Original Articles

MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 2, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0033

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Establishment of a Novel Cancer-Specific Anti-HER2
Monoclonal Antibody H2Mab-250/H2CasMab-2
for Breast Cancers

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Mika K. Kaneko, Hiroyuki Suzuki, and Yukinari Kato

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is an
important target for monoclonal antibody (mAb) therapy. All therapeutic mAbs, including anti-HER2 mAbs,
exhibit adverse effects probably due to the recognition of antigens expressed in normal cells. Therefore, tumorselective or specific mAbs can be beneficial in reducing the adverse effects. In this study, we established a novel
cancer-specific anti-HER2 monoclonal antibody, named H2Mab-250/H2CasMab-2 (IgG1, kappa). H2Mab-250
reacted with HER2-positive breast cancer BT-474 and SK-BR-3 cells. Importantly, H2Mab-250 did not reactwith nontransformed normal epithelial cells (HaCaT and MCF 10A) and immortalized normal epithelial cells inflow cytometry. In contrast, most anti-HER2 mAbs, such as H2Mab-119 and trastuzumab reacted with bothcancer and normal epithelial cells. Immunohistochemical analysis demonstrated that H2Mab-250 possesses
much higher reactivity to the HER2-positive breast cancer tissues compared with H2Mab-119, and did not reactwith normal tissues, including heart, breast, stomach, lung, colon, kidney, and esophagus. The epitope mapping demonstrated that the Trp614 of HER2 domain IV mainly contributes to the recognition by H2Mab-250.
H2Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody–drug conjugates
without adverse effects for breast cancer therapy.
Keywords: HER2, cancer-specific monoclonal antibody, screening, epitope, flow cytometry

and overall survival in HER2-positive breast cancer patientswith metastasis.6 Trastuzumab has become the standard
treatment for HER2-positive breast cancers7 and HER2positive gastric cancers.8 Trastuzumab has been the mosteffective therapy for HER2-positive breast cancer for morethan 20 years.9
The major adverse effect associated with anti-HER2 therapeutic mAbs is cardiotoxicity, thereby necessitating routine
cardiac monitoring in clinics.10 Furthermore, mice lacking
ErbB2 (ortholog of HER2) displayed embryonic lethality due
to the dysfunctions associated with a lack of cardiac trabeculae.11 Ventricular-restricted ErbB2-deficient mice showed thefeatures of dilated cardiomyopathy.12 These results indicatethat HER2 is vital for normal heart development and homeostasis. Therefore, more selective anti-HER2 mAbs against
tumors, which can reduce heart failures are required.

IntroductionHuman epidermal growth factor receptor 2(HER2) is included in the receptor tyrosine kinase
family of human epidermal growth factor receptors. To activate the downstream signaling, HER2 must either form
heterodimers with other HER members and their specific ligands or self-assemble into ligand-independent homodimers
when overexpressed.1 The HER2 overexpression is observedin *20% of breast cancers2 and 20% of gastric cancers,3which are associated with higher rates of recurrence, poor
prognosis, and shorter overall survival. A monoclonal antibody (mAb) against HER2, trastuzumab, exhibited an antiproliferating effect in vitro and a potent antitumor effectin vivo.4,5 The addition of trastuzumab to chemotherapy
improves objective response rates, progression-free survival,

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
This article was previously published in preprint.org (doi: https://www.preprints.org/manuscript/202309.0906/v2?specify=1).

