Article

Defucosylated Monoclonal Antibody (H2Mab-139-mG2a-f)
Exerted Antitumor Activities in Mouse Xenograft Models ofBreast Cancers against Human Epidermal Growth Factor
Receptor 2
Hiroyuki Suzuki 1,2,*,†, Tomokazu Ohishi 3,4,†, Ren Nanamiya 1, Manabu Kawada 4, Mika K. Kaneko 1,2
and Yukinari Kato 1,2,*
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan; ren.nanamiya.p5@dc.tohoku.ac.jp (R.N.);
k.mika@med.tohoku.ac.jp (M.K.K.)
2 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan
3 Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
18-24 Miyamoto, Numazu-shi 410-0301, Shizuoka, Japan; ohishit@bikaken.or.jp
4 Institute of Microbial Chemistry (BIKAKEN), Laboratory of Oncology, Microbial Chemistry Research Foundation, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan; kawadam@bikaken.or.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.)† These authors contributed equally to this work.
1

Citation: Suzuki, H.; Ohishi, T.;
Nanamiya, R.; Kawada, M.; Kaneko,
M.K.; Kato, Y. Defucosylated
Monoclonal Antibody (H2Mab-139mG2a-f) Exerted Antitumor Activities
in Mouse Xenograft Models ofBreast Cancers against Human
Epidermal Growth Factor Receptor
2. Curr. Issues Mol. Biol. 2023, 45,7734–7748. https://doi.org/10.3390/
cimb45100488
Academic Editor: Simona Gurzu
Received: 30 August 2023
Revised: 18 September 2023
Accepted: 21 September 2023

Abstract: The clinically approved human epidermal growth factor receptor 2 (HER2)-targeting
monoclonal antibodies (mAbs), trastuzumab, and pertuzumab, target domains IV and II, respectively. Trastuzumab is now the standard treatment for HER2-overexpressed breast and gastric cancers, and trastuzumab in combination with pertuzumab showed clinical benefit. However, there
still exist patients who do not respond to the therapy. Furthermore, HER2 mutants that cannot berecognized by pertuzumab were found in tumors. Therefore, novel anti-HER2 mAbs and modalities
have been desired. In our previous study, we developed a novel anti-HER2 domain I mAb, H2Mab139 (mouse IgG1, kappa). We herein produced a defucosylated mouse IgG2a type of mAb against
HER2 (H2Mab-139-mG2a-f) to enhance antibody-dependent cellular cytotoxicity (ADCC)-mediatedantitumor activity. H2Mab-139-mG2a-f exhibits a high binding affinity in flow cytometry with thedissociation constant (KD) determined to be 3.9 × 10−9 M and 7.7 × 10−9 M against HER2-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/HER2) and HER2-positive BT-474 cells, respectively. Moreover, we showed that H2Mab-139-mG2a-f exerted ADCC and complement-dependent
cytotoxicity against CHO/HER2 and BT-474 in vitro and exhibited potent antitumor activities inmouse xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects
against HER2-positive human breast cancers and is useful as an antibody treatment for HER2-positive human cancers.

Published: 23 September 2023

Keywords: antibody-dependent cellular cytotoxicity; breast cancer; HER2; monoclonal antibody;
antitumor activities; mouse tumor model
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

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license(https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Human epidermal growth factor receptor 2 (HER2) is included in the receptor tyrosine kinase family of human epidermal growth factor receptor (EGFR). The HER activation is controlled by EGF-family ligands under physiological conditions. The formationof multiple combinations of HER homo- and heterodimers is induced by ligand binding,Curr. Issues Mol. Biol. 2023, 45, 7734–7748. https://doi.org/10.3390/cimb45100488

