 (scale bar, 1 cm). (G,H) The body weight of BT-474 (G) and SK-BR-3(H)Discussion
xenograft-bearing mice treated with trastuzumab, H2 Mab-250-hG1 , or a control hIgG.
3.

Most therapeutic mAbs including anti-HER2 mAbs possess adverse eﬀects, probably
due to the recognition of antigens in normal cells [29]. Therefore, tumor-selective or -specific mAbs would minimize the adverse eﬀects. We developed CasMabs against
podoplanin (LpMab-2 and LpMab-23 [30]), podocalyxin (PcMab-6 [31]), and HER2
(H2Mab-250 [22]) by evaluating the reactivity against cancer and normal cells in flow

Int. J. Mol. Sci. 2024, 25, 1941

10 of 16

The BT-474 tumors from the H2 Mab-250-hG1 - and trastuzumab-treated mice weighed
significantly less than those from the control human IgG-treated mice (59% and 57%
reduction, respectively; p < 0.01, Figure 6C,E). The SK-BR-3 tumors from the H2 Mab-250hG1 and trastuzumab-treated mice weighed significantly less than those from the control
human IgG-treated mice (48% and 48% reduction, respectively; p < 0.01, Figure 6D,F). There
was no significant difference between H2 Mab-250-hG1 - and trastuzumab-treated tumors.
The body weight loss was not observed in H2 Mab-250-hG1 - and trastuzumab-treated
BT-474 and SK-BR-3 xenograft-bearing mice (Figure 6G,H).
3. Discussion
Most therapeutic mAbs including anti-HER2 mAbs possess adverse effects, probably
due to the recognition of antigens in normal cells [29]. Therefore, tumor-selective or -specificmAbs would minimize the adverse effects. We developed CasMabs against podoplanin(LpMab-2 and LpMab-23 [30]), podocalyxin (PcMab-6 [31]), and HER2 (H2 Mab-250 [22]) by
evaluating the reactivity against cancer and normal cells in flow cytometry. We also showed
the in vivo antitumor effect of the recombinant mAbs (mouse IgG2a or human IgG1 types)
derived from the abovementioned mAbs [30–32]. In this study, we evaluated the antitumor effect of H2 Mab-250-mG2a and H2 Mab-250-hG1 , which exhibited a cancer specificity
compared to tras-mG2a and trastuzumab, respectively (Figure 1). Although H2 Mab-250
possesses ≈10-fold lower affinity (Figure 2) and 100~1000-fold lower effect for effector activation than trastuzumab (Figure 3), mouse IgG2a -type and human IgG1 -type H2 Mab-250
possess comparable antitumor effects with those of trastuzumab (Figures 5 and 6).
The benefit of low-affinity mAbs for therapeutic applications has been discussed. A
low-affinity anti-EGFR mAb (KD : 3.4 × 10−7 M) was efficiently taken up by cancer cells but
not normal cells, which resulted in sufficient efficacy against tumor cells, but low toxicity
against normal keratinocytes [33]. An anti-HER3 mAb, Ab562, possesses ≈10-fold lower
affinity (KD : 2~3 × 10−8 M) than patritumab. The ADC AMT-562 exhibited sufficient
antitumor effects with minimizing potential toxicity [34]. Since H2 Mab-250 exhibited noreactivity to normal epithelial cells in flow cytometry (Figure 1) and IHC [22], H2 Mab-250
or H2 Mab-250-ADC could exhibit antitumor efficacy with lower side effects.
H2 Mab-250-mG2a and H2 Mab-250-hG1 could trigger the ADCC activity to BT-474
and SK-BR-3 cells; however, the effects of H2 Mab-250-mG2a and H2 Mab-250-hG1 werealso lower than that of tras-mG2a and trastuzumab, respectively (Figure 3). In contrast,
H2 Mab-250 exhibited a superior reactivity to HER2-positive breast cancer tissue in immunohistochemistry (Figure 4). H2 Mab-250 also recognized the HER2-positive breast cancer
tissue in the absence of antigen retrieval (Figure S1). We previously identified the H2 Mab250 epitope as 613- IWKFP-617 in the HER2 domain IV. The epitope sequence is partially
included with the wider binding epitope of trastuzumab (residues 579–625) [35]. Furthermore, we identified Trp614 as a central amino acid in the recognition by H2 Mab-250 [22].
Since the similar in vivo antitumor efficacy of H2 Mab-250-mG2a and H2 Mab-250-hG1 wasshown compared with tras-mG2a and trastuzumab, respectively (Figures 5 and 6), the613- IWKFP-617 sequence may be highly accessible by H2 Mab-250 in vivo due to the unknown mechanism. Compared to the in vitro cell culture condition, in vivo tumor cells
received various stresses such as hypoxia [36], nutrient deprivation [37], and abnormal
redox state [38]. Further studies are required for the influence of those stresses on therecognition by H2 Mab-250 in cancer cells.
Chimeric antigen receptor (CAR)-T cell therapy against HER2 has been evaluated inclinical trials [15]. CAR-T cells against CD19 have improved outcomes for patients with
B-cell lymphoma. However, disease relapse commonly occurs in many patients [39,40].
Although different mechanisms of immune escape have been demonstrated [41], the tumor
cells from many relapsed patients did not exhibit the features of immune escape. Recently,
additional mechanisms, such as trogocytosis, have been proposed [42]. When CAR-T cells,
possessing the high-affinity anti-CD19 FMC63-based CAR, are co-cultured with CD19positive lymphoma cells, the CAR-T cells strip CD19 from lymphoma cells and incorporate