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We previously established several anti-HER2 mAbs, suchas H2Mab-19 (IgG2b, kappa),13 H2Mab-41 (IgG2b, kappa),14
H2Mab-77 (IgG1, kappa),15 H2Mab-119 (IgG1, kappa),16
H2Mab-139 (IgG1, kappa),17 and H2Mab-181 (IgG1, kappa)18
by the immunization of HER2 ectodomain (HER2ec). We
further engineered the mAbs into the mouse IgG2a type
(H2Mab-77-mG2a, H2Mab-119-mG2a, and H2Mab-139mG2a, respectively), and produced the core fucose-deficient
types (H2Mab-77-mG2a-f, H2Mab-119-mG2a-f, and H2Mab139-mG2a-f, respectively) to potentiate the antibodydependent cellular cytotoxicity (ADCC) and antitumor effectin vivo.19–21 In this study, we developed and characterized anovel HER2 mAb, named H2Mab-250/H2CasMab-2.
Materials and Methods

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Cell culture

Chinese hamster ovaries, (CHO)-K1, BT-474, SK-BR-3,
MDA-MB-468, MCF 10A, hTERT TIGKs, HBEC3-KT,
hTERT-HME1, RPTEC/TERT1, and P3X63Ag8U.1 (P3U1),
were obtained from the American Type Culture Collection(ATCC, Manassas, VA). Human keratinocyte HaCaT waspurchased from Cell Lines Service GmbH (Eppelheim,
Germany). The hTCEpi, hTEC/SVTERT24-B, and HCEC1CT were purchased from EVERCYTE (Vienna, Austria).
The cDNA of HER2 (wild type; WT) and deletion mutants(dN218, dN342, and dN511) were cloned into the pCAGnPA16 vector (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan). A HER2 point mutant (W614A) and HER2
WT were cloned into the pCAG-nPA-cRAPMAP vector(FUJIFILM Wako Pure Chemical Corporation). CHO-K1
cells were transfected with the abovementioned vectors usinga Neon transfection system (Thermo Fisher Scientific, Inc.,
Waltham, MA). A few days after transfection, PA tagpositive cells were sorted by the cell sorter (SH800; Sony
Corp., Tokyo, Japan) using NZ-1, which was originally developed as an anti-human podoplanin mAb.22 Finally, CHO/
HER2 and CHO/HER2 (dN218, dN342, dN511, and W614A)
cell lines were established.
CHO-K1, CHO/HER2 (WT, deletion, and point mutants),
and P3U1 were cultured in Roswell Park Memorial Institute1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), andBT-474, SK-BR-3, MDA-MB-468, HEK293T, and HaCaT
were cultured in Dulbecco’s modified Eagle’s medium(DMEM) (Nacalai Tesque, Inc.), supplemented with 10%
heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin, 100 lg/mL streptomycin,
and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.).
Mammary epithelial cell line, MCF 10A was cultured in
Mammary Epithelial Cell Basal Medium BulletKit (Lonza,
Basel, Switzerland) supplemented with 100 ng/mL cholera
toxin (Sigma-Aldrich Corp., St. Louis, MO).
Immortalized normal epithelial cell lines were maintained
as follows; hTERT TIGKs, Dermal Cell Basal Medium and
Keratinocyte Growth Kit (ATCC); HBEC3-KT, Airway
Epithelial Cell Basal Medium and Bronchial Epithelial Cell
Growth Kit (ATCC); hTERT-HME1, Mammary Epithelial
Cell Basal Medium BulletKit without GA-1000 (Lonza);
hTCEpi, KGMTM-2 BulletKit (Lonza); hTEC/SVTERT24-B,
OptiPRO SFM, and GlutaMAX-I (Thermo Fisher Scientific, Inc.); RPTEC/TERT1, DMEM/F-12, and hTERT
Immortalized RPTEC Growth Kit with supplement A and B

KANEKO ET AL.

(ATCC); HCEC-1CT, DMEM/M199 (4:1, Thermo Fisher
Scientific, Inc.), 2% Cosmic Calf Serum (Cytiva, Marlborough,MA), 20 ng/mL hEGF (Sigma-Aldrich Corp.), 10 lg/mL insulin (Sigma-Aldrich Corp.), 2 lg/mL apo-transferrin (SigmaAldrich Corp.), 5 nM sodium selenite (Sigma-Aldrich Corp.),1 lg/mL hydrocortisone (Sigma-Aldrich Corp.).
All cell lines were cultured at 37C in a humidified atmosphere with 5% CO2 and 95% air.
Development of hybridomas

