Establishment of a Novel Cancer-Speciﬁc Anti-HER2 Monoc...

1 / 18https://www.liebertpub.com/doi/10.1089/mab.2023.0033) Monoclonal Antibodies in Immunodiagnosis and Immunotherapy > Ahead of Print
Research Article. FULL ACCESS

Published Online: 2 April 2024

Establishment of a Novel Cancer-Specific Anti-HER2 Monoclonal
Antibody H 2 Mab-250/H 2 CasMab-2 for Breast Cancers
Authors: Mika K. Kaneko, Hiroyuki Suzuki, and Yukinari Kato * +AUTHORS INFO & AFFILIATIONS

Publication: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Abstract
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,
tumor-selective or speciDc mAbs can be beneDcial in reducing the adverse effects. In this study, weestablished a novel cancer-speciDc 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 react with nontransformed normal epithelial cells (HaCaT and MCF 10A) andimmortalized normal epithelial cells in Vow cytometry. In contrast, most anti-HER2 mAbs, such as
H2Mab-119 and trastuzumab reacted with both cancer 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 react with 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 thedevelopment of chimeric antigen receptor-T or antibody–drug conjugates without adverse effects for breast
cancer therapy.

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Introduction
Human epidermal growth factor receptor 2 (HER2) IS included in the receptor tyrosine kinase family ofhuman epidermal growth factor receptors. To activate the downstream signaling, HER2 must either form
heterodimers with other HER members and their speciDc ligands or self-assemble into ligand-independent
homodimers when overexpressed.1 The HER2 overexpression is observed in ∼20% of breast cancers2 and
20% of gastric cancers,3 which 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 effect in vivo.4,5 The addition of trastuzumab to chemotherapy
improves objective response rates, progression-free survival, and overall survival in HER2-positive breast
cancer patients with metastasis.6 Trastuzumab has become the standard treatment for HER2-positive
breast cancers7 and HER2-positive gastric cancers.8 Trastuzumab has been the most effective therapy for
HER2-positive breast cancer for more than 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-deDcient mice showed the features of dilated cardiomyopathy.12 These results
indicate that HER2 is vital for normal heart development and homeostasis. Therefore, more selective antiHER2 mAbs against tumors, which can reduce heart failures are required.
We previously established several 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 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-139-mG2a, respectively), andproduced the core fucose-deDcient types (H2Mab-77-mG2a-f, H2Mab-119-mG2a-f, and H2Mab-139-mG2a-f,
respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC) and antitumor effect in vivo.
19–21 In this study, we developed and characterized a novel HER2 mAb, named H Mab-250/H CasMab-2.
22Materials and Methods
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

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Collection (ATCC, Manassas, VA). Human keratinocyte HaCaT was purchased from Cell Lines Service GmbH
(Eppelheim, Germany). The hTCEpi, hTEC/SVTERT24-B, and HCEC-1CT were purchased from EVERCYTE(Vienna, Austria).
The cDNA of HER2 (wild type; WT) and deletion mutants (dN218, dN342, and dN511) were cloned into thepCAG-nPA16 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 using a Neon transfection
system (Thermo Fisher ScientiDc, Inc., Waltham, MA). A few days after transfection, PA tag-positive 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, andW614A) cell lines were established.
CHO-K1, CHO/HER2 (WT, deletion, and point mutants), and P3U1 were cultured in Roswell Park Memorial
Institute-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and BT-474, SK-BR-3, MDA-MB-468, HEK293T,
and HaCaT were cultured in Dulbecco's modiDed Eagle's medium (DMEM) (Nacalai Tesque, Inc.),
supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher ScientiDc, Inc.), 100 U/mL of
penicillin, 100 μg/mL streptomycin, and 0.25 μg/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 ScientiDc, Inc.); RPTEC/TERT1, DMEM/F-12, and hTERT Immortalized RPTEC Growth Kit with
supplement A and B (ATCC); HCEC-1CT, DMEM/M199 (4:1, Thermo Fisher ScientiDc, Inc.), 2% Cosmic Calf
Serum (Cytiva, Marlborough, MA), 20 ng/mL hEGF (Sigma-Aldrich Corp.), 10 μg/mL insulin (Sigma-AldrichCorp.), 2 μg/mL apo-transferrin (Sigma-Aldrich Corp.), 5 nM sodium selenite (Sigma-Aldrich Corp.), 1 μg/mL
hydrocortisone (Sigma-Aldrich Corp.).
All cell lines were cultured at 37°C in a humidiDed atmosphere with 5% CO2 and 95% air.