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it into their plasma membrane [42]. This is called “trogocytosis”, which results in theemergence of antigen-negative target cells. Furthermore, the CAR-T cells that acquired
CD19 by trogocytosis can be killed by the other CD19 CAR-T cells [42]. A promising
approach to limit CAR-T cell-mediated trogocytosis is the reduction of CAR affinity [43].
CD19-targeting CAR with ≈40-fold lower affinity (KD : 1.4 × 10−8 M) than the clinicallyapproved FMC63-based CAR (KD : 3.3 × 10−10 M) was developed [44]. The reduced
affinity CAR-T cells exhibited higher efficacy and persistence than FMC63-based CAR-T
cells in a mouse model [44], as well as robust antitumor efficacy and persistence in two
clinical trials [44,45]. These data show that it is possible to significantly limit trogocytosis
by reducing CAR affinity while maintaining antitumor activity as well as clinical efficacy.
The property of H2 Mab-250 could contribute to the development of HER2-targeting CAR-T
cells (now in a clinical phase I study in the US) by limiting trogocytosis and maintaining
cancer specificity.
4. Materials and Methods
4.1. Cell Culture
CHO-K1, BT-474, SK-BR-3, MDA-MB-468, MCF 10A, HBEC3-KT, hTERT-HME1, and
RPTEC/TERT1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human keratinocyte HaCaT was purchased from the Cell Lines Service
GmbH (Eppelheim, Germany). hTCEpi, hTEC/SVTERT24-B, and HCEC-1CT were purchased from EVERCYTE (Vienna, Austria).
CHO-K1 and CHO/HER2 were cultured in Roswell Park Memorial Institute (RPMI)1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and BT-474, SK-BR-3, MDA-MB-468, andHaCaT were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque,Inc.), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher
Scientific Inc., Waltham, MA, USA), 100 units/mL of penicillin, 100 µg/mL streptomycin,
and 0.25 µg/mL amphotericin B (Nacalai Tesque, Inc.). The mammary epithelial cell line
MCF 10A was cultured in Mammary Epithelial Cell Basal Medium BulletKitTM (Lonza,
Basel, Switzerland) supplemented with 100 ng/mL cholera toxin (Sigma-Aldrich Corp.,
St. Louis, MO, USA).
Immortalized normal epithelial cell lines were maintained as follows; HBEC3-KT,
Airway Epithelial Cell Basal Medium and Bronchial Epithelial Cell Growth Kit (ATCC);hTERT-HME1, Mammary Epithelial Cell Basal Medium BulletKitTM without GA-1000(Lonza); hTCEpi, KGMTM-2 BulletKitTM (Lonza); hTEC/SVTERT24-B, OptiPROTM SFM
and GlutaMAXTM -I (Thermo Fisher Scientific Inc.); RPTEC/TERT1, DMEM/F-12, andhTERT Immortalized RPTEC Growth Kit with supplement A and B (ATCC); and HCEC1CT, DMEM/M199 (4:1, Thermo Fisher Scientific Inc.), 2% Cosmic Calf Serum (Cytiva,
Marlborough, MA, USA), 20 ng/mL hEGF (Sigma-Aldrich Corp.), 10 µg/mL insulin (SigmaAldrich Corp.), 2 µg/mL apo-transferrin (Sigma-Aldrich Corp.), 5 nM sodium-selenite(Sigma-Aldrich Corp.), and 1 µg/mL hydrocortisone (Sigma-Aldrich Corp.).
All cell lines were cultured at 37 ◦ C in a humidified atmosphere with 5% CO2 and
95% air.
4.2. Production of Recombinant mAbs
To generate recombinant H2 Mab-250, the VH cDNAs and the CH cDNA of mouse
IgG1 were cloned into the pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan). The VL cDNAs and CL cDNA of the mouse kappa light chain were also
cloned into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation). The vectors
were transfected into ExpiCHO-S cells using the ExpiCHO Expression System (Thermo
Fisher Scientific, Inc.), and Ab-Capcher (ProteNova Co., Ltd., Kagawa, Japan) was used topurify the recombinant H2 Mab-250.
To generate mouse IgG2a -type H2 Mab-250 (H2 Mab-250-mG2a ), we cloned the VH
cDNA of H2 Mab-250 and CH of mouse IgG2a into the pCAG-Ble vector. The mouse kappalight chain vector of H2 Mab-250 was described above. To generate a mouse IgG2a type