www.mdpi.com/journal/cimb

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which triggers the activation of the cytoplasmic tyrosine kinase domain. The activation ofseveral downstream signaling pathways, such as the RAS/RAF/MAPK and PI3K/AKT
pathways [1], is induced by the autophosphorylation of specific tyrosine residues. HER2
does not have ligands and cannot form ligand-dependent homodimers, unlike EGFR,
HER3, and HER4. To activate the downstream signaling, HER2 must either form heterodimers with other HER members and their specific ligands or self-assemble into ligandindependent homodimers when overexpressed. HER2 possesses four extracellular domains (I–IV) [2]. Domain II is known to be essential for the heterodimer formation withother HER members, such as EGFR, HER3, and HER4 in the presence of their ligands,
such as EGF [3] and neuregulin 1 (NRG1, a HER3 ligand) [4].
HER2 is overexpressed in approximately 18% of breast cancers and is associated withhigher rates of recurrence, poor prognosis, and shorter overall survival [5]. HER2 overexpression is also observed in ~20% of gastric cancers [6]. A monoclonal antibody (mAb)against domain IV of HER2, trastuzumab, exhibited an anti-proliferating effect in vitro anda potent antitumor effect in vivo [7,8]. The addition of trastuzumab to chemotherapy improves objective response rates, progression-free survival, and overall survival in HER2positive breast cancer patients with metastasis [9]. Trastuzumab has become the standard
treatment for HER2-positive breast cancers [10] and HER2-positive gastric cancers [11].
For more than 20 years, trastuzumab has been the most effective therapy for HER2-positive breast cancer [12].
Clinically, the efficacy of trastuzumab involves immunologic engagement [8]. The Fc
domain of trastuzumab mediates engagement with Fcγ receptors (FcγRs) on various immune cells. The binding of trastuzumab to FcγR facilitates the phagocytosis of antibodybound tumor cells, a process known as antibody-dependent cellular phagocytosis(ADCP). The FcγR engagement also activates macrophages, dendritic cells, and neutrophils, that change adaptive immune responses by antigen presentation, cytokine production, and chemotaxis. Moreover, the FcγR engagement activates natural killer (NK) cells,
which can result in the lysis of the target tumor cells, termed antibody-dependent cellularcytotoxicity (ADCC) [13]. To improve the FcγRIIIA engagement and ADCC activity, margetuximab was developed by introducing several optimization mutations of trastuzumab[14]. Margetuximab was approved by the U.S. Food and Drug Administration (FDA) andshowed significant improvement in progression-free survival in heavily pretreated patients [15,16]. Moreover, the Fc domain of these mAbs can exert complement-dependent
cytotoxicity (CDC) [17,18].
Another clinically approved HER2-targeting mAb, pertuzumab, binds to the domain
II and prevents NRG1-induced heterodimerization with HER3 and intracellular signaling[19]. The heterodimerization is known to be an important mechanism for resistance totrastuzumab [19]. Therefore, pertuzumab is considered to possess a complementarymechanism to trastuzumab [20]. The first-line treatment combining trastuzumab, pertuzumab, and chemotherapy has been evaluated and demonstrated clinical benefits [21].
The double anti-HER2 blockade has been the standard therapy in the initial managementof metastatic HER2-positive breast cancer [9]. However, HER2 (S310F/Y) is the most frequent oncogenic missense mutation which cannot be recognized by pertuzumab [22].
In our previous studies, we established anti-HER2 mAbs including H2Mab-139 (IgG1,
kappa) [23] by the immunization of HER2 ectodomain. Those mAbs have been revealed
to recognize the domain I of HER2, and are available for flow cytometry, western blotting,and immunohistochemistry (IHC) [23]. Mouse IgG1 cannot bind to mouse FcγRIV whichis essential for the activation of effector cells such as macrophage. In contrast, mouse IgG2a
or IgG2b can bind to it with high affinity [24]. Furthermore, a core fucose deficiency on the
Fc N-glycan has been shown to enhance the binding to Fc receptors on effector cells [25].
Therefore, we have demonstrated that class-switched (from IgG1 to IgG2a) and defucosylated IgG2a mAbs exhibited a superior ability to activate effector cells and exerted potent
antitumor effects in several mouse xenograft models [26]. The defucosylated recombinantCurr. Issues Mol. Biol. 2023, 45