The animal was housed under specific pathogen-free
conditions. All animal experiments were approved by the
Animal Care and Use Committee of Tohoku University(Permit number: 2022MdA-001).
Anti-HER2 mAbs, such as H2Mab-19 (IgG2b, kappa),13
H2Mab-41 (IgG2b, kappa),14 H2Mab-77 (IgG1, kappa),15
H2Mab-119 (IgG1, kappa),16 H2Mab-139 (IgG1, kappa),17
and H2Mab-181 (IgG1, kappa),18 were established previously. In this study, H2Mab-193 (IgG1, kappa), H2Mab-215(IgG1, kappa), and H2Mab-250 (IgG1, kappa) were established by the same strategy. Briefly, BALB/c mice were
immunized with recombinant HER2ec produced by LN229
cells together with Imject Alum (Thermo Fisher Scientific,Inc.). After several additional immunizations, spleen cells
were fused with P3U1 cells. The culture supernatants ofhybridomas were screened using enzyme-linked immunosorbent assay (ELISA) with recombinant HER2ec and flow
cytometry with cell lines.
Production of recombinant mAb

To generate recombinant H2Mab-250 and H2Mab-119,
their VH cDNAs and the CH cDNA of mouse IgG1 werecloned into the pCAG-Neo vector (FUJIFILM Wako Pure
Chemical Corporation). The VL cDNAs and CL cDNA of themouse 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, Kagawa, Japan) was used
to purify the recombinant H2Mab-250 and H2Mab-119.
To generate mouse IgG2a-type H2Mab-250 (H2Mab-250mG2a), we cloned the VH cDNA of H2Mab-250 and the CH ofmouse IgG2a into the pCAG-Neo vector. The mouse kappalight chain vector of H2Mab-250 is described above. To produce the defucosylated form (H2Mab-250-mG2a-f), the vectors were transfected into BINDS-09 (fucosyltransferase
8-knockout ExpiCHO-S) cells using the ExpiCHO Expression
System. H2Mab-250-mG2a-f was purified using Ab-Capcher.
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 mouse antiHER2 mAbs (10 lg/mL), trastuzumab (10 lg/mL), NZ-1
(10 lg/mL), or blocking buffer (control; 0.1% bovine serum
albumin [BSA] in phosphate-buffered saline [PBS]) for 30
minutes at 4C. Next, the cells were treated with Alexa Fluor
488-conjugated anti-mouse IgG (1:1000; Cell Signaling
Technology, Danvers, MA) for mouse anti-HER2 mAbs,
Alexa Fluor 488-conjugated anti-human IgG (1:1000;

CANCER-SPECIFIC ANTI-HER2 MAB
ADCC reporter bioassay

The ADCC reporter bioassay was performed using
an ADCC Reporter Bioassay Kit (Promega Corporation,
Madison, WI), according to the manufacturer’s instructions.
Target cells (BT-474 and HaCaT, 12,500 cells per well) were

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Sigma-Aldrich, Corp.) for trastuzumab, or Alexa Fluor 488conjugated anti-rat IgG (1:1000; Cell Signaling Technology)
for NZ-1 for 30 minutes at 4C. The fluorescence data
were collected using EC800 or SA3800 Cell Analyzer(Sony Corp), and the data were analyzed using FlowJo(BD Biosciences, Franklin Lakes, NJ).

37

FIG. 1. Flow cytometry using anti-HER2 mAbs. (A) Flow cytometry using H2Mab-250 (10 mg/mL; red line) and H2Mab-119(10 mg/mL; blue 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 nontransformed normal epithelial cells (HaCaT and MCF 10A). (B) Flow cytometry using
H2Mab-250 (10 mg/mL; red line) and H2Mab-119 (10 mg/mL; blue line) against immortalized normal epithelial cells, including
hTERT TIGKs (gingiva), HBEC3-KT (lung bronchus), hTERT-HME1 (mammary gland), hTCEpi (corneal), hTEC/
SVTERT24-B (thymus), RPTEC/TERT1 (kidney proximal tubule), and HCEC-1CT (colon). The black line represents thenegative control (blocking buffer). CHO, Chinese hamster ovary; HER2, human epidermal growth factor receptor 2.