Int. J. Mol. Sci. 2024, 25, 1941

12 of 16of trastuzumab (tras-mG2a ), the VH cDNA of trastuzumab and the CH cDNA of mouse
IgG2a were cloned into the pCAG-Neo vector, and the VL cDNA of trastuzumab and the
CL cDNA of mouse kappa light chain were cloned into the pCAG-Ble vector.
To generate the recombinant PMab-231, we cloned heavy and light chains of PMab231 [46] into the pCAG-Neo and pCAG-Ble vectors, respectively. To generate a mousehuman chimeric mAb (H2 Mab-250-hG1 ), VH of H2 Mab-250 and CH of human IgG1 werecloned into the pCAG-Ble vector. The VL of H2 Mab-250 and CL of the human kappalight chain were cloned into the pCAG-Neo vector. The vectors were transfected into
BINDS-09 (fucosyltransferase 8-knockout ExpiCHO-S) cells using the ExpiCHO Expression
System (Thermo Fisher Scientific, Inc.). H2 Mab-250-mG2a , tras-mG2a , H2 Mab-250-hG1 ,trastuzumab, and PMab-231 were purified using Ab-Capcher.
Normal human IgG was purchased from Sigma-Aldrich Corp.
4.3. Flow Cytometry
Cells were collected using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid
(EDTA; Nacalai Tesque, Inc.). The cells (1 × 105 cells/sample) were treated with primary mAbs (10 µg/mL) or blocking buffer (control; 0.1% bovine serum albumin (BSA) in
phosphate-buffered saline (PBS)) for 30 min at 4 ◦ C. Next, the cells were treated with Alexa
Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Danvers, MA,
USA) or FITC-conjugated anti-human IgG (1:1000; Sigma-Aldrich Corp.) for 30 min at 4 ◦ C.
The fluorescence data were collected using an SA3800 Cell Analyzer (Sony Corp., Tokyo,
Japan), and the data were analyzed using FlowJo v10.8.1 (BD Biosciences, Franklin Lakes,
NJ, USA).
4.4. ELISAHER2ec [22] was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo
Fisher Scientific Inc.) at a concentration of 1 µg/mL for 30 min at 37 ◦ C. After washingwith PBS containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.), wells were blockedwith 1% (w/v) BSA-containing PBST for 30 min at 37 ◦ C. The serially diluted H2 Mab-250,
H2 Mab-250-mG2a , H2 Mab-250-hG1 , tras-mG2a , and trastuzumab (0.0006–10 µg/mL) wereadded to each well, followed by peroxidase-conjugated anti-mouse immunoglobulins(1:3000 diluted; Agilent Technologies Inc., Santa Clara, CA, USA) or peroxidase-conjugatedanti-human immunoglobulins (1:3000 diluted; Sigma-Aldrich Corp.). Enzymatic reactions
were conducted using an ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.), followedby the measurement of the optical density at 655 nm, using an iMark microplate reader(Bio-Rad Laboratories, Inc., Berkeley, CA, USA). The binding isotherms were fitted into thebuilt-in, one-site binding model in GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla,
CA, USA) to calculate the dissociation constant (KD ).
4.5. ADCC Reporter Bioassay
The ADCC reporter bioassay was performed using an ADCC Reporter Bioassay kit(Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions.
Target cells (BT-474 and SK-BR-3, 12,500 cells per well) were cultured in a 96-well whitesolid plate. H2 Mab-250-mG2a , H2 Mab-250-hG1 , tras-mG2a , and trastuzumab were seriallydiluted and added to the target cells. Jurkat cells stably expressing the human FcγRIIIa
receptor and a NFAT-response element driving firefly luciferase were used as effector cells.
The engineered Jurkat cells (75,000 cells in 25 µL) were then added and co-cultured with
antibody-treated target cells at 37 ◦ C for 6 h. Luminescence using the Bio-Glo Luciferase
Assay System was measured using a GloMax luminometer (Promega Corporation).
4.6. Immunohistochemical Analysis
FFPE tissue of HER2-positive breast cancer was obtained from the Sendai Medical
Center [20]. Informed consent for sample procurement and subsequent data analyses
was obtained from the patient or the patient’s guardian at the Sendai Medical Center. A