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mAbs can be produced using fucosyltransferase 8 (FUT8)-knockout (KO) Expi-CHO-S
cells [27].
In this study, we produced a defucosylated IgG2a type of anti-HER2 mAb (H2Mab139-mG2a-f) and evaluated the ability to induce ADCC/CDC in vitro or antitumor efficacy
in vivo against HER2-positive and HER2-negative breast cancer cells.
2. Materials and Methods
2.1. Cell Lines
LN229, BT-474, MDA-MB-468, HEK-293T, and Chinese hamster ovary (CHO)-K1 celllines were obtained from the American Type Culture Collection (ATCC, Manassas, VA,
USA). LN229/HER2 and CHO/HER2 were established as described previously [23]. The
HEK-293T/HER2-KO cell line (BINDS-23) was generated by transfecting a CRISPR/Cas9
plasmid that targets HER2 [Thermo Fisher Scientific, Inc. (Thermo), Waltham, MA, USA].
CHO-K1 and CHO/HER2 cell lines were cultured in RPMI-1640 medium [Nacalai Tesque,Inc. (Nacalai), Kyoto, Japan]. LN229, BT-474, BINDS-23, and MDA-MB-468 cell lines were
cultured in a DMEM medium (Nacalai). Both media were supplemented with 100
units/mL of penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B (Nacalai), and
10% fetal bovine serum (FBS, Thermo, Waltham, MA, USA). All cell lines were cultured at
37 °C in a humidified atmosphere with 5% CO2 and 95% air.
2.2. Recombinant mAb Production
Anti-HER2 mAb H2Mab-139 was established as previously described [23]. To
generate H2Mab-139-mG2a-f, VH cDNA of H2Mab-139 and CH of mouse IgG2a were cloned
into the pCAG-Ble vector [FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka,
Japan]. VL cDNA of H2Mab-139 and CL cDNA of mouse kappa light chain were also cloned
into the pCAG-Neo vector (Wako). The vectors were transfected into FUT8-knockout
ExpiCHO-S (BINDS-09) cells as described previously [28]. H2Mab-139-mG2a-f was
purified using Ab-Capcher (ProteNova Co., Ltd., Kagawa, Japan).
2.3. Animal Experiments for ADCC Assay and Mice Xenograft Model
Every animal experiment for ADCC and antitumor activity by H2Mab-139-mG2a-f
was approved by the Institutional Committee for Experiments of the Institute of Microbial
Chemistry (Numazu, Japan; approval no. 2022-056, 2023-001, and 2023-018). Mice weremonitored and maintained as described previously [28].
2.4. Flow Cytometry
CHO-K1, CHO/HER2, LN229, LN229/HER2, BT-474, and MDA-MB-468 were collected using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA) (Nacalai).
The cells (1 × 105 cells/sample) were incubated with blocking buffer (control) (0.1% BSA in
PBS) or H2Mab-139-mG2a-f for 30 min at 4 °C. Next, the cells were incubated with Alexa
Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers,
MA, USA) for 30 min at 4 °C. To confirm the isotype of H2Mab-139-mG2a-f, the mAbtreated LN229/HER2 cells were incubated with fluorescein-conjugated anti-mouse heavy
chains (IgG1 and IgG2a; SouthernBiotech, Birmingham, AL, USA) or Alexa Fluor 488-conjugated anti-mouse immunoglobulins for 30 min at 4 °C. SA3800 Cell Analyzer (SonyCorp., Tokyo, Japan) was used to collect the fluorescence data, which were analyzed using
FlowJo [BD Biosciences (BD), Franklin Lakes, NJ, USA].
2.5. Determination of Dissociation Constant (KD) via Flow Cytometry
CHO/HER2 and BT-474 were suspended in 100 μL of serially diluted H2Mab-139mG2a-f (600 pg/mL–10 μg/mL). After the washing step, those cells were reacted with Alexa
Fluor 488-conjugated anti-mouse IgG at a ratio of 1:200 (n = 3). Fluorescence data wereCurr. Issues Mol. Biol. 2023, 45

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detected using the SA3800 Cell Analyzer. The apparent dissociation constant (KD) was calculated using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) as described previously [26].
2.6. Western Blot Analysis
The cell lysates (10 μg) were treated with sodium dodecyl sulfate (SDS) sample buffer(Nacalai) for 5 min at 95 °C. The proteins were electrophoresed using 5–20% polyacrylamide precast gels for electrophoresis (Wako) and transferred to polyvinylidene difluoride(PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked
using 4% skim milk (Nacalai) in PBS with 0.05% Tween 20 and were treated with 1 μg/mL
of H2Mab-139-mG2a-f or 1 μg/mL of an anti-isocitrate dehydrogenase 1 (IDH1) mAb(RcMab-1). The membranes were then incubated with anti-mouse immunoglobulins conjugated with peroxidase [diluted 1:1000; Agilent Technologies, Inc. (Agilent), Santa Clara,
CA, USA, for H2Mab-139-mG2a-f], or with anti-rat immunoglobulins conjugated with peroxidase (diluted 1:1000; Agilent, for RcMab-1). Finally, the signals were detected using achemiluminescence reagent, ImmunoStar LD (Wako) using a Sayaca-Imager (DRC Co.,
Ltd., Tokyo, Japan).
2.7. IHC Analysis
A paraffin-embedded breast cancer tissue microarray (T8235721-5, BioChain InstituteInc., Eureka Drive Newark, CA, USA) was autoclaved for 20 min using Envision FLEXTARGET RETRIEVAL SOLUTION High pH. We used SuperBlock T20 (Thermo) for blocking to inhibit the non-specific binding of mAbs to sections. The sections were treated with
10 μg/mL of H2Mab-139-mG2a-f for 1 h at room temperature and then incubated with the
EnVision+ Kit for mouse (Agilent) for 30 min. The chromogenic reaction and counterstaining were performed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent) andhematoxylin (Wako), respectively.
2.8. ADCC
The splenocytes were obtained from female BALB/c nude mice (five-week-old,
Charles River Laboratories, Inc., Tokyo, Japan) as described previously [28], and wereused as effector cells. The ADCC by H2Mab-139-mG2a-f was assayed as follows. CHO-K1,
CHO/HER2, BT-474, and MDA-MB-468 were labeled with 10 μg/mL Calcein AM(Thermo). The Calcein AM-labeled target cells (2 × 104 cells) were mixed with the effector
cells (effector/target cells ratio, 50), 100 μg/mL of H2Mab-139-mG2a-f or control mouse
IgG2a (mIgG2a, Sigma-Aldrich, St. Louis, MO, USA). We incubated them for 4.5 h at 37 °C,
and measured the Calcein release into the medium using a microplate reader (Power Scan
HT; BioTek Instruments, Inc., Winooski, VT, USA).
We determined the cytotoxicity (% lysis) as follows:% lysis = (E − S)/(M − S) × 100“E” indicates the fluorescence in cultures of both effector and target cells,“S” indicates the spontaneous fluorescence of only target cells,“M” indicates the maximum fluorescence following the treatment with a lysis buffer [10mM Tris-HCl (pH 7.4), 10 mM EDTA, and 0.5% Triton X-100].
2.9. CDC
We plated the Calcein AM-labeled target cells (CHO-K1, CHO/HER2, BT-474, and
MDA-MB-468) and mixed those cells with rabbit complement (final dilution 1:10, LowTox-M Rabbit Complement; Cedarlane Laboratories, Hornby, ON, Canada) and 100
μg/mL of control mIgG2a or H2Mab-139-mG2a-f. We incubated them for 4.5 h at 37 °C, andmeasured the Calcein release into the medium, as described above.