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cultured in a 96-well white solid plate. H2Mab-250-mG2a-f
and trastuzumab (Herceptin; Chugai Pharmaceutical Co., Ltd,Tokyo, Japan) were serially diluted and added to the target
cells. Jurkat cells stably expressing the human FccRIIIa receptor and a nuclear factor of activated T cell (NFAT) response element driving firefly luciferase, were used as effector
cells. The engineered Jurkat cells (75,000 cells in 25 lL) werethen added and cocultured with antibody-treated target cells at
37C for 6 hours. Luminescence using the Bio-Glo Luciferase
Assay System was measured using a GloMax luminometer(Promega Corporation).

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Immunohistochemical analysis

Formalin-fixed paraffin-embedded tissue of HER2positive breast cancer was obtained from Sendai Medical
Center.15 Informed consent for sample procurement andsubsequent data analyses was obtained from the patient or thepatient’s guardian at Sendai Medical Center. Normal tissues
were purchased from BioChain Institute, Inc. (Eureka Drive
Newark, CA) or Cybrdi, Inc. (Frederick, MD). The tissue
sections were autoclaved in citrate buffer (pH 6.0; Nichirei
Biosciences, Inc., Tokyo, Japan) for 20 minutes. The blocking was performed using SuperBlock T20 (Thermo Fisher
Scientific, Inc.). The sections were incubated with H2Mab250 (1, 0.5, or 0.1 lg/mL) and H2Mab-119 (0.5 or
0.1 lg/mL), and then treated with the EnVision+ Kit formouse (Agilent Technologies, Inc., Santa Clara, CA). The
chromogenic reaction was performed using 3,3¢diaminobenzidine tetrahydrochloride (Agilent Technologies,Inc.). Counterstaining was performed using Hematoxylin

KANEKO ET AL.

(FUJIFILM Wako Pure Chemical Corporation) and Leica
DMD108 (Leica Microsystems GmbH, Wetzlar, Germany)
was used to obtain images and examine the sections.
ELISA

Synthesized peptides covering the HER2 extracellulardomain IV and point mutant peptides were synthesized by
Sigma-Aldrich Corp. The peptides (10 lg/mL) and HER2ec(1 lg/mL) were immobilized on Nunc Maxisorp 96-wellimmunoplates (Thermo Fisher Scientific, Inc.). Platewashing was performed with PBS containing 0.05% (v/v)
Tween 20 (PBST; Nacalai Tesque, Inc.). After blocking
with 1% (w/v) BSA in PBST, H2Mab-250 (10 lg/mL) was
added to each well. Then, the wells were further incubatedwith peroxidase-conjugated anti-mouse immunoglobulins(1:2000 dilution; Agilent Technologies, Inc.). Enzymatic
reactions were conducted using One-Step Ultra TMB. The
optical density at 655 nm was measured using an iMark
microplate reader.
Determination of KD through surface
plasmon resonance

Measurement of KD between H2Mab-250 and the HER2
peptides was performed using surface plasmon resonance(SPR). H2Mab-250 was immobilized on the sensor chip CM5in accordance with the manufacturer’s protocol by Cytiva.
Immobilization of H2Mab-250 (10 mg/mL in acetate buffer(pH 4.0); Cytiva) was carried out using an amine coupling
reaction. The surface of the flow cell 2 of the sensor chip

FIG. 2. ADCC reporter assay by H2Mab-250-mG2a-f and trastuzumab in the presence of BT-474 and HaCaT cells. Target
cells such as BT-474 (A) or HaCaT (B) were cultured in a 96-well white solid plate. H2Mab-250-mG2a-f and trastuzumab
were serially diluted and added to the target cells. The engineered Jurkat cells were then added and cocultured with
antibody-treated target cells. Luminescence using the Bio-Glo Luciferase Assay System was measured using a GloMax
luminometer. Error bars represent means – SDs. ADCC, antibody-dependent cellular cytotoxicity; N.D., not determined;
SD, standard deviation.