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normal tissue array (B901064) was purchased from BioChain Institute Inc. (Eureka Drive,
Newark, CA, USA). The antigen retrieval was performed by autoclave in citrate buffer(pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min. The blocking was performed
using SuperBlock T20 (Thermo Fisher Scientific Inc.). The sections were incubated with
H2 Mab-250 (10, 1, 0.5 or 0.1 µg/mL) and tras-mG2a (10 µg/mL) and then treated with
the EnVision+ Kit for mouse (Agilent Technologies, Inc., Santa Clara, CA, USA). The
chromogenic reaction was performed using 3,3′ -diaminobenzidine tetrahydrochloride(DAB; Agilent Technologies, Inc.). Counterstaining was performed using hematoxylin(FUJIFILM Wako Pure Chemical Corporation), and Leica DMD108 (Leica Microsystems
GmbH, Wetzlar, Germany) was used to obtain images and examine the sections.
4.7. Antitumor Activities of H2 Mab-250-mG2a , Tras-mG2a , H2 Mab-250-hG1 , and Trastuzumab in
Breast Cancer Xenograft Models
To examine the antitumor effect of H2 Mab-250-mG2a tras-mG2a , H2 Mab-250-hG1 ,and trastuzumab, animal experiments were approved by the Institutional Committee
for Experiments of the Institute of Microbial Chemistry (approval no. 2023-060 or 2023066). During the experimental period, we monitored mice maintained in a pathogen-freeenvironment on an 11 h light/13 h dark cycle with food and water supplied ad libitum.
Mice were monitored for health and weight every one or five days. We identified bodyweight loss exceeding 25% and maximum tumor size exceeding 3000 mm3 as humane
endpoints and terminated the experiments.
BT-474 and SK-BR-3 cells were suspended in 0.3 mL of 1.33 × 108 cells/mL using
DMEM and mixed with 0.5 mL of BD Matrigel Matrix Growth Factor Reduced (BD Biosciences, San Jose, CA, USA). Then, BALB/c nude mice (Jackson Laboratory Japan, Kanagawa, Japan) were injected subcutaneously in the left flank with 100 µL of the suspension(5 × 106 cells). On day 6 post-inoculation, 100 µg of H2 Mab-250-mG2a (n = 8), tras-mG2a(n = 8), or control (PMab-231; n = 8) in 100 µL PBS was intraperitoneally injected. On
days 13 and 20, additional antibody injections were performed. The tumor diameter wasmeasured on days 6, 13, 20, and 27 after the inoculation of cells.
For the evaluation of H2 Mab-250-hG1 and trastuzumab, we injected the mice with
100 µg of H2 Mab-250-hG1 (n = 8), trastuzumab (n = 8), and control human IgG (n = 8)
in 100 µL of PBS through intraperitoneal injection on day 7 post-inoculation. Additional
antibodies were injected on days 14 and 21. Furthermore, human NK cells (8.0 × 105 cells,
Takara Bio, Inc., Shiga, Japan) were injected around the tumors on days 7, 14, and 21. The
tumor diameter was measured on days 7, 14, 21, and 28 after inoculation with cells.
The tumor volume was calculated using the following formula: volume = W2 × L/2,
where W is the short diameter and L is the long diameter. All mice were euthanized bycervical dislocation.
5. Conclusions
A cancer-specific anti-HER2 mAb, H2 Mab-250, exhibited antitumor efficacy in vivo.
In the future, H2 Mab-250 could contribute to the development of CAR-T or ADCs without
adverse effects for breast cancer therapy.
Supplementary Materials: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms25031941/s1.
Author Contributions: M.K.K., H.S., T.O. and T.N. performed the experiments. M.K.K. and Y.K.
designed the experiments. H.S., T.T. and Y.K. analyzed the data. H.S. 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 JP23ama121008 (to Y.K.), JP23am0401013 (to Y.K.), JP23bm1123027(to Y.K.), JP23ck0106730 (to Y.K.), JP18am0301010 (to Y.K.), and JP21am0101078 (to Y.K.).