Curr. Issues Mol. Biol. 2023, 457738

2.10. Antitumor Activity of H2Mab-139-mG2a-f in Xenografts of CHO-K1, CHO/HER2, BT474, and MDA-MB-468 Cells
We resuspended CHO/HER2 (5 × 106 cells) in DMEM and mixed them with BD Matrigel Matrix Growth Factor Reduced (BD). We injected them subcutaneously into the left
flank of BALB/c nude mice (female, 5 weeks old, Charles River Laboratories, Inc.). On day
8 post-inoculation, 100 μg of H2Mab-139-mG2a-f (n = 8) or control mouse IgG (mIgG, Wako)(n = 8) in 100 μL PBS were intraperitoneally injected. On days 14 and 22, additional antibody injections were performed. The tumor volume was measured on days 8, 12, 14, 19,
22, and 26 after the inoculation of cells.
We injected BT-474 and MDA-MB-468 (5 × 106 cells) subcutaneously into the left flankof BALB/c nude mice, as indicated above. On day 7 post-inoculation, 100 μg of H2Mab139-mG2a-f (n = 8) or control mIgG (n = 8) in 100 μL PBS was intraperitoneally injected. On
days 14 and 21, additional antibody injections were performed. The tumor volume wasmeasured on days 7, 10, 14, 16, 21, 24, and 28 after the inoculation of cells.
2.11. Statistical Analyses
All data are shown as mean ± standard error of the mean (SEM). Welch’s t-test wasused for the statistical analyses in ADCC, CDC, and tumor weight. ANOVA with Sidak’s
post hoc test was used in tumor volume and mouse weight. GraphPad Prism 8 (GraphPad
Software, Inc.) was utilized for the calculations. A p < 0.05 was considered to indicate astatistically significant difference.
3. Results
3.1. Detection of HER2 Using H2Mab-139-mG2a-f by Flow Cytometry
We previously established an anti-HER2 mAb (H2Mab-139, IgG1, kappa) by the immunization of the HER2 ectodomain produced by glioblastoma LN229 cells [23]. H2Mab139 was advantageous for western blotting, flow cytometry, and IHC [23]. Here, we engineered a class-switched and defucosylated H2Mab-139 (H2Mab-139-mG2a-f) by fusing the
VH chains of H2Mab-139 with the CH chains of mouse IgG2a (Figure 1A). We confirmed that
H2Mab-139-mG2a-f was selectively recognized by anti-IgG2a, but not anti-IgG1 secondary
antibodies in flow cytometry (Figure 1B). H2Mab-139-mG2a-f detected CHO/HER2 cells,
but not parental CHO-K1 cells (Figure 1C). The reactivity did not change compared to theoriginal H2Mab-139 (Figure 1C). Furthermore, H2Mab-139-mG2a-f reacted with HEK-293T
cells, but not with HER2-KO HEK293T (BINDS-23) cells (Figure 1D). We next investigated
the reactivity of H2Mab-139-mG2a-f against breast cancer cell lines. As shown in Figure 1E,
H2Mab-139-mG2a-f reacted HER2-positive breast cancer cell line, BT-474, but not the triplenegative breast cancer (TNBC) cell line, MDA-MB-468.
A kinetic analysis of the interactions of H2Mab-139-mG2a-f with CHO/HER2 and BT474 was performed by flow cytometry. The apparent KD for the interaction of H2Mab-139mG2a-f with CHO/HER2 and BT-474 were 3.9 × 10−9 M and 7.7 × 10−9 M, respectively. These
results suggest that H2Mab-139-mG2a-f demonstrates a high affinity for HER2-expressing
cells.