CANCER-SPECIFIC ANTI-HER2 MAB

constant (kd) and KD. The affinity constant (KA) at equilibrium was calculated as 1/KD.
Results
Establishment of H2Mab-250

We immunized mice with HER2ec produced by LN229
cells. The culture supernatants of hybridomas were screened

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CM5 was treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, followed by the
injection of H2Mab-250. The KD between H2Mab-250 andthe peptides was determined using Biacore X100 (Cytiva).
The binding signals were measured using a single-cycle kinetics method. Data were analyzed by 1:1 binding kinetics
using Biacore X100 evaluation software (Cytiva) to determine the association rate constant (ka) and dissociation rate

39

FIG. 3. Immunohistochemical analysis of H2Mab-250 in breast cancer and normal epithelium. (A) The HER2-positive
breast cancer tissue sections were treated with H2Mab-250 or H2Mab-119 (0.1 or 0.5 mg/mL). (B) A normal heart section
was treated with H2Mab-250 (1 mg/mL). (C) Sections of normal breast, stomach, lung, colon, kidney, and esophagus weretreated with H2Mab-250 (0.1 mg/mL). The sections were then treated with the Envision+ kit. The chromogenic reaction was
performed using DAB, and the sections were counterstained with Hematoxylin. Scale bar = 100 mm. DAB, 3,3¢diaminobenzidine tetrahydrochloride.

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using ELISA with HER2ec. We further screened the reactivity
to HER2-positive breast cancers (BT-474 and SK-BR-3) andnontransformed normal epithelial cells, including HaCaT(keratinocyte) and MCF 10A (mammary gland) using flow
cytometry. One of the established hybridomas, H2Mab-250,
reacted with CHO/HER2, HER2-positive BT-474, and SKBR-3 cells, but not with triple-negative MDA-MB-468 cells.
H2Mab-250 did not react with HaCaT and MCF 10A cells(Fig. 1A). In contrast, H2Mab-119 showed similar reactivity
to both cancer and normal epithelial cells (Fig. 1A).
We next investigated the difference in the reactivity
to immortalized normal epithelial cells, including hTERTTIGKs (gingiva), HBEC3-KT (lung bronchus), hTERT-HME1(mammary gland), hTCEpi (corneal), hTEC/SVTERT24-B(thymus), RPTEC/TERT1 (kidney proximal tubule), and
HCEC-1CT (colon). H2Mab-250 did not react with thosenormal cells, while H2Mab-119 was reactive with all immortalized normal epithelial cells (Fig. 1B), indicating that H2Mab250 possesses cancer-specific reactivity against HER2.
We further compared the reactivity of H2Mab-250 with ourestablished anti-HER2 mAbs and trastuzumab. As shown in
Supplementary Figures S1 and S2, anti-HER2 domain I mAbs
(H2Mab-7715 and H2Mab-13917), anti-HER2 domain II mAbs
(H2Mab-193 and H2Mab-215), anti-HER2 domain III mAbs
(H2Mab-1913 and H2Mab-18118), and anti-HER2 domain IV
mAbs (H2Mab-4114 and trastuzumab) reacted with HER2positive breast cancers, nontransformed normal epithelial
cells, and immortalized normal epithelial cells.16 These results
indicated that H2Mab-250 exhibits an exceptional reactivity
compared with other anti-HER2 mAbs.
The ability of effector cell activation by H2Mab-250
and trastuzumab

The ADCC reporter bioassay is a bioluminescent reporter
gene assay to quantify the biological activity of the antibody
through FccRIIIa-mediated pathway activation in an ADCC
mechanism of action.23 We next produced H2Mab-250mG2a-f, the core-fucose deleted IgG2a version of H2Mab-250
using fucosyltransferase 8-deficient ExpiCHO-S (BINDS09) cells and examined whether H2Mab-250-mG2a-f could
activate ADCC program in the presence of BT-474 and HaCaT
cells. To compare the ADCC pathway activation by H2Mab250-mG2a-f and trastuzumab, we treated BT-474 and HaCaT
cells with serially diluted mAbs, and then incubated with effector Jurkat cells, which express the human FccRIIIa receptor
and an NFAT response element driving firefly luciferase.