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Institutional Review Board Statement: The animal experiment to examine the antitumor effect wasapproved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry(approval nos. 2023-060 and 2023-066).
Informed Consent Statement: Informed consent for sample procurement and subsequent data
analyses was obtained from the patient or the patient’s guardian at the Sendai Medical Center.
Data Availability Statement: The data presented in this study are available in the article.
Conflicts of Interest: The authors declare no conflicts of interest.

References
1.
2.

3.
4.
5.
6.

7.

8.
9.

10.
11.
12.

13.
14.
15.
16.

17.
18.
19.
20.

Slamon, D.J.; Clark, G.M.; Wong, S.G.; Levin, W.J.; Ullrich, A.; McGuire, W.L. Human breast cancer: Correlation of relapse andsurvival with amplification of the HER-2/neu oncogene. Science 1987, 235, 177–182. [CrossRef]
Van Cutsem, E.; Bang, Y.J.; Feng-Yi, F.; Xu, J.M.; Lee, K.W.; Jiao, S.C.; Chong, J.L.; López-Sanchez, R.I.; Price, T.; Gladkov, O.;et al. HER2 screening data from ToGA: Targeting HER2 in gastric and gastroesophageal junction cancer. Gastric Cancer 2015, 18,
476–484. [CrossRef]
Yarden, Y.; Sliwkowski, M.X. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2001, 2, 127–137. [CrossRef]
Tsao, L.C.; Force, J.; Hartman, Z.C. Mechanisms of Therapeutic Antitumor Monoclonal Antibodies. Cancer Res. 2021, 81, 4641–4651.
[CrossRef]
Gschwind, A.; Fischer, O.M.; Ullrich, A. The discovery of receptor tyrosine kinases: Targets for cancer therapy. Nat. Rev. Cancer
2004, 4, 361–370. [CrossRef]
Cardoso, F.; Paluch-Shimon, S.; Senkus, E.; Curigliano, G.; Aapro, M.S.; André, F.; Barrios, C.H.; Bergh, J.; Bhattacharyya, G.S.;
Biganzoli, L.; et al. 5th ESO-ESMO international consensus guidelines for advanced breast cancer (ABC 5). Ann. Oncol. 2020, 31,
1623–1649. [CrossRef] [PubMed]
Slamon, D.J.; Leyland-Jones, B.; Shak, S.; Fuchs, H.; Paton, V.; Bajamonde, A.; Fleming, T.; Eiermann, W.; Wolter, J.; Pegram, M.;
et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.
N. Engl. J. Med. 2001, 344, 783–792. [CrossRef] [PubMed]
Essadi, I.; Benbrahim, Z.; Kaakoua, M.; Reverdy, T.; Corbaux, P.; Freyer, G. HER2-Positive Metastatic Breast Cancer: Available
Treatments and Current Developments. Cancers 2023, 15, 1738. [CrossRef] [PubMed]
Bang, Y.J.; Van Cutsem, E.; Feyereislova, A.; Chung, H.C.; Shen, L.; Sawaki, A.; Lordick, F.; Ohtsu, A.; Omuro, Y.; Satoh, T.; et al.
Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastricor gastro-oesophageal junction cancer (ToGA): A phase 3, open-label, randomised controlled trial. Lancet 2010, 376, 687–697.