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Figure 1. Flow cytometry using H2Mab-139-mG2a-f. (A) A core-fucose-deficient mouse IgG2a mAb,
H2Mab-139-mG2a-f was produced from H2Mab-139 (mouse IgG1). (B) LN229/HER2 cells weretreated with 1 μg/mL of H2Mab-139-mG2a-f (red) or buffer control (filled gray), followed by Alexa
Fluor 488-conjugated anti-mouse IgG or Fluorescein-conjugated anti-mouse heavy chains (IgG1 andIgG2a). (C) CHO-K1 and CHO/HER2 cells were treated with 10 μg/mL of H2Mab-139-mG2a-f (red),
H2Mab-139 (red), or buffer control (filled gray), followed by Alexa Fluor 488-conjugated anti-mouse
IgG. (D) HEK293T and HER2-KO HEK293T (BINDS-23) cells were treated with 10 μg/mL of H2Mab139-mG2a-f (red) or buffer control (filled gray), followed by Alexa Fluor 488-conjugated anti-mouse
IgG. (E) Breast cancer cell lines, BT-474 and MDA-MB-468 cells were treated with 10 μg/mL of
H2Mab-139-mG2a-f (red) or buffer control (filled gray), followed by Alexa Fluor 488-conjugated antimouse IgG.

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3.2. Detection of HER2 Using H2Mab-139-mG2a-f in Western Blot and IHC Analyses
We next performed western blot analysis using H2Mab-139-mG2a-f. As shown in Figure 2A, H2Mab-139-mG2a-f strongly detected HER2 as more than 180-kDa bands in
LN229/HER2 and BT-474 cells. H2Mab-139-mG2a-f faintly detected endogenous HER2 in
LN229 cells, but not MDA-MB-468 cells. The expression of IDH1 detected by RcMab-1 wasused as an internal control (Figure 2B). These results indicated that H2Mab-139-mG2a-f
could detect exogenous and endogenous HER2 in western blot analysis.
Next, IHC analyses against the formalin-fixed paraffin-embedded (FFPE) sections ofbreast cancer tissue were performed using H2Mab-139-mG2a-f. As shown in Figure 2C–H,
H2Mab-139-mG2a-f could distinguish HER2-strong positive (Figure 2C,D), moderate (Figure 2E,F), and negative (Figure 2G,H) breast cancers. The HER2-positive staining was
mainly observed on the plasma membrane. We summarized the results of HER2 expression in breast cancer tissue array in supplementary Table S1. H2Mab-139-mG2a-f stained
10 out of 63 cases (16%) of breast cancers. These results indicated that H2Mab-139-mG2a-f
is also available for IHC analysis of FFPE tumor sections.

Figure 2. Western blot and IHC analyses using H2Mab-139-mG2a-f. (A,B) Western blotting using
H2Mab-139-mG2a-f. The cell lysates of LN229, LN229/HER2, BT-474, and MDA-MB-468 were electrophoresed and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with 1 μg/mL of H2Mab-139-mG2a-f (A) or 1 μg/mL of RcMab-1 (an anti-IDH mAb) (B). The
arrow indicates the predicted size of HER2 (~180 kDa). An arrow indicates the band of HER2. (C–H) IHC analysis of a breast cancer tissue array using H2Mab-139-mG2a-f. Scale bar = 100 μm.

Curr. Issues Mol. Biol. 2023, 457741

3.3. ADCC and CDC by H2Mab-139-mG2a-f against CHO/HER2 Cells
We next investigated whether H2Mab-139-mG2a-f could exert ADCC against
CHO/HER2 cells. H2Mab-139-mG2a-f showed ADCC (54.1% cytotoxicity) against
CHO/HER2 cells more effectively than the control mouse IgG2a (18.8% cytotoxicity; p <0.05) (Figure 3A). No difference was observed between H2Mab-139-mG2a-f and control
mIgG2a about ADCC against CHO-K1 (Figure 3B).
We then examined whether H2Mab-139-mG2a-f could exhibit CDC against
CHO/HER2 cells. As shown in Figure 3C, H2Mab-139-mG2a-f elicited a higher degree of
CDC (62.5% cytotoxicity) in CHO/HER2 cells compared with that elicited by control
mIgG2a (15.6% cytotoxicity; p < 0.05). There was no difference between H2Mab-139-mG2a-f
and control mIgG2a in CDC for CHO-K1 (Figure 3D). These results showed that H2Mab139-mG2a-f exerted significantly high levels of ADCC/CDC against CHO/HER2 cells.