KANEKO ET AL.

As shown in Figure 2A, H2Mab-250-mG2a-f could activate
the effector (EC50: 9.9 lg/mL), but it was less effective than
trastuzumab (EC50: 0.053 lg/mL). Importantly, H2Mab-250mG2a-f did not activate the effector in the presence of HaCaT
cells. In contrast, trastuzumab activated the effector withsimilar EC50 (0.036 lg/mL) to BT-474 cells (Fig. 2B). These
results indicated that H2Mab-250-mG2a-f selectively activates the effector cells against breast cancer cells.
Immunohistochemical analysis of H2Mab-250 in breast
cancer and normal epithelium

Immunohistochemical analysis was performed to examine
the reactivity of H2Mab-250 with normal and tumor tissue
sections. In contrast to flow cytometry, H2Mab-250 exhibited
more potent reactivity to the HER2-positive breast cancer
section than H2Mab-119 (Fig. 3A). Since all anti-HER2
therapeutic mAbs are associated with cardiotoxicity, a major
adverse effect,10 the reactivity of H2Mab-250 to a normal
heart was further investigated. Even with higher concentrations of H2Mab-250 (1 lg/mL), no reactivity with the normal
heart could be detected (Fig. 3B). Finally, the reactivity of
H2Mab-250 to other normal tissues was investigated. As
shown in Figure 3C, no reactivity of H2Mab-250 with any
normal tissues, including breast, stomach, lung, colon, kidney, and esophagus could be observed.
Epitope identification for H2Mab-250

To determine the epitope for H2Mab-250, we examined thereactivity to CHO/HER2 (WT) and the N-terminal HER2
deletion mutant (dN218, dN342, and dN511)-expressed CHOK1 cells (Fig. 4A, left). H2Mab-250 reacted with dN218,
dN342, dN511, and HER2 (WT). In contrast, H2Mab-119 reacted with only WT, but not with dN218, dN342, and dN511.
Since HER2 (WT) and the deletion mutants possess PA16 tagat the N-terminus, all expressions on the cell surface could be
confirmed by anti-PA16 tag mAb, NZ-1 (Fig. 4A, right). These
results suggest that H2Mab-250 and H2Mab-119 recognize thedomain IV and domain I, respectively.
For further assessment of the H2Mab-250 epitope, ELISA
was performed using synthetic peptides that cover the HER2
domain IV. As shown in Figure 4B, H2Mab-250 reacted with
HER2 domain IV peptide, amino acids 603–622 and 613–
632, but not with 593–612, 623–642, and 633–652, indicatingthat H2Mab-250 recognizes the 613–622 of HER2 domain
IV. We further used alanine-substituted peptides of the 603–
622 in HER2 domain IV. A potent reduction of the reactivity‰
FIG. 4. Epitope identification for H2Mab-250. (A) Epitope determination of H2Mab-250 and H2Mab-119 using flow
cytometry. The schematic representation of HER2 and the deletion mutants (left). Flow cytometry using H2Mab-250(10 mg/mL; red line) and H2Mab-119 (10 mg/mL; blue line) against CHO/HER2 (WT and deletion mutants). The cell surface
expression was confirmed by an anti-PA tag mAb, NZ-1 (10 mg/mL; green). The cells were treated with Alexa Fluor 488conjugated anti-mouse IgG for H2Mab-250 and H2Mab-119 or Alexa Fluor 488-conjugated anti-rat IgG for NZ-1. The blackline represents the negative control (blocking buffer). (B, C) Determination of H2Mab-250 epitope by ELISA. Five synthesizedpeptides that cover the HER2 domain IV (B), alanine-substituted peptides of HER2 domain IV (603–622) (C), HER2ec, orbuffer control (NC) were immobilized on immunoplates. The plates were incubated with H2Mab-250 (10 mg/mL), followed byincubation with peroxidase-conjugated anti-mouse immunoglobulins. Optical density was measured at 655 nm. Error bars
represent means – SDs. (D) Flow cytometry using H2Mab-250 (10 mg/mL; red line) against CHO/HER2 (WT and W614A). The
cell surface expression was confirmed by an anti-PA tag mAb, NZ-1 (10 mg/mL; red line). The black line represents the negative
control (blocking buffer). (E) Surface plasmon resonance analysis between H2Mab-250 and HER2 domain IV (603–622)
peptides. The affinity constant (KA) at equilibrium was calculated as 1/KD. ELISA, enzyme-linked immunosorbent assay;
HER2ec, HER2 ectodomain; mAb, monoclonal antibody; NC, negative control; SPR, surface plasmon resonance; WT, wild type.