[CrossRef] [PubMed]
Mark, C.; Lee, J.S.; Cui, X.; Yuan, Y. Antibody-Drug Conjugates in Breast Cancer: Current Status and Future Directions. Int. J. Mol.
Sci. 2023, 24, 13726. [CrossRef] [PubMed]
Modi, S.; Saura, C.; Yamashita, T.; Park, Y.H.; Kim, S.B.; Tamura, K.; Andre, F.; Iwata, H.; Ito, Y.; Tsurutani, J.; et al. Trastuzumab
Deruxtecan in Previously Treated HER2-Positive Breast Cancer. N. Engl. J. Med. 2020, 382, 610–621. [CrossRef] [PubMed]
Shitara, K.; Bang, Y.J.; Iwasa, S.; Sugimoto, N.; Ryu, M.H.; Sakai, D.; Chung, H.C.; Kawakami, H.; Yabusaki, H.; Lee, J.; et al.
Trastuzumab Deruxtecan in Previously Treated HER2-Positive Gastric Cancer. N. Engl. J. Med. 2020, 382, 2419–2430. [CrossRef][PubMed]Li, B.T.; Smit, E.F.; Goto, Y.; Nakagawa, K.; Udagawa, H.; Mazières, J.; Nagasaka, M.; Bazhenova, L.; Saltos, A.N.; Felip, E.; et al.
Trastuzumab Deruxtecan in HER2-Mutant Non-Small-Cell Lung Cancer. N. Engl. J. Med. 2022, 386, 241–251. [CrossRef]
Modi, S.; Jacot, W.; Yamashita, T.; Sohn, J.; Vidal, M.; Tokunaga, E.; Tsurutani, J.; Ueno, N.T.; Prat, A.; Chae, Y.S.; et al. Trastuzumab
Deruxtecan in Previously Treated HER2-Low Advanced Breast Cancer. N. Engl. J. Med. 2022, 387, 9–20. [CrossRef]
Mercogliano, M.F.; Bruni, S.; Mauro, F.L.; Schillaci, R. Emerging Targeted Therapies for HER2-Positive Breast Cancer. Cancers
2023, 15, 1978. [CrossRef]
Soares, L.R.; Vilbert, M.; Rosa, V.D.L.; Oliveira, J.L.; Deus, M.M.; Freitas-Junior, R. Incidence of interstitial lung disease andcardiotoxicity with trastuzumab deruxtecan in breast cancer patients: A systematic review and single-arm meta-analysis. ESMOOpen 2023, 8, 101613. [CrossRef]
Copeland-Halperin, R.S.; Liu, J.E.; Yu, A.F. Cardiotoxicity of HER2-targeted therapies. Curr. Opin. Cardiol. 2019, 34, 451–458.
[CrossRef] [PubMed]
Lee, K.F.; Simon, H.; Chen, H.; Bates, B.; Hung, M.C.; Hauser, C. Requirement for neuregulin receptor erbB2 in neural and cardiac
development. Nature 1995, 378, 394–398. [CrossRef]
Crone, S.A.; Zhao, Y.Y.; Fan, L.; Gu, Y.; Minamisawa, S.; Liu, Y.; Peterson, K.L.; Chen, J.; Kahn, R.; Condorelli, G.; et al. ErbB2 isessential in the prevention of dilated cardiomyopathy. Nat. Med. 2002, 8, 459–465. [CrossRef]
Itai, S.; Fujii, Y.; Kaneko, M.K.; Yamada, S.; Nakamura, T.; Yanaka, M.; Saidoh, N.; Chang, Y.W.; Handa, S.; Takahashi, M.; et al.
H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer. Monoclon. Antib. Immunodiagn.
Immunother. 2017, 36, 143–148. [CrossRef]

Int. J. Mol. Sci. 2024, 25, 1941

21.
22.
23.
24.
25.

26.

27.

28.

29.
30.
31.
32.

33.

34.

35.
36.
37.
38.
39.

40.

41.
42.

43.

44.