Figure 3. H2Mab-139-mG2a-f-mediated ADCC and CDC activities in CHO/HER2 and CHO-K1 cells.
(A,B) ADCC induced by H2Mab-139-mG2a-f or control mouse IgG2a (mIgG2a) against CHO/HER2 (A)and CHO-K1 (B) cells. (C,D) CDC induced by H2Mab-139-mG2a-f or control mIgG2a against
CHO/HER2 (C) and CHO-K1 (D) cells. Values are shown as mean ± SEM. Asterisks indicate statistical significance (* p < 0.05; Welch’s t-test). n.s., not significant.

3.4. Antitumor Effects of H2Mab-139-mG2a-f in the Mouse Xenografts of CHO/HER2 Cells
Following the inoculation of CHO/HER2, we injected H2Mab-139-mG2a-f and control
mIgG intraperitoneally into CHO/HER2 xenograft tumor-bearing mice on days 8, 14, and
22. On days 8, 12, 14, 19, 22, and 26 after the tumor inoculation, we measured the tumor
volume. The H2Mab-139-mG2a-f administration reduced the tumor volume on days 19 (p< 0.01), 22 (p < 0.01), and 26 (p < 0.01) compared with that of mIgG (Figure 4A). The H2Mab139-mG2a-f administration led to a 52% reduction of the tumor volume compared withthat of the control mIgG on day 26 post-injection.
The weight of CHO/HER2 tumors treated with H2Mab-139-mG2a-f was significantly
lower than that treated with mIgG (66% reduction; p < 0.05; Figure 4B). CHO/HER2 tumorsthat were resected from mice on day 26 are demonstrated in Figure 4C.
The body weight loss and skin disorders were not detected in CHO/HER2 tumorbearing mice (Figure 4D). The mice on day 26 were shown in supplementary Figure S1.

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Figure 4. Antitumor activity of H2Mab-139-mG2a-f against CHO/HER2 xenograft. (A) CHO/HER2
cells were subcutaneously injected into BALB/c nude mice (day 0). On day 8, 100 μg of H2Mab-139mG2a-f or control normal mouse IgG (mIgG) was injected intraperitoneally into mice. Additional
antibodies were injected on days 14 and 22. We measured the tumor volume on days 8, 12, 14, 19,
22, and 26. Values are presented as the mean ± SEM. ** p < 0.01 (ANOVA and Sidak’s multiple comparisons test). (B) Tumor weight of CHO/HER2 xenograft tumors on day 28. Values are presented
as the mean ± SEM. * p < 0.05 (Welch’s t-test). (C) The CHO/HER2 xenograft tumors on day 28 (scalebar, 1 cm). (D) Body weight in mIgG and H2Mab-139-mG2a-f treated mice. n.s., not significant.

3.5. ADCC and CDC by H2Mab-139-mG2a-f against BT-474 and MDA-MB-468 Cells
It was investigated whether H2Mab-139-mG2a-f was capable of mediating ADCC
against BT-474 and MDA-MB-468 cells. As revealed in Figure 5A, H2Mab-139-mG2a-f
showed ADCC (37.6% cytotoxicity) against BT-474 cells more potently than did the control mIgG2a (3.8% cytotoxicity; p < 0.01). We next investigated whether H2Mab-139-mG2a-f
exhibited CDC against BT-474 cells. H2Mab-139-mG2a-f showed a significantly high CDC(70.6% cytotoxicity) in BT-474 cells compared with that induced by control mIgG2a (34.6%
cytotoxicity; p < 0.01) (Figure 5B). However, there was no difference between H2Mab-139mG2a-f and control mIgG2a in ADCC (Figure 5C) and CDC (Figure 5D) against MDA-MB468. These results demonstrated that H2Mab-139-mG2a-f exhibited higher levels of ADCC
and CDC against HER2-positive BT-474 cells.

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Figure 5. H2Mab-139-mG2a-f-mediated ADCC and CDC activities in BT-474 (HER2-positive) and
MDA-MB-468 (TNBC) cells. (A,C) ADCC induced by H2Mab-139-mG2a-f or control mIgG2a against
BT-474 (A) and MDA-MB-468 (C) cells. (B,D) CDC induced by H2Mab-139-mG2a-f or control mIgG2aagainst BT-474 (B) and MDA-MB-468 (D) cells. Values are shown as mean ± SEM. Asterisks indicate
statistical significance (** p < 0.01; Welch’s t-test). n.s., not significant.