Development of hybridomas
The animal was housed under speciDc pathogen-free conditions. All animal experiments were approved by

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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 establishedpreviously. In this study, H2Mab-193 (IgG1, kappa), H2Mab-215 (IgG1, kappa), and H2Mab-250 (IgG1, kappa)
were established by the same strategy. BrieVy, BALB/c mice were immunized with recombinant HER2ec
produced by LN229 cells together with Imject Alum (Thermo Fisher ScientiDc, Inc.). After several additional
immunizations, spleen cells were fused with P3U1 cells. The culture supernatants of hybridomas werescreened using enzyme-linked immunosorbent assay (ELISA) with recombinant HER2ec and Vow 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 cDNAof 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 ScientiDc, Inc.), and Ab-Capcher (ProteNova, Kagawa, Japan) was used to purify therecombinant H2Mab-250 and H2Mab-119.
To generate mouse IgG2a-type H2Mab-250 (H2Mab-250-mG2a), we cloned the VH cDNA of H2Mab-250 and
the CH of mouse IgG2a into the pCAG-Neo vector. The mouse kappa light 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 puriDed 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 anti-HER2 mAbs (10 μg/mL), trastuzumab(10 μg/mL), NZ-1 (10 μg/mL), or blocking buffer (control; 0.1% bovine serum albumin [BSA] in phosphatebuffered saline [PBS]) for 30 minutes at 4°C. Next, the cells were treated with Alexa Fluor 488-conjugatedanti-mouse IgG (1:1000; Cell Signaling Technology, Danvers, MA) for mouse anti-HER2 mAbs, Alexa Fluor
488-conjugated anti-human IgG (1:1000; 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 Vuorescencedata were collected using EC800 or SA3800 Cell Analyzer (Sony Corp), and the data were analyzed using

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FlowJo (BD Biosciences, Franklin Lakes, NJ).

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 perwell) were 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 FcγRIIIa receptor and a nuclear factor of activated T cell (NFAT) response element
driving DreVy luciferase, were used as effector cells. The engineered Jurkat cells (75,000 cells in 25 μL) 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).

Immunohistochemical analysis
Formalin-Dxed parasn-embedded tissue of HER2-positive breast cancer was obtained from Sendai Medical
Center.15 Informed consent for sample procurement and subsequent data analyses was obtained from thepatient or the patient'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 autoclavedin citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 minutes. The blocking was
performed using SuperBlock T20 (Thermo Fisher ScientiDc, Inc.). The sections were incubated with
H2Mab-250 (1, 0.5, or 0.1 μg/mL) and H2Mab-119 (0.5 or 0.1 μg/mL), and then treated with the EnVision+ Kit
for mouse (Agilent Technologies, Inc., Santa Clara, CA). The chromogenic reaction was performed using 3,3′diaminobenzidine tetrahydrochloride (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.

ELISA
Synthesized peptides covering the HER2 extracellular domain IV and point mutant peptides weresynthesized by Sigma-Aldrich Corp. The peptides (10 μg/mL) and HER2ec (1 μg/mL) were immobilized on
Nunc Maxisorp 96-well immunoplates (Thermo Fisher ScientiDc, Inc.). Plate washing 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 μg/mL) was added to each well. Then, the wells were further incubated withperoxidase-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

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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 CM5 in accordance with the
manufacturer's protocol by Cytiva. Immobilization of H2Mab-250 (10 μg/mL in acetate buffer (pH 4.0);
Cytiva) was carried out using an amine coupling reaction. The surface of the Vow cell 2 of the sensor chip
CM5 was treated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide, followedby the injection of H2Mab-250. The KD between H2Mab-250 and the 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 constant (kd) and KD. The asnity constant (KA) at equilibrium wascalculated as 1/KD.

Results
Establishment of H2Mab-250
We immunized mice with HER2ec produced by LN229 cells. The culture supernatants of hybridomas werescreened using ELISA with HER2ec. We further screened the reactivity to HER2-positive breast cancers(BT-474 and SK-BR-3) and nontransformed normal epithelial cells, including HaCaT (keratinocyte) and MCF
10A (mammary gland) using Vow cytometry. One of the established hybridomas, H2Mab-250, reacted with
CHO/HER2, HER2-positive BT-474, and SK-BR-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).

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FIG. 1. Flow cytometry using anti-HER2 mAbs. (A) Flow cytometry using H2Mab-250 (10 μg/mL; red line) andH2Mab-119 (10 μg/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 μg/mL; red line) and H2Mab-119 (10 μg/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 the negative control (blocking buffer). CHO, Chinese hamster ovary; HER2, human epidermal
growth factor receptor 2.