15 of 16

Takei, J.; Kaneko, M.K.; Ohishi, T.; Kawada, M.; Harada, H.; Kato, Y. H(2)Mab-19, an anti-human epidermal growth factor receptor
2 monoclonal antibody exerts antitumor activity in mouse oral cancer xenografts. Exp. Ther. Med. 2020, 20, 846–853. [CrossRef]
Kaneko, M.K.; Suzuki, H.; Kato, Y. Establishment of a Novel Cancer-specific Anti-HER2 Monoclonal Antibody H2Mab250/H2CasMab-2 for breast cancers. Preprint 2023, 2023090906. [CrossRef]
Galvez-Cancino, F.; Simpson, A.P.; Costoya, C.; Matos, I.; Qian, D.; Peggs, K.S.; Litchfield, K.; Quezada, S.A. Fcγ receptors andimmunomodulatory antibodies in cancer. Nat. Rev. Cancer 2024, 24, 51–71. [CrossRef] [PubMed]
Bruhns, P.; Jönsson, F. Mouse and human FcR effector functions. Immunol. Rev. 2015, 268, 25–51. [CrossRef] [PubMed]
Shinkawa, T.; Nakamura, K.; Yamane, N.; Shoji-Hosaka, E.; Kanda, Y.; Sakurada, M.; Uchida, K.; Anazawa, H.; Satoh, M.;
Yamasaki, M.; et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1
complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J. Biol. Chem. 2003,
278, 3466–3473. [CrossRef]
Niwa, R.; Shoji-Hosaka, E.; Sakurada, M.; Shinkawa, T.; Uchida, K.; Nakamura, K.; Matsushima, K.; Ueda, R.; Hanai, N.; Shitara,
K. Defucosylated chimeric anti-CC chemokine receptor 4 IgG1 with enhanced antibody-dependent cellular cytotoxicity shows
potent therapeutic activity to T-cell leukemia and lymphoma. Cancer Res. 2004, 64, 2127–2133. [CrossRef] [PubMed]
Yamane-Ohnuki, N.; Kinoshita, S.; Inoue-Urakubo, M.; Kusunoki, M.; Iida, S.; Nakano, R.; Wakitani, M.; Niwa, R.; Sakurada, M.;
Uchida, K.; et al. Establishment of FUT8 knockout Chinese hamster ovary cells: An ideal host cell line for producing completely
defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol. Bioeng. 2004, 87, 614–622. [CrossRef][PubMed]Garvin, D.; Stecha, P.; Gilden, J.; Wang, J.; Grailer, J.; Hartnett, J.; Fan, F.; Cong, M.; Cheng, Z.J. Determining ADCC Activityof Antibody-Based Therapeutic Molecules using Two Bioluminescent Reporter-Based Bioassays. Curr. Protoc. 2021, 1, e296.
[CrossRef] [PubMed]
Gogia, P.; Ashraf, H.; Bhasin, S.; Xu, Y. Antibody-Drug Conjugates: A Review of Approved Drugs and Their Clinical Level of
Evidence. Cancers 2023, 15, 3886. [CrossRef]
Suzuki, H.; Kaneko, M.K.; Kato, Y. Roles of Podoplanin in Malignant Progression of Tumor. Cells 2022, 11, 575. [CrossRef]
Suzuki, H.; Ohishi, T.; Tanaka, T.; Kaneko, M.K.; Kato, Y. A Cancer-Specific Monoclonal Antibody against Podocalyxin Exerted
Antitumor Activities in Pancreatic Cancer Xenografts. Int. J. Mol. Sci. 2023, 25, 161. [CrossRef] [PubMed]
Suzuki, H.; Ohishi, T.; Kaneko, M.K.; Kato, Y. A Humanized and Defucosylated Antibody against Podoplanin (humLpMab-23-f)
Exerts Antitumor Activities in Human Lung Cancer and Glioblastoma Xenograft Models. Cancers 2023, 15, 5080. [CrossRef][PubMed]Wong, O.K.; Tran, T.T.; Ho, W.H.; Casas, M.G.; Au, M.; Bateman, M.; Lindquist, K.C.; Rajpal, A.; Shelton, D.L.; Strop, P.;
et al. RN765C, a low affinity EGFR antibody drug conjugate with potent anti-tumor activity in preclinical solid tumor models.
Oncotarget 2018, 9, 33446–33458. [CrossRef] [PubMed]