3.6. Antitumor Effects of H2Mab-139-mG2a-f in BT-474 and MDA-MB-468 Xenografts
In the BT-474 xenograft models, we injected H2Mab-139-mG2a-f and control mIgG intraperitoneally on days 7, 14, and 21 after BT-474 inoculation. We measured the tumor
volume on days 7, 10, 14, 16, 21, 24, and 28 following the inoculation. The H2Mab-139mG2a-f administration led to a significant reduction in BT-474 xenograft on days 21 (p <0.01), 24 (p < 0.01), and 28 (p < 0.01) compared with that of the control mIgG (Figure 6A).
The H2Mab-139-mG2a-f administration resulted in a 36% reduction of tumor volume compared with that of the control mIgG on day 28.
Tumors from the H2Mab-139-mG2a-f-treated mice weighed significantly less thanthose from the control mIgG-treated mice (45% reduction; p < 0.01, Figure 6C). We resected
tumors from mice on day 28 (Figure 6E).
In the MDA-MB-468 xenograft models, H2Mab-139-mG2a-f and control mIgG were
injected into mice on days 7, 14, and 21 intraperitoneally after the inoculation of MDAMB-468 cells. The tumor volume was measured on days 7, 10, 14, 16, 21, 24, and 28. No
difference was observed between H2Mab-139-mG2a-f and control mIgG about MDA-MB468 xenograft volume (Figure 6B) and weight (Figure 6D). MDA-MB-468 tumors that were
resected from mice on day 28 are depicted in Figure 6F.
The body weight loss was not detected in both BT-474 and MDA-MB-468 xenograftbearing mice (Figure 6G,H). The mice on day 28 about BT-474 and MDA-MB-468 xenograft were demonstrated in supplementary Figure S2.

Curr. Issues Mol. Biol. 2023, 45

7744

Figure 6. Antitumor activity of H2Mab-139-mG2a-f against BT-474 and MDA-MB-468 xenografts.
(A,B) BT-474 (A) and MDA-MB-468 (B) cells were injected into BALB/c nude mice (day 0). On day
7, 100 μg of H2Mab-139-mG2a-f or control normal mouse IgG (mIgG) were injected into mice. On
days 14 and 21, additional antibodies were injected. On days 7, 10, 14, 16, 21, 24, and 28, the tumor
volume was measured. Values are presented as the mean ± SEM. ** p < 0.01 (ANOVA and Sidak’s
multiple comparisons test). (C,D) Tumor weight of BT-474 (C) and MDA-MB-468 (D) xenograft tumors on day 28. Values are presented as the mean ± SEM. ** p < 0.01 (Welch’s t-test). (E,F) The BT474 (E) and MDA-MB-468 (F) xenograft tumors on day 28. Scale bar, 1 cm. (G,H) The body weightof BT-474 (G) and MDA-MB-468 (H) xenograft-bearing mice treated with mIgG and H2Mab-139mG2a-f. n.s., not significant.

Curr. Issues Mol. Biol. 2023, 457745

4. Discussion
Trastuzumab is clinically administered for patients with HER2-overexpressing metastatic breast cancers, which are defined by strong and complete IHC membranous staining of more than 10% of cells (IHC 3+) and/or in situ hybridization (ISH)-amplified. Basedon clinical studies, the 5th European School of Oncology and the European Society of
Medical Oncology guidelines for advanced breast cancer (ABC 5) and the National Comprehensive Cancer Network guidelines consider trastuzumab (anti-HER2 domain IV
mAb), pertuzumab (anti-HER2 domain II mAb), and docetaxel as the standard of care forfirst-line treatment of HER2-positive metastatic breast cancer [29]. However, most deathsin the study were due to breast cancer [21]. Therefore, better treatments including novel
combination therapies and novel modalities are still needed. In this study, we evaluated anovel anti-HER2 domain I mAb, H2Mab-139-mG2a-f, and showed the ADCC activity invitro (Figures 3 and 5) and antitumor effect in vivo (Figures 4 and 6). Therefore, H2Mab139-mG2a-f could be an antibody treatment regimen for HER2-positive breast cancer.
The structures of the HER2–HER3–NRG1β complex, revealed by cryo-EM, exhibit adynamic dimer interface. In the complex, the NRG1β-bound HER3 dimerization arm remains unresolved due to the lack of a ligand-induced conformational change in the apo
HER2 monomer, which is essential for the formation of the HER3 dimerization arm-binding pocket [22]. In contrast, the most frequent oncogenic HER2 mutation (S310F/Y) was
found primarily in cancers without HER2 overexpression. The HER2 S310 is localized in
the dimerization arm-binding pocket of domain II [30]. The structures of HER2 (S310F)–
HER3–NRG1β complex exhibited stabilizing interactions with the HER3 dimerizationarm and compensate for the inability of HER2 to undergo a needed conformationalchange [22]. Furthermore, HER2–HER3 and HER2 (S310F)–HER3 retain the ability to bind
to trastuzumab, but the mutant complex does not bind to pertuzumab [22]. These results
suggest that pertuzumab is less effective at targeting cancers driven by HER2 (S310F), anddifferent epitope-possessing anti-HER2 mAbs including H2Mab-139 could be required forthe combination therapy with trastuzumab.
Trastuzumab-based antibody-drug conjugates (ADCs) including trastuzumabderuxtecan (T-DXd) have been evaluated. These ADCs rely on the direct cytotoxicity of
the released DXd (a DNA topoisomerase I inhibitor) following endocytosis of the HER2bound mAbs-drug conjugate [31]. T-DXd initially exhibited beneficial outcomes in patients with metastatic breast cancer, who had undergone multiple anti-HER2-targeting
treatments [32]. Currently, various clinical trials are evaluating the efficacy of T-DXd.
Based on studies, T-DXd has been approved in not only HER2-positive breast cancer [33–35], but also HER2-mutant lung cancer [34] and HER2-low (IHC 1+ or IHC 2+/ISH-nonamplified) advanced breast cancer [33].
Given that approximately half of all breast cancers are classifiable as HER2-low [36],a greater number of patients may benefit from T-DXd therapy. These results have had asignificant impact on the field of breast oncology, particularly in the future clinical diagnostics of HER2-low breast cancer. As a result, future treatment algorithms for both hormone receptor-positive and TNBC are anticipated to change [37]. There are several challenges in elucidating the biological roles and pathological significance of HER2-low [38].
Since our H2Mab-139 is applicable for IHC (Figure 2), it would be valuable to compare its
reactivity with approved anti-HER2 diagnostic mAbs such as HercepTestTM and PATHWAY®.
We achieved increased ADCC activity of H2Mab-139-mG2a-f through class switching
and a core fucose deficiency on the N-glycan in the Fc region, which promotes the bindingof Fc to FcγRIIIa on effector cells [25]. This technique is also applied to mogamulizumab(Poteligeo), a defucosylated humanized mAb targeting CCR4 [39]. In contrast, margetuximab is derived from trastuzumab and shares the same epitope with HER2. Five aminoacid substitutions in the Fc domain of margetuximab (human IgG1) achieve increased
binding to FcγRIIIa and reduced binding to an inhibitory FcγR, FcγRIIb, when comparedCurr. Issues Mol. Biol. 2023, 457746