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KANEKO ET AL.

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Table 1. Identification of H2Mab-250 Epitope
Using Point Mutants by Biacore
PeptideSequence

KD (M)

603–622
(WT)
G603A
V604A
K605A
P606A
D607A
L608A
S609A
Y610A
M611A
P612A
I613A
W614A
K615A
F616A
P617A
D618A
E619A
E620A
G621A
A622G

GVKPDLSYMPIWKFPDEEGA

5.8 · 10-9

AVKPDLSYMPIWKFPDEEGAGAKPDLSYMPIWKFPDEEGAGVAPDLSYMPIWKFPDEEGA
GVKADLSYMPIWKFPDEEGAGVKPALSYMPIWKFPDEEGAGVKPDASYMPIWKFPDEEGAGVKPDLAYMPIWKFPDEEGAGVKPDLSAMPIWKFPDEEGAGVKPDLSYAPIWKFPDEEGAGVKPDLSYMAIWKFPDEEGAGVKPDLSYMPAWKFPDEEGAGVKPDLSYMPIAKFPDEEGAGVKPDLSYMPIWAFPDEEGAGVKPDLSYMPIWKAPDEEGAGVKPDLSYMPIWKFADEEGAGVKPDLSYMPIWKFPAEEGAGVKPDLSYMPIWKFPDAEGAGVKPDLSYMPIWKFPDEAGAGVKPDLSYMPIWKFPDEEAAGVKPDLSYMPIWKFPDEEGG

5.9 · 10-9
6.5 · 10-9
6.5 · 10-9
5.3 · 10-9
7.1 · 10-9
8.8 · 10-9
6.5 · 10-9
7.9 · 10-9
7.5 · 10-9
9.5 · 10-9
9.4 · 10-8
1.1 · 10-3
3.4 · 10-7
2.0 · 10-7
2.1 · 10-7
5.8 · 10-9
6.3 · 10-9
8.0 · 10-9
6.9 · 10-9
6.9 · 10-9

WT, wild type.

was observed in the W614A peptide (Fig. 4C). We confirmedthat the reactivity of H2Mab-250 completely disappeared in
CHO/HER2 W614A cells in flow cytometry (Fig. 4D).
The KD of H2Mab-250 with the alanine-substituted peptides of HER2 domain IV (603–622) was measured using
Biacore X100 (Table 1). The affinity constant (KA) at equilibrium was calculated as 1/KD (Fig. 4E). Compared with the
KA of the 603–622 (WT) peptide, decreased KA values wereobserved from the 613–617 region, suggesting that the 613–
617 region is involved in the binding to H2Mab-250. A remarkable reduction was measured in the W614A peptide,
indicating that Trp614 is mainly involved in the recognitionby H2Mab-250.
Discussion