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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 notreact with those normal cells, while H2Mab-119 was reactive with all immortalized normal epithelial cells(Fig. 1B), indicating that H2Mab-250 possesses cancer-speciDc reactivity against HER2.
We further compared the reactivity of H2Mab-250 with our established anti-HER2 mAbs and trastuzumab. As
shown in Supplementary Figures S1 and S2, anti-HER2 domain I mAbs (H2Mab-7715 and H2Mab-13917), antiHER2 domain II mAbs (H2Mab-193 and H2Mab-215), anti-HER2 domain III mAbs (H2Mab-1913 andH2Mab-18118), and anti-HER2 domain IV mAbs (H2Mab-4114 and trastuzumab) reacted with HER2-positive
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 theantibody through FcγRIIIa-mediated pathway activation in an ADCC mechanism of action.23 We next
produced H2Mab-250-mG2a-f, the core-fucose deleted IgG2a version of H2Mab-250 using fucosyltransferase
8-deDcient ExpiCHO-S (BINDS-09) 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
H2Mab-250-mG2a-f and trastuzumab, we treated BT-474 and HaCaT cells with serially diluted mAbs, and thenincubated with effector Jurkat cells, which express the human FcγRIIIa receptor and an NFAT response
element driving DreVy luciferase.
As shown in Figure 2A, H2Mab-250-mG2a-f could activate the effector (EC50: 9.9 μg/mL), but it was lesseffective than trastuzumab (EC50: 0.053 μg/mL). Importantly, H2Mab-250-mG2a-f did not activate the effector
in the presence of HaCaT cells. In contrast, trastuzumab activated the effector with similar EC50 (0.036 μg/mL) to BT-474 cells (Fig. 2B). These results indicated that H2Mab-250-mG2a-f selectively activates theeffector cells against breast cancer cells.

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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.

Immunohistochemical analysis of H2Mab-250 in breast cancer and normal epithelium
Immunohistochemical analysis was performed to examine the reactivity of H2Mab-250 with normal andtumor tissue sections. In contrast to Vow 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 areassociated 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 μg/mL), no reactivity with the normal

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heart could be detected (Fig. 3B). Finally, the reactivity of H2Mab-250 to other normal tissues wasinvestigated. 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.

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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 μg/mL). (B) A normal heart
section was treated with H2Mab-250 (1 μg/mL). (C) Sections of normal breast, stomach, lung, colon, kidney, andesophagus were treated with H2Mab-250 (0.1 μg/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 μm. DAB, 3,3′-diaminobenzidine tetrahydrochloride.

Epitope identiMcation for H2Mab-250
To determine the epitope for H2Mab-250, we examined the reactivity to CHO/HER2 (WT) and the N-terminal
HER2 deletion mutant (dN218, dN342, and dN511)-expressed CHO-K1 cells (Fig. 4A, left). H2Mab-250
reacted with dN218, dN342, dN511, and HER2 (WT). In contrast, H2Mab-119 reacted with only WT, but notwith dN218, dN342, and dN511. Since HER2 (WT) and the deletion mutants possess PA16 tag at the Nterminus, all expressions on the cell surface could be conDrmed by anti-PA16 tag mAb, NZ-1 (Fig. 4A, right).
These results suggest that H2Mab-250 and H2Mab-119 recognize the domain IV and domain I, respectively.

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FIG. 4. Epitope identiDcation for H2Mab-250. (A) Epitope determination of H2Mab-250 and H2Mab-119 using Vow
cytometry. The schematic representation of HER2 and the deletion mutants (left). Flow cytometry using H2Mab-250(10 μg/mL; red line) and H2Mab-119 (10 μg/mL; blue line) against CHO/HER2 (WT and deletion mutants). The cellsurface expression was conDrmed by an anti-PA tag mAb, NZ-1 (10 μg/mL; green). The cells were treated with Alexa
Fluor 488-conjugated anti-mouse IgG for H2Mab-250 and H2Mab-119 or Alexa Fluor 488-conjugated anti-rat IgG for
NZ-1. The black line represents the negative control (blocking buffer). (B, C) Determination of H2Mab-250 epitope by
ELISA. Five synthesized peptides that cover the HER2 domain IV (B), alanine-substituted peptides of HER2 domain IV(603–622) (C), HER2ec, or buffer control (NC) were immobilized on immunoplates. The plates were incubated with
H2Mab-250 (10 μg/mL), followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins. Opticaldensity was measured at 655 nm. Error bars represent means ± SDs. (D) Flow cytometry using H2Mab-250 (10 μg/mL;
red line) against CHO/HER2 (WT and W614A). The cell surface expression was conDrmed by an anti-PA tag mAb, NZ-1
(10 μg/mL; red line). The black line represents the negative control (blocking buffer). (E) Surface plasmon resonanceanalysis between H2Mab-250 and HER2 domain IV (603–622) peptides. The asnity constant (KA) at equilibrium wascalculated as 1/KD. ELISA, enzyme-linked immunosorbent assay; HER2ec, HER2 ectodomain; mAb, monoclonal
antibody; NC, negative control; SPR, surface plasmon resonance; WT, wild type.

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, indicating that 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 was observed in the W614A peptide (Fig. 4C). We
conDrmed that the reactivity of H2Mab-250 completely disappeared in CHO/HER2 W614A cells in Vow
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 asnity constant (KA) at equilibrium was calculated as 1/KD (Fig. 4E).
Compared with the KA of the 603–622 (WT) peptide, decreased KA values were observed 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 recognition by
H2Mab-250.

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

PeptideSequence

KD (M)

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Peptidehttps://www.liebertpub.com/doi/10.1089/mab.2023.0033

Sequence

KD (M)

603–622 (WT)GVKPDLSYMPIWKFPDEEGA

5.8 × 10−9

G603A

AVKPDLSYMPIWKFPDEEGA

5.9 × 10−9

V604A

GAKPDLSYMPIWKFPDEEGA

6.5 × 10−9

K605A

GVAPDLSYMPIWKFPDEEGA

6.5 × 10−9

P606A

GVKADLSYMPIWKFPDEEGA

5.3 × 10−9

D607A

GVKPALSYMPIWKFPDEEGA

7.1 × 10−9

L608A

GVKPDASYMPIWKFPDEEGA

8.8 × 10−9

S609A

GVKPDLAYMPIWKFPDEEGA

6.5 × 10−9Y610A

GVKPDLSAMPIWKFPDEEGA

7.9 × 10−9

M611A

GVKPDLSYAPIWKFPDEEGA

7.5 × 10−9

P612A

GVKPDLSYMAIWKFPDEEGA

9.5 × 10−9

I613A

GVKPDLSYMPAWKFPDEEGA

9.4 × 10−8

W614A

GVKPDLSYMPIAKFPDEEGA

1.1 × 10−3

K615A

GVKPDLSYMPIWAFPDEEGA

3.4 × 10−7

F616A

GVKPDLSYMPIWKAPDEEGA

2.0 × 10−7

P617A

GVKPDLSYMPIWKFADEEGA

2.1 × 10−7

D618A

GVKPDLSYMPIWKFPAEEGA

5.8 × 10−9

E619A

GVKPDLSYMPIWKFPDAEGA

6.3 × 10−9

E620A

GVKPDLSYMPIWKFPDEAGA

8.0 × 10−9

G621A

GVKPDLSYMPIWKFPDEEAA

6.9 × 10−9

A622G

GVKPDLSYMPIWKFPDEEGG

6.9 × 10−9

WT, wild type.

Discussion
In this study, we developed a cancer-speciDc mAb targeting HER2. H2Mab-250 can recognize breast cancer
cells, but not normal cells in Vow cytometry (Fig. 1) and immunohistochemistry (Fig. 3). H2Mab-250-mG2a-f

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could activate ADCC against breast cancer cells, but not against normal epithelial cells (Fig. 2). We also
identiDed 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 was observed between H2Mab-250 and CHO/HER2 W614A in Vow cytometry (Fig.
4), indicating that Trp614 plays a central role in recognition by H2Mab-250. Although H2Mab-250 possesses
a high asnity to epitope-containing peptide (603–622) in SPR analysis, the recognition in Vow cytometry
using cell lines was lower compared with H2Mab-119 (Fig. 1). In contrast, H2Mab-250 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 that the epitope sequence is partially exposed in cancer
cells, but not in normal cells in clinical cancer tissues. The mechanism of recognition by H2Mab-250 should
be further investigated in future studies.
For the clinical treatment of metastatic breast cancer, trastuzumab is administered in patients with HER2overexpressing tumors, which are deDned by strong and complete IHC membranous staining of more than
10% of cells (IHC 3+) and/or in situ hybridization (ISH) ampliDed. Furthermore, trastuzumab-based
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 approved in not only HER2-positive breast cancers,25,26
but also HER2-mutant lung cancer27 and HER2-low (IHC 1+ or IHC 2+/ISH-nonampliDed) advanced breast
cancer.28 A signiDcant number of patients can beneDt from T-DXd therapy since approximately half of allbreast cancers are classiDable as HER2-low.29 Meanwhile, cardiotoxicity is the most signiDcant 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
H2Mab-250-mG2a-f is lower compared with trastuzumab, we should consider that effector Jurkat cells
express human FcγRIIIa 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 thecancer speciDcity of H2Mab-250 scFv and the escacy of CAR-T against HER2-positive tumors in futurestudies. In the future, H2Mab-250 could contribute to the development of CAR-T or ADCs without adverse
effects for breast cancer therapy.

Supplementary Material

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