to trastuzumab [9]. We are going to apply the strategy to potentiate the ADCC activity
when we generate the humanized H2Mab-139 mAb.
We previously produced a bispecific Ab against EGFR and HER2 from our established anti-EGFR mAb (EMab-134) and an anti-HER2 mAb (H2Mab-77) [40]. The
bispecific Ab possesses the tetravalent structure by fusing the single chain Fv of H2Mab77 at the light chains of EMab-134 and showed the antitumor effect in the mouse xenograftmodel [40]. Since we can produce the different types of bispecific Abs and have various
clones of anti-HER2 mAbs including H2Mab-139 (see Supplementary Materials), we will
investigate the activity in future studies.
Previously, we established H2Mab-139 using cancer cell-produced HER2 ectodomain
as an immunogen. This methodology is essential for the development of cancer-specificmAbs (CasMabs). We have developed CasMabs that target podoplanin (PDPN) [41],which recognize the aberrant glycosylation patterns typical of cancer cells [42]. AntiPDPN-CasMabs are currently applied to CAR-T therapy in preclinical models [43,44]. For
the development of anti-HER2 CasMab, we need to perform further screening of our already established anti-HER2 mAbs (more than 200 clones), comparing their reactivity
against normal cells [45,46]. Anti-HER2 CasMabs could be employed in designing modalities including ADCs and CAR-T.
Supplementary Materials: The following supporting information can be downloaded at:https://www.mdpi.com/article/10.3390/cimb45100488/s1, Supplementary Figure S1, Body appearance in CHO/HER2 xenografts-implanted mice treated with mIgG and H2Mab-139-mG2a-f on day
26. Supplementary Figure S2, Body appearance in (A) BT-474 and (B) MDA-MB-468 xenograftsimplanted mice treated with mIgG and H2Mab-139-mG2a-f on day 28. Supplementary Table S1, IHC
analysis using H2Mab-139-mG2a-f against breast cancer tissue array.
Author Contributions: H.S., T.O. and R.N. performed the experiments. M.K., M.K.K. and Y.K. designed the experiments. H.S., R.N. 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.),
23bm1123027h0001 (to Y.K.), and JP23ck0106730 (to Y.K.), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant nos. 22K06995 (toH.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Institutional Review Board Statement: Animal experiments were approved by the Institutional
Committee for Experiments of the Institute of Microbial Chemistry (approval no. 2022-056, 2023001, and 2023-018).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article and supplementary material.
Acknowledgments: The authors would like to thank Shun-ichi Ohba and Akiko Harakawa (Institute of Microbial Chemistry [BIKAKEN], Numazu, the Microbial Chemistry Research Foundation)
for technical assistance with animal experiments.
Conflicts of Interest: The authors have no conflict of interest to declare.

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