In this study, we developed a cancer-specific mAb targeting
HER2. H2Mab-250 can recognize breast cancer cells, but notnormal cells in flow cytometry (Fig. 1) and immunohistochemistry (Fig. 3). H2Mab-250-mG2a-f could activate ADCC against
breast cancer cells, but not against normal epithelial cells(Fig. 2). We also identified the H2Mab-250 epitope sequence(613-IWKFP-617) by SPR analysis (Fig. 4). The 613-IWKFP-617
sequence is partially included with the wider binding epitope of
trastuzumab (residues 579–625).24 Furthermore, no reaction wasobserved between H2Mab-250 and CHO/HER2 W614A in flow
cytometry (Fig. 4), indicating that Trp614 plays a central role inrecognition by H2Mab-250. Although H2Mab-250 possesses ahigh affinity to epitope-containing peptide (603–622) in SPR
analysis, the recognition in flow cytometry using cell lines was
lower compared with H2Mab-119 (Fig. 1). In contrast, H2Mab250 exhibited a higher reactivity than H2Mab-119 in the immunohistochemical analysis using breast cancer tissues (Fig. 3).
This discrepancy might be induced by the possibility thatthe epitope sequence is partially exposed in cancer cells, but

not in normal cells in clinical cancer tissues. The mechanismof recognition by H2Mab-250 should be further investigatedin future studies.
For the clinical treatment of metastatic breast cancer, trastuzumab is administered in patients with HER2-overexpressing
tumors, which are defined by strong and complete IHC membranous staining of more than 10% of cells (IHC 3+) and/orin situ hybridization (ISH) amplified. Furthermore, trastuzumabbased antibody–drug conjugates (ADCs), such as trastuzumab–
deruxtecan (T-DXd) have been evaluated in various clinical
trials. Based on the studies, T-DXd has been approvedin not only HER2-positive breast cancers,25,26 but also
HER2-mutant lung cancer27 and HER2-low (IHC 1+ or IHC
2+/ISH-nonamplified) advanced breast cancer.28 A significant number of patients can benefit from T-DXd therapy
since approximately half of all breast cancers are classifiable
as HER2-low.29 Meanwhile, cardiotoxicity is the most significant toxicity associated with T-DXd.30 Further studies are
essential to evaluate in vivo toxicities of H2Mab-250.
H2Mab-250-mG2a-f could trigger the ADCC activity to
BT-474 selectively (Fig. 2). Although the effect of H2Mab250-mG2a-f is lower compared with trastuzumab, we should
consider that effector Jurkat cells express human FcgRIIIa receptor. In contrast, H2Mab-250 exhibited a superior reactivity
to HER2-positive breast cancer tissue sections in immunohistochemistry (Fig. 3). Chimeric antigen receptor (CAR)-T celltherapy against HER2 has been evaluated in clinical studies.29
It would be worthwhile to investigate the cancer specificity of
H2Mab-250 scFv and the efficacy of CAR-T against HER2positive tumors in future studies. In the future, H2Mab-250
could contribute to the development of CAR-T or ADCs
without adverse effects for breast cancer therapy.
Authors’ Contributions

M.K.K. and H.S. performed the experiments. M.K.K. and
Y.K. designed the experiments. H.S. and M.K.K. analyzed
the data. H.S. and Y.K. wrote the article. All authors haveread and agreed to the published version of the article.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by the Japan Agency
for Medical Research and Development (AMED) under grantnos.: JP23ama121008 (to Y.K.), JP23am0401013 (to Y.K.),
23bm1123027h0001 (to Y.K.), and JP23ck0106730(to Y.K.), and by the Japan Society for the Promotionof Science ( JSPS) Grants-in-Aid for Scientific Research(KAKENHI) grant nos. 22K06995 (to H.S.), 21K07168(to M.K.K.), and 22K07224 (to Y.K.).
Supplementary Material

Supplementary Figure S1
Supplementary Figure S2
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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: December 27, 2023
Accepted: February 5, 2024