Weng, W.; Meng, T.; Pu, J.; Ma, L.; Shen, Y.; Wang, Z.; Pan, R.; Wang, M.; Chen, C.; Wang, L.; et al. AMT-562, a Novel HER3targeting Antibody-Drug Conjugate, Demonstrates a Potential to Broaden Therapeutic Opportunities for HER3-expressing
Tumors. Mol. Cancer Ther. 2023, 22, 1013–1027. [CrossRef] [PubMed]
Diwanji, D.; Trenker, R.; Thaker, T.M.; Wang, F.; Agard, D.A.; Verba, K.A.; Jura, N. Structures of the HER2-HER3-NRG1β complex
reveal a dynamic dimer interface. Nature 2021, 600, 339–343. [CrossRef] [PubMed]
Chen, Z.; Han, F.; Du, Y.; Shi, H.; Zhou, W. Hypoxic microenvironment in cancer: Molecular mechanisms and therapeuticinterventions. Signal Transduct. Target. Ther. 2023, 8, 70. [CrossRef] [PubMed]
Lobel, G.P.; Jiang, Y.; Simon, M.C. Tumor microenvironmental nutrients, cellular responses, and cancer. Cell Chem. Biol. 2023, 30,
1015–1032. [CrossRef] [PubMed]
Forman, H.J.; Zhang, H. Targeting oxidative stress in disease: Promise and limitations of antioxidant therapy. Nat. Rev. Drug
Discov. 2021, 20, 689–709. [CrossRef]
Wudhikarn, K.; Flynn, J.R.; Rivière, I.; Gönen, M.; Wang, X.; Senechal, B.; Curran, K.J.; Roshal, M.; Maslak, P.G.; Geyer, M.B.; et al.
Interventions and outcomes of adult patients with B-ALL progressing after CD19 chimeric antigen receptor T-cell therapy. Blood
2021, 138, 531–543. [CrossRef]
Pan, J.; Tan, Y.; Deng, B.; Tong, C.; Hua, L.; Ling, Z.; Song, W.; Xu, J.; Duan, J.; Wang, Z.; et al. Frequent occurrence of
CD19-negative relapse after CD19 CAR T and consolidation therapy in 14 TP53-mutated r/r B-ALL children. Leukemia 2020, 34,
3382–3387. [CrossRef]
Shah, N.N.; Fry, T.J. Mechanisms of resistance to CAR T cell therapy. Nat. Rev. Clin. Oncol. 2019, 16, 372–385. [CrossRef] [PubMed]
Hamieh, M.; Dobrin, A.; Cabriolu, A.; van der Stegen, S.J.C.; Giavridis, T.; Mansilla-Soto, J.; Eyquem, J.; Zhao, Z.; Whitlock, B.M.;
Miele, M.M.; et al. CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape. Nature 2019, 568, 112–116.
[CrossRef] [PubMed]
Olson, M.L.; Mause, E.R.V.; Radhakrishnan, S.V.; Brody, J.D.; Rapoport, A.P.; Welm, A.L.; Atanackovic, D.; Luetkens, T. Lowaffinity CAR T cells exhibit reduced trogocytosis, preventing rapid antigen loss, and increasing CAR T cell expansion. Leukemia
2022, 36, 1943–1946. [CrossRef] [PubMed]
Ghorashian, S.; Kramer, A.M.; Onuoha, S.; Wright, G.; Bartram, J.; Richardson, R.; Albon, S.J.; Casanovas-Company, J.; Castro,F.; Popova, B.; et al. Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a
low-affinity CD19 CAR. Nat. Med. 2019, 25, 1408–1414. [CrossRef]

Int. J. Mol. Sci. 2024, 25, 1941

45.

46.

16 of 16

Roddie, C.; Dias, J.; O’Reilly, M.A.; Abbasian, M.; Cadinanos-Garai, A.; Vispute, K.; Bosshard-Carter, L.; Mitsikakou, M.; Mehra,
V.; Roddy, H.; et al. Durable Responses and Low Toxicity After Fast Off-Rate CD19 Chimeric Antigen Receptor-T Therapy in
Adults With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia. J. Clin. Oncol. 2021, 39, 3352–3363. [CrossRef]
Furusawa, Y.; Kaneko, M.K.; Nakamura, T.; Itai, S.; Fukui, M.; Harada, H.; Yamada, S.; Kato, Y. Establishment of a Monoclonal
Antibody PMab-231 for Tiger Podoplanin. Monoclon. Antib. Immunodiagn. Immunother. 2019, 38, 89–95. [CrossRef